Étude de l'implication de l'acide lysophosphatidique par

© Stephan Hasse, 2021

Étude de l'implication de l'acide lysophosphatidique par la production de vésicules extracellulaires vasculaires

dans les dommages associés aux maladies rhumatismales auto-immunes systémiques

Thèse

Stephan Hasse

Doctorat en microbiologie-immunologie

Philosophiæ doctor (Ph. D.)

Québec, Canada

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Résumé

L’acide lysophosphatidique (LPA) est un lipide bioactif qui est formé dans le sang par

l’autotaxine. Le LPA est un médiateur important du système vasculaire, notamment par sa

modulation de l’immunité et de l’inflammation. Plusieurs espèces moléculaires de LPA

existent en fonction de leur acide gras. Les espèces moléculaires de LPA ont des affinités

différentes pour les récepteurs aux LPA. Il en résulte que les espèces moléculaires de LPA

peuvent avoir des effets différents, même si elles ciblent une même cellule.

Parmi ses nombreux effets, le LPA induit l’activation des plaquettes et est le seul activateur

endogène connu des globules rouges (GR). L’activation des plaquettes et des GR induit la

libération de vésicules extracellulaires (EV). Les EV de plaquettes (PEV) et les EV de GR

(REV) ont des effets pro-inflammatoires et sont des acteurs importants de la coagulation.

Le LPA est connu pour promouvoir la pathophysiologie de la polyarthrite rhumatoïde (PAR),

une maladie rhumatismale auto-immune systémique (MRAS). Les patients touchés par les

MRAS comme la PAR ou le lupus érythémateux disséminé (LED) présentent une

inflammation vasculaire importante et sont plus à même de développer des maladies

cardiovasculaires comme l’athérosclérose. Les maladies cardiovasculaires sont la première

cause de mortalité chez ces patients. Le LPA et les EV promeuvent tous deux l’inflammation

vasculaire et le développement de maladies cardiovasculaires. Ils sont également impliqués

dans la coagulation. L’hypothèse à l’origine des travaux de cette thèse est que le LPA via

l’activation des GR peut promouvoir l’inflammation vasculaire et participer aux dommages

vasculaires associés aux MRAS comme l’athérosclérose et la thrombose.

Dans cette thèse, nous avons d’abord étudié l’action des principales espèces moléculaires de

LPA trouvées dans le plasma sur les GR. Par des approches de cytométrie en flux à haute

sensibilité, nous avons montré que certaines espèces moléculaires de LPA induisent

l’exposition de la phosphatidylsérine (PS) par les GR et la libération de REV PS- et PS+

similaire à celles trouvées dans le plasma de patients LED. Cependant, d’autres espèces

moléculaires de LPA inhibent l’activation des GR. J’ai établi les principales voies de

signalisation impliquées dans l’activation et l’inhibition des GR. De plus, nous avons mis en

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évidence que, même si elle est possible dans le plasma, l’activation des GR par le LPA

dépend de son environnement.

Notre deuxième focus était centré les potentielles associations entre l’autotaxine et les EV

avec le risque de thrombose et le développement de l’athérosclérose chez des patients LED.

Nous avons montré que l’autotaxine n’était pas augmentée ni associée avec l’activité de la

maladie chez les patients LED. Et bien que les patients LED présentaient des quantités très

importantes de PEV et de REV, elles n’étaient pas associées avec l’activité de la maladie.

Cependant, les quantités de REV PS+ sont associées avec un risque plus élevé de thrombose

chez les patients SLE. De plus, le groupe de patients avec des quantités élevées de REV PS+

présentait également des concentration d’autotaxine plasmatique plus élevées.

Le travail présenté dans cette thèse approfondit la compréhension de l’effet du LPA sur

l’activation des GR et leur libération de REV. Il met également en évidence l’implication

potentielle du LPA et des REV dans les thromboses associées aux patients MRAS.

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Abstract

The lysophosphatidic acid (LPA) is a bioactive lipid which is formed by autotaxin in blood.

LPA is an important mediator in the vascular system mainly through its modulation of

immunity and inflammation. Several LPA species exist depending on the fatty acid. LPA

species varies in their affinity for the LPA receptors, which means that LPA species may

have different effects, even if they target a same cell.

Among its numerous biological actions, LPA induces platelet activation and is the only

known endogenous activator of red blood cells (RBCs). Both platelet and RBC activation

lead to the liberation of extracellular vesicles (EVs). Platelet EVs (PEVs) and RBC EVs

(REVs) are the two main populations of EVs found in blood. Both PEVs and REVs have

been described as pro-inflammatory mediators and are important actors of the coagulation.

LPA is a known promoter of the pathophysiology of rheumatoid arthritis (RA), a systemic

autoimmune rheumatic disease (SARD). Patients affected by SARDs such as RA and

systemic lupus erythematosus (SLE) present high vascular inflammation and are more prone

to develop cardiovascular diseases for instance atherosclerosis. Cardiovascular diseases are

the first cause of mortality for these patients. LPA and EVs are two mediators which

promotes vascular inflammation and the development of cardiovascular diseases. Also, both

are pro-coagulant factors. The hypothesis driving this thesis is that LPA through the

activation of RBCs promotes vascular inflammation and participate to the vascular damages

associated with MRAS patients such as atherosclerosis and thrombosis.

In this thesis, we first focused our interest to study the action of major blood LPA species on

RBCs. Through high sensitivity flow cytometry, we found that some LPA species induces

the exposition of phosphatidylserine (PS) by RBCs and the liberation of PS- and PS+ REVs

similar to those found in the plasma of LED patients. However, other species were inhibitors

of RBC activation. We have established the main LPA’s signaling pathways involved in the

activation and inhibition as well that even if it is possible in the plasma, RBC activation by

LPA is affected by the environment.

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Our second focus was on the potential associations of autotaxin and EVs with thrombotic

risk and the development of atherosclerosis in SLE patients. We found that autotaxin were

not elevated in SLE patients nor associated with the disease activity. Even though, SLE

patients presented high quantities of PEVs and REVs, they were not associated with the

disease activity. However, we showed that the quantities of PS+ REVs were associated with

a higher risk of thrombosis in SLE patients. Moreover, the group of patients with high

quantities of PS+ REVs also presented higher quantities of plasmatic autotaxin.

The work presented in this thesis brings a better understanding of LPA impact on RBC

activation and REV liberation. It also highlights the potential implication of both LPA and

REVs in thrombosis associated with SARD patients.

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Table des matières

Résumé _________________________________________________________________ ii Abstract ________________________________________________________________ iv Table des matières ________________________________________________________ vi Liste des figures __________________________________________________________ ix Liste des tableaux _________________________________________________________ x

Liste des abréviations ______________________________________________________ xi Remerciements __________________________________________________________ xv Avant-propos __________________________________________________________ xvii Introduction _____________________________________________________________ 1

1 L’axe autotaxine / acide lysophosphatidique / lipide-phosphate phosphatase _____ 1 1.1 L’acide lysophosphatidique ______________________________________________________ 2

1.1.1 Structure, espèce et nomenclature _____________________________________________ 2 1.1.2 L’isolation et l’identification ________________________________________________ 3 1.1.3 L’acide lysophosphatidique chez les mammifères ________________________________ 4

1.2 Synthèse de l’acide lysophosphatidique _____________________________________________ 5 1.2.1 Synthèse intracellulaire _____________________________________________________ 6 1.2.2 Synthèse extracellulaire ____________________________________________________ 6

1.3 L’autotaxine __________________________________________________________________ 7 1.3.1 Isoformes _______________________________________________________________ 8 1.3.2 Structure et expression _____________________________________________________ 9 1.3.3 Régulation de l’expression _________________________________________________ 10

1.4 L’acide lysophosphatidique vasculaire _____________________________________________ 11 1.5 Signalisation dépendante de l’acide lysophosphatidique _______________________________ 14

1.5.1 La famille EDG des récepteurs couplés aux protéines G : LPA1, 2 et 3 ______________ 15 1.5.1.1 LPA1/Edg2 ________________________________________________________ 15 1.5.1.2 LPA2/Edg4 ________________________________________________________ 18 1.5.1.3 LPA3/Edg7 ________________________________________________________ 21

1.5.2 Récepteurs couplés aux protéines G de type non-EDG ___________________________ 24 1.5.2.1 LPA4/P2Y9 ________________________________________________________ 24 1.5.2.2 LPA5 _____________________________________________________________ 25 1.5.2.3 LPA6/P2Y5 ________________________________________________________ 27 1.5.2.4 GPR87 ____________________________________________________________ 28

1.5.3 Récepteurs non couplés à des protéines G _____________________________________ 28 1.5.3.1 TRPV1____________________________________________________________ 28 1.5.3.2 TREK-1/-2 _________________________________________________________ 29 1.5.3.3 PPARγ ____________________________________________________________ 29 1.5.3.4 Activation des cibles intracellulaires par le LPA ___________________________ 29

1.6 Régulation de l’activité du LPA : les lipide-phosphate phosphatases _____________________ 30 2 Vésicules extracellulaires _______________________________________________ 32

2.1 Diversité et formation des EV ___________________________________________________ 32 2.1.1 Les classes : exosomes, microvésicules, corps apoptotiques _______________________ 32 2.1.2 Isolation et étude _________________________________________________________ 35

2.2 Vésicules extracellulaires de plaquettes ____________________________________________ 37 2.2.1 Présentation de la plaquette ________________________________________________ 37 2.2.2 Description générale ______________________________________________________ 39 2.2.3 Fonctions_______________________________________________________________ 40

2.3 Vésicules extracellulaires de globules rouges _______________________________________ 40 2.3.1 Présentation des globules rouges ____________________________________________ 40 2.3.2 Description générale ______________________________________________________ 43 2.3.3 Fonctions_______________________________________________________________ 44

3 L’acide lysophosphatidique et les vésicules extracellulaires dans les maladies

rhumatismales auto-immunes systémiques _______________________________________ 45

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3.1 Polyarthrite rhumatoïde ________________________________________________________ 45 3.2 Lupus érythémateux disséminé __________________________________________________ 46 3.3 Comorbidité : athérosclérose ____________________________________________________ 48

4 Objectif _____________________________________________________________ 52 Chapitre 1 : Interplay between LPA2 and LPA3 in LPA-mediated phosphatidylserine cell

surface exposure and extracellular vesicles release by erythrocytes ________________ 53 1 Résumé ______________________________________________________________ 53 2 Abstract _____________________________________________________________ 55 3 Introduction __________________________________________________________ 56 4 Material and methods __________________________________________________ 57

4.1 Products ____________________________________________________________________ 57 4.2 Human plasma samples ________________________________________________________ 58 4.3 RBC isolation and activation ____________________________________________________ 58 4.4 Platelet and EV-free plasma preparation ___________________________________________ 59 4.5 RBC and REV labeling for flow cytometry _________________________________________ 59 4.6 Control for REV detection by flow cytometry _______________________________________ 60 4.7 Analysis and statistics _________________________________________________________ 60

5 Results ______________________________________________________________ 61 5.1 Detection of activated RBCs and REVs by flow cytometry _____________________________ 61 5.2 LPA species differentially activate RBCs. __________________________________________ 61 5.3 Characterization of RBC activation by LPA 18:1. ____________________________________ 62 5.4 LPA3 receptor induce RBC activation. ____________________________________________ 63 5.5 LPA2 receptor inhibits PS- REV formation. _________________________________________ 63 5.6 LPA 20:4 inhibits both RBC PS exposure and the production of PS- REVs. ________________ 64 5.7 RBC activation by LPA in physiological condition. __________________________________ 65

6 Discussion ___________________________________________________________ 66 7 References ___________________________________________________________ 69 8 Figures and legends ___________________________________________________ 74

Chapitre 2 : Plasma level of red blood cell-derived phosphatidylserine positive

extracellular vesicles are associated with thrombosis in systemic erythematous lupus

patients ________________________________________________________________ 83 1 Résumé ______________________________________________________________ 83 2 Abstract _____________________________________________________________ 85 3 Introduction __________________________________________________________ 86 4 Material and methods __________________________________________________ 87

4.1 SLE patients and healthy donors _________________________________________________ 87 4.2 SARD-BDB protocol __________________________________________________________ 87 4.3 Flow cytometry_______________________________________________________________ 88

4.3.1 Detection of platelet activation ______________________________________________ 88 4.3.2 Detection of plasmatic EVs ________________________________________________ 88

4.4 Autotaxin measurement ________________________________________________________ 89 4.5 Analysis and Statistics _________________________________________________________ 89

5 Results ______________________________________________________________ 89 5.1 Patient’s characteristics ________________________________________________________ 89 5.2 SLE patients present higher platelet activation and plasma EV levels at baseline ____________ 90 5.3 Prevalent and incident SLE patients show similar levels of plasma EVs and platelet activation. 90 5.4 Platelet activation is associated with the SLEDAI score in incident cases of SLE. ___________ 91 5.5 Higher PS+ REVs are associated with vascular damages in SLE patients. __________________ 91

6 Discussion ___________________________________________________________ 93 7 References ___________________________________________________________ 97 8 Figures, legends and tables ____________________________________________ 101

Discussion ____________________________________________________________ 110 1 Mise en contexte _____________________________________________________ 110

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2 Impact des limitations techniques dans l’analyse des vésicules extracellulaires __ 110 3 Résumé des travaux et discussion _______________________________________ 112

3.1 L’acide lysophosphatidique et les vésicules extracellulaires de globules rouges ____________ 112 3.2 Les vésicules extracellulaires de globules rouges dans le lupus érythémateux disséminée ____ 115

4 Perspectives _________________________________________________________ 117 Conclusion ____________________________________________________________ 119 Bibliographie __________________________________________________________ 121 Annexe I : Targeting the autotaxin - Lysophosphatidic acid receptor axis in cardiovascular

diseases _______________________________________________________________ 170 1 Abstract ____________________________________________________________ 171 2 Graphical abstract ___________________________________________________ 172 3 Lysophosphatidic acid and its receptors __________________________________ 173 4 LPA production pathways _____________________________________________ 175 5 The LPA-induced responses in cells of the cardiovascular system ____________ 176 6 The ATX-LPA axis in cardiovascular diseases ____________________________ 180 7 Targeted ATX-LPA therapy ___________________________________________ 184 8 Conclusions _________________________________________________________ 187 9 References __________________________________________________________ 187

Annexe II :Phosphatidylserine-specific phospholipase A1: A friend or the devil in disguise

_____________________________________________________________________ 196 1 Abstract ____________________________________________________________ 197 2 General introduction _________________________________________________ 198 3 Expression of PLA1A and lysoPS receptors in cells ________________________ 205 4 Expression of PLA1A in disease states ___________________________________ 207 5 Other enzymes regulating serine phospholipid metabolism in neural system ___ 216 6 Conclusions _________________________________________________________ 217 7 References __________________________________________________________ 217

Annexe III: Platelet-derived extracellular vesicles contain an active proteasome involved

in protein processing for antigen presentation via class I major histocompatibility

molecules _____________________________________________________________ 225 1 Abstract ____________________________________________________________ 227 2 Introduction _________________________________________________________ 228 3 Material and methods _________________________________________________ 229 4 Results _____________________________________________________________ 230 5 Discussion __________________________________________________________ 237 6 References: _________________________________________________________ 241 7 Figures _____________________________________________________________ 246

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Liste des figures

Introduction

Figure 1 : Structure biochimique du LPA. ................................................................... 2

Figure 2 : Sites d’hydrolyses des phospholipases de type A, B, C et D. ........................... 5

Figure 3 : Structure des isoformes d’autotaxine. .......................................................... 9

Figure 4 : Synthèse, signalisation et dégradation du LPA. ............................................13

Figure 5 : Récapitulatif du contenu trouvé dans les EV à l’exception des organelles. ......33

Figure 6 : Libération des différentes vésicules extracellulaires. ....................................34

Figure 7 : Les différentes méthodes d’isolation des EV. ..............................................35

Figure 8 : Interaction des globules rouges pour leur élimination par les macrophages. ....42

Figure 9 : Progression d’une plaque d’athérosclérose. .................................................50

Chapitre 1 :

Figure 1 : RBC activation and REV detection by high-sensitivity flow cytometry. .........74

Figure 2 : RBC activation by LPA varies depending of the fatty acid. ...........................75

Figure 3 : LPA induced RBC activation leads to two distinct REV populations. .............76

Figure 4 : LPA1/3 mediates RBC activation by LPA. ..................................................77

Figure 5 : LPA2 inhibits PS- REV production. ...........................................................78

Figure 6 : LPA 20:4 inhibits PS- REV production through LPA2 and PS exposure by RBCs.

...............................................................................................................................79

Figure 7 : LPA 18:1 induces PS+ REVs in platelet-free and EV-free plasma from healthy

donors. ....................................................................................................................80

Figure 8 : High plasmatic quantities of PS+ and PS- REV are present in SLE patients. ....81

Figure 9 : LPA signaling in RBCs. ............................................................................82

Chapitre 2 :

Figure 1 : High platelet activation and EV quantities are found in incident and prevalent SLE

patients. . ............................................................................................................... 105

Supplementary Figure 1 : Platelet activation and EV detection by high-sensitivity flow

cytometry. . ............................................................................................................ 107

Conclusion

Figure 1 : Récapitulatif des contributions des travaux de cette thèse. .......................... 120

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Liste des tableaux

Chapitre 2 :

Table 1 : characteristics for SLE patients included in the study at baseline. ................. 101

Table 2 : High platelet activation and EV quantities are found in SLE patients at baseline.

............................................................................................................................. 103

Table 3 : Spearman correlation between our measurement and the total SLEDAI score for

SLE patients. .......................................................................................................... 103

Table 4 : Comparison of SLE patients with low and high PS+ REVs. .......................... 104

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Liste des abréviations

ACR American College of Rheumatology

ADN Acide désoxyribonucléique

ADP Adénosine diphosphate

AGPTA Acylglycerophosphate acyltransférase

ARF6 (ADP-ribosylation factor 6)

ARN Acide ribonucléique

ATP Adénosine triphosphate

ATX Autotaxine

CCL (chemokine CC ligands)

CD (cluster of differentiation)

CMH Complexe majeur d’histocompatibilité

CXCL (chemokine CXC ligands)

DAGK Diacylglycerols kinases

DLD (Deterministic lateral displacement)

Edg Gènes de différentiation endothéliales (endothelial differentiation gene)

EGF Facteurs de croissance épidermique (Epidermal Growth Factor)

EGFR Récepteur des EGF (EGF receptor)

ELISA (enzyme-linked immunosorbent assay)

ESCRT Complexe de tri endosomal nécessaire au transport (Endosomal Sorting

Complex Required for Transport)

EULAR European Alliance of Associations for Rheumatology (anciennement

EUropean League Against Rheumatism)

EV Vésicule extracellulaire (Extracellular Vesicle)

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Fc receptors Récepteurs aux fragments cristallisables

FGF Facteurs de croissance des fibroblastes (fibroblast growth factor)

GIPC Protéine d’interaction en C-terminus de type Gα (Galpha-interacting protein

C-terminus)

GPAT Glycerophosphates acyltransferases

GPR Récepteur couplé au protéine G (G Protein-Coupled Receptor)

GR Globules rouges

Ig Immunoglobuline

IL Interleukine

IP3 Inositol trisphosphate

ISEV Société internationale pour les vésicules extracellulaires (International

Society for Extracellular Vesicles)

LDL Lipoprotéine de basse densité (low density lipoprotein)

LED Lupus érythémateux disséminé

LPA Acide lysophosphatidique (lysophosphatidic acid)

LPAR Récepteur au LPA (LPA receptor)

LPP Lipide-phosphate phosphatases

Lyso-PS Lysophosphatidylsérine

MAGI-3 Guanylate kinase associé à la membrane avec une orientation inversé 3

(membrane-associated guanylate kinase with inverted orientation-3)

MAGK Monoacylglycerols kinases

MAP kinases (Mitogen-activated protein kinases)

MRAS Maladie rhumatismale auto-immune systémique

Nabs Auto-anticorps naturels (natural antibodies)

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NFTA1 Facteur nucléaire des lymphocytes T activés 1 (Nuclear factor of activated T

cells 1)

NFκB Facteur nucléaire κ B (Nuclear factor κ B)

NHERF2 facteur 2 de régulation des échanges sodium hydrogène (sodium/hydrogen

exchanger regulatory factor 2)

oxLDL LDL oxydé (oxidized LDL)

PAR Polyarthrite rhumatoïde

PCR réaction de polymérisation en chaîne (Polymerase chain reaction)

PDZ (post synaptic density protein (PSD95), Drosophila disc large tumor

suppressor (Dlg1), and zonula occludens-1 protein (zo-1))

PEV Vésicules extracellulaires de plaquettes (platelet-derived EV)

PI3K Phosphoinositide 3-kinase

PKC Protéine kinase C

PLA/B/C/D Phospholipase de type A/B/C/D

PMA (Phorbol myristate acetate)

PPARγ récepteur intracellulaire activé par les proliférateurs de peroxysomes

(peroxisome proliferator-activated receptor gamma)

PRP Plasma riche en plaquettes

PS Phosphatidylsérine

RAB (RAS-related protein in brain)

REV EV de globule rouge (red blood cell EVs)

RhoA (Ras homolog family member A)

RhoGEF facteur d’échange de nucléotide guanine spécifique à RhoA (Rho-specific

guanine nucleotide exchange factors)

Rnase Ribonucléase

xiv

ROCK (Rho-associated protein kinase)

RT-PCR Transcription inverse PCR (reverse transcription-PCR)

SIMPLE (small integral membrane protein of lysosomes and late endosomes)

SIRPα (Signal regulatory protein α)

SLEDAI Indice d’activité de la maladie LED (SLE disease activity index)

SLICC Systemic Lupus International Collaborating Clinics

STAT3 Signal de transduction et d’activation de transcription 3 (Signal transducer

and activator of transcription 3)

TAP Protéines de transport associées à l’antigène (Transporters associated with

Antigen Processing)

TAZ (PDZ-binding motif)

TGF (Transforming growth factor)

TLR Récepteurs de type Toll (Toll-like receptors)

TNF Facteur de nécrose tumorale (Tumor necrosis factor)

TREK-1/-2 Canaux à ion de potassium apparenté TWIK-1 et -2 (TWIK related K+

channel-1 and -2)

TRIP6 protéine d’interaction au récepteur de la thyroïde 6 (thyroid receptor-

interacting protein 6)

TRPV1 Récepteur transitoire à potentiel vanilloïde 1 (transient receptor potential

vanilloid 1)

YAP (yes-associated protein 1)

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Remerciements

Je remercie mon directeur de thèse, le docteur Sylvain Bourgoin pour avoir m’avoir

accompagné tout au long de cette thèse. J’ai énormément apprécié notre relation de travail

que ce soit l’autonomie et la liberté qu’il m’a accordées pour gérer mes projets ou sa patience

et son acceptation de mes résultats négatifs.

Je remercie également l’ensemble des membres de notre laboratoire. Merci Lynn Davis, cela

a toujours été un plaisir de travailler et de discuter avec toi. Merci Chenqi Zhao, j’ai toujours

apprécié ta gentillesse et ton aide sur mes projets. Enfin merci Myriam pour ton esprit et ta

gentillesse ainsi que pour m’avoir permis mettre le point final à mes expériences.

Je remercie également l’ensemble des membres de l’équipe Boilard et Fernandez avec

lesquelles j’ai beaucoup interagi au cours de mon doctorat. Je pense tout particulièrement à

Isabelle Allaeys, Tania Lévesque, et Anne Zufferey. Vous avez été une source précieuse

d’aide et de conseil ainsi que de la bonne humeur dans nos rangées de travail. Mes

compétences ne seraient pas ce qu’elles sont sans votre apport.

Je remercie aussi les nombreux étudiants, post-doctorants et professionnels de recherche que

j’ai côtoyé. Je ne serai jamais suffisamment reconnaissant pour avoir croisé le chemin de

Geneviève, Julien, Anne, Pepito, Patate, Tania, Yann, Katerina, Oona, Aurélie, Marine,

Régis, Andréa, et Anthony. Vous avez tous été au long de ces cinq années une source de joie,

de conseil, d’encouragement et d’inspiration. Et, je suis sûr que vous continuerez de l’être.

Je ne permettrai pas d’oublier mes colocataires, Camille, Yann, Abde, Bibi et Guinness ainsi

que Mathieu et Laurence qui me permirent de me changer régulièrement les idées autour d’un

repas, d’un jeu de société ou d’un beigne.

Je remercie mes amis clermontois, Lucas, Loïc et Mazière pour ne citer qu’eux, qui malgré

leurs habitudes, ont eu la retenue et la délicatesse de ne pas me demander trop souvent quand

je finissais et ce que j’allais faire après. Merci à ma famille de m’avoir soutenu et d’avoir

compris la démarche qui nous a séparés par un océan. Et merci à mon frère de m’avoir

accueilli pour une partie de l’écriture de cette thèse.

xvi

Enfin, je ne remercierai jamais assez ma compagne qui, malgré la distance, m’a soutenu,

encouragé et a été une source de bien-être et de calme au quotidien. Ta compétence et ta

passion pour la science m’impressionne et me stimule pour améliorer mon travail. Merci.

xvii

Avant-propos

Le Dr Sylvain G Bourgoin a conçu et dirigé le projet de recherche. Il a participé à l’analyse

des données et corrigé les articles de cette thèse. Le Dr Éric Boilard a apporté son expertise

au projet de recherche. Le Dr Paul Fortin a contribué et à l’analyse des données obtenues sur

les échantillons de la biobanque MRAS du CHU de Québec – Université Laval.

J’ai conçu et réalisé les expériences, analysé et interprété les données, réalisé les analyses

statistiques et écrit les manuscrits des articles présentés aux chapitre 2 et 3 de ce document

en collaboration avec les auteurs mentionnés ci-dessous.

L’article qui constitue le chapitre 1, intitulé Interplay between LPA2 and LPA3 in LPA-

mediated phosphatidylserine cell surface exposure and extracellular vesicles release by

erythrocytes, a été publié dans le journal Biochemical Pharmacology en 2021:

Hasse S, Duchez AC, Fortin P, Boilard E, Bourgoin SG. (2021) Interplay between LPA2 and

LPA3 in LPA-mediated phosphatidylserine cell surface exposure and extracellular vesicles

release by erythrocytes. Biochem Pharmacol. 2021 Jun 30;192:114667. doi:

10.1016/j.bcp.2021.114667. Online ahead of print. PMID: 34216604

L’article qui constitue le chapitre 2, intitulé Plasma level of red blood cell-derived

phosphatidylserine positive extracellular vesicles are associated with thrombosis in

systemic erythematous lupus patients, a été soumis au journal Lupus Science & Medicine

pour publication (lupus-2021-000605).

Hasse S, Julien AS, Duchez AC, Chenqi Zhao C, Fortin P, Boilard E, Bourgoin SG.

Au cours de mon doctorat, j’ai collaboré à la rédaction de deux revues de littérature. La

première en tant que co-premier auteur qui est intitulée Targeting the autotaxin -

Lysophosphatidic acid receptor axis in cardiovascular diseases est présentée en

Annexe I :

xviii

Zhao Y, Hasse S, Zhao C, Bourgoin SG. (2019) Targeting the autotaxin - Lysophosphatidic

acid receptor axis in cardiovascular diseases. Biochem Pharmacol. 2019 Jun;164:74-81. doi:

10.1016/j.bcp.2019.03.035. Epub 2019 Mar 27. PMID: 30928673

La seconde, intitulée Phosphatidylserine-specific phospholipase A1: A friend or the devil in

disguise est présentée en Annexe II :

Zhao Y, Hasse S, Bourgoin SG. (2021) Phosphatidylserine-specific phospholipase A1: A

friend or the devil in disguise. Prog Lipid Res. 2021 Jun 22;83:101112. doi:

10.1016/j.plipres.2021.101112. Online ahead of print. PMID: 34166709

J’ai aussi collaboré significativement à plusieurs projets qui ne sont pas inclus dans cette

thèse. J’ai participé significativement à l’imagerie en microscopie à transmission ainsi qu’à

la rédaction et aux analyses statistiques dans cet article en Annexe III qui vient d’être publié

dans le journal Blood :

Marcoux G, Laroche A, Hasse S, Bellio M, Mbarik M, Tamagne M, Allaeys I, Zufferey A,

Lévesque T, Rebetz J, Karakeussian-Rimbaud A, Turgeon J, Bourgoin SG, Hamzeh-

Cognasse H, Cognasse F, Kapur R, Semple JW, Hebert MJ, Pirenne F, Overkleeft H, Florea

B, Dieude M, Vingert B and Boilard E. Platelet EVs contain an active proteasome involved

in protein processing for antigen presentation via MHC-I molecules. Blood. 2021 Jul

22:blood.2020009957. doi: 10.1182/blood.2020009957. Epub ahead of print. PMID:

34293122

J’ai également assisté significativement la réalisation d’expérience de l’article intitulé

Phospholipase A1 member A activates fibroblast-like synoviocytes through the autotax-

in-lysophosphatidic acid receptor axis, qui est en cours de révision par le journal

International Journal of Molecular Sciences.

Zhao Y, Hasse S, Vaillancourt M, Zhao C, Davis L, Boilard E, Fortin P, Di Battista J,

Poubelle PE, Bourgoin SG. Phospholipase A1 member A activates fibroblast-like

synoviocytes through the autotax-in-lysophosphatidic acid receptor axis. (ijms-1418430)

Seule la numérotation des titres a été changé par rapport aux versions publiées des articles.

1

Introduction

1 L’axe autotaxine / acide lysophosphatidique / lipide-

phosphate phosphatase

Les lipides qui présentent un groupement phosphate sont nommés phospholipides. Ils sont

essentiels aux structures membranaires ainsi qu’au transport des protéines. Bien que leur

identification et leur caractérisation datent du milieu du 19e siècle, l’identification d’une

activité biologique propre aux phospholipides ne débute que dans les années 1950. L’intérêt

pour l’acide lysophosphatidique (lysophosphatidic acid, LPA) découle du travail sur les

facteurs de Darmstoff1 et d’Arneil2. Vogt associe un facteur de stimulation des muscles lisses

qu’il nomme Darmstoff à des extraits de lipides d’intestins de mammifères et

d’amphibiens1,3. En 1963, Vogt montre les effets contractiles de différents lipides sur des

duodénums de lapins, dont celui du LPA1. Dans ces mêmes années, l’équipe d’Arneil met en

évidence un facteur vasoconstricteur dans le plasma humain qui bien que peu détectable lors

de la préparation du plasma, s’accumule quand il est entreposé à température ambiante2. De

plus, il montre par des approches de chromatographie et de traitement par des phospholipases

que ce facteur, dit d’Arneil, est proche de la lysophosphatidylcholine4. Il faut attendre

l’année 1979 pour que cette substance soit formellement identifiée comme le LPA5. Les

premiers travaux sur le LPA se concentrent sur son effet vasoconstricteur et sur les

plaquettes5,6. La découverte, au début des années 90, d’un récepteur au LPA va accélérer son

étude7. Depuis que l’importance du LPA a été établie dans des processus variés aussi bien

physiologiques, du développement embryonnaire, au recrutement lymphocytaire ou encore à

la pousse des cheveux que pathologiques comme dans le cancer, les maladies

cardiovasculaires ou les maladies rhumatismales auto-immunes systémiques (MRAS).

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1.1 L’acide lysophosphatidique

1.1.1 Structure, espèce et nomenclature

Le LPA regroupe l’ensemble des lipides formés par un squelette de glycérol avec un

groupement phosphate en position sn-3 et un acide gras en position sn-1 ou 2 (Figure 1) et

fait partie de la famille des médiateurs lipidiques bioactifs. L’acide gras forme une queue

hydrophobe connectée par le glycérol à un groupement phosphate hydrophile.

Les LPA sont divisés en trois classes en fonction de la liaison de l’acide gras au squelette de

glycérol. Les trois classes de LPA possibles sont les acyl-, alkyl- ou alkenyl-, soit

respectivement une liaison ester, éther ou vinyl éther. Les LPA vont être ensuite subdivisés

en différentes espèces selon de la nature de la chaîne carbonée de l’acide gras présent,

considérant sa longueur et son nombre d’insaturations. La longueur de la chaine est

importante pour l’activité du LPA. Les LPA à chaine courte, en dessous de 14 carbones, ne

présentent pas d’activité biologique, contrairement aux LPA à chaine longue, de 14 à 26

carbones8. Enfin, un dernier élément est la position de l’acide gras sur le squelette de glycérol

qui est soit en position sn-1 ou sn-2. Les LPA avec l’acide gras en position sn-2 sont instables

et peuvent spontanément migrer en position sn-1 à un ratio de 1 pour 99. Ces trois éléments,

illustrés en Figure 1, modifient la conformation en 3 dimensions du LPA et donc son activité

biologique8,10.

Je vais utiliser la nomenclature suivante pour définir les espèces de LPA: la position de

l’acide gras sur le glycérol- la liaison entre le glycérol et l’acide gras- LPA la longueur : le

Figure 1: Structure biochimique du LPA. L’acide gras se présente le plus souvent sous forme acyl- en

position sn-1 du glycérol. Quand l’acide gras est présent sur la position sn-2 du glycérol il peut migrer

spontanément en position sn-1.

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nombre d’insaturations de l’acide gras. Par exemple, un 1-acyl-LPA 18:1, est un LPA dont

l’acide gras est en position sn-1 du glycérol par une liaison acyl et à une chaîne de 18 carbones

qui contient une insaturation.

1.1.2 L’isolation et l’identification

L’isolation et l’extraction de lipides reposent sur leurs propriétés de solvatation. L’acide gras

des lipides est lipophile tandis que le groupement de tête est hydrophile. Donc plus l’acide

gras présente une longue chaine carbonée, plus le lipide sera soluble dans des solvants

organiques et insoluble dans l’eau. À l’inverse plus le groupement de tête est important, plus

le lipide sera soluble dans l’eau11. Les phospholipides, ce qui comprend le LPA, présentent

une solubilité intermédiaire dans la balance lipophile/hydrophile. Historiquement, l’isolation

et l’extraction des lipides avec une solubilité intermédiaire se font avec la méthode de Folch12

ou celle de Bligh et Dyer13. Ces deux méthodes reposent sur le potentiel de ces lipides à être

soluble dans un mélange de méthanol/chloroforme. Le mélange de l’échantillon avec le

méthanol, le chloroforme et une solution aqueuse va permettre l’obtention d’une phase

aqueuse et d’une phase méthanol/chloroforme qui contient les lipides. Ces deux méthodes

utilisent une solution aqueuse différente et effectuent la séparation de la phase organique et

aqueuse selon des techniques différentes. La méthode Bligh et Dyer utilise de l’eau et

l’obtention des 2 phases se fait par ajout d’eau et de chloroforme au mélange de

méthanol/chloroforme/eau13. La méthode Folch utilise du sérum physiologique comme

solution aqueuse et la séparation des 2 phases se fait sans ajout de solvant organique ou

aqueux12.

De nombreuses adaptations existent pour certaines applications, notamment pour des raisons

de sécurité et pour cibler certains glycérophospholipides difficilement isolables avec les

méthodes d’origine. Les modifications sont principalement l’utilisation de solvant moins

toxiques ou plus adapté à l’automatisation par exemple, ou servent à modifier les conditions

comme le pH ou la température. Une méthode à base de butanol a été développée pour

l’isolation et l’extraction du LPA. Les échantillons sont mélangés au 1-butanol et du 1-

butanol saturé en eau (2:1, v/v). Une fois formée, la phase organique contient le LPA14,15.

Cette méthode présente un taux de récupération supérieur à 95 %14.

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Historiquement, l’étude des lipides se faisait par chromatographies sur couche mince puis à

l’aide de la chromatographie en phase liquide à haute performance. Actuellement, la

technique la plus répandue est la chromatographie en phase liquide à haute performance

associée à la spectrométrie de masse. Elle permet d’une part la détection des espèces

moléculaires de LPA et leur quantification relative14,16-18. D’autre part, la quantification

absolue du LPA total et de ses espèces est également possible à l’aide de standards internes16.

La manipulation des lipides peut engendrer des modifications qui vont affecter la nature des

espèces et des lipides identifiés. Pour limiter la perte, les modifications et la dégradation des

lipides au cours du processus d’isolation et d’identification, il est recommandé de travailler

avec de la verrerie et de limiter l’exposition à l’air et à la lumière16,18.

1.1.3 L’acide lysophosphatidique chez les mammifères

Les classes acyl-, alkyl- et alkenyl-LPA sont détectées dans les fluides biologiques humains19

et dans les tissus de rat20-22. Les acyl-LPA sont toutefois la forme prépondérante19-22. Les

formes acyl-LPA 16:0, 18:0, 18:1 et 20:4 sont les quatre espèces présentes en quantités

élevées dans les tissus de rat. La proportion de chacune de ces espèces varie significativement

d’un tissu à l’autre20,23. Les espèces saturées sont celles les plus abondantes avec, notamment,

les acyl-LPA 16:0 et 18:0 qui peuvent représenter jusqu’à 30% et 60%, respectivement du

LPA total détecté dans les tissus22.

Le LPA est trouvé dans le milieu extracellulaire24,25 et dans le milieu intracellulaire26 au

niveau du noyau27, du réticulum endoplasmique28 et des mitochondries29. L’activité

biologique du LPA est principalement médiée par le LPA extracellulaire et les récepteurs qui

y sont associés. Le LPA intracellulaire est avant tout un intermédiaire dans le métabolisme

des glycérophospholipides. Sa formation intracellulaire a aussi été décrite dans le contexte

d’inhibition de l’activité de l’acide phosphatidique. Cependant, l’activation d’un récepteur

intracellulaire par le LPA intracellulaire a été mis en évidence30,31 et l’accumulation de LPA

intracellulaire module la survie et la migration de cellules tumorales32. Une activité

biologique propre au LPA intracellulaire reste encore débattue33. Bien que le LPA ait un rôle

dans le milieu extra- et intracellulaire, il n’y a pas d’évidence qu’il soit transporté à travers

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la membrane plasmique26. La synthèse du LPA extra- et intracellulaire est faite par des

mécanismes distincts.

1.2 Synthèse de l’acide lysophosphatidique

Différents mécanismes conduisent à la formation de LPA intracellulaire et extracellulaire. La

seule voie commune qui peut conduire à la production de LPA intracellulaire et

extracellulaire est l’oxydation de phospholipide sous l’action d’oxydants et de radicaux

libres. L’oxydation de lipoprotéine de base densité permet la production d’acyl- et d’alkyl-

LPA34,35. Les autres voies de synthèse du LPA sont spécifiques soit aux compartiments intra

ou extracellulaires et font intervenir diverses phospholipases.

Les phospholipases sont la classe d’enzyme capable d’hydrolyser certaine liaison ester dans

les lipides qui présentent un groupement phosphate. Il existe quatre classes de phospholipases

en fonction de la position de la liaison qu’elles hydrolysent (Figure 2)36. Les phospholipases

de type A (PLA) sont capables d’hydrolyser la liaison de l’acide gras position sn-1 ou sn-2

du squelette de glycérol et forment un acide gras et un lysophospholipide. Deux sous-classes

existent, les PLA1 qui hydrolysent la liaison en position sn-1 et les PLA2 qui hydrolysent la

liaison en position sn-2. Les phospholipases de type B (PLB) sont capables d’hydrolyser la

liaison entre les acides gras et le squelette de glycérol en position sn-1 et sn-2. Les

phospholipases de type C (PLC) et D (PLD) hydrolysent les liaisons de chaque côté du

groupement phosphate. Les PLC hydrolysent la liaison entre le groupement phosphate et le

squelette de glycérol. Enfin, les PLD hydrolysent la liaison entre le groupement phosphate et

le groupement de tête qui peut être une choline, une sérine ou une éthanolamine.

Figure 2 : Sites d’hydrolyses des phospholipases de type A, B, C et D.

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1.2.1 Synthèse intracellulaire

Le LPA intracellulaire peut être formé selon trois substrats différents : le monoacylglycérol,

le glycérol-3-phosphate et l’acide phosphatidique (Figure 4, partie supérieure)37. Le LPA

peut être formé par l’hydrolyse d’un acide gras de l’acide phosphatidique par une PLA1/238.

L’acide phosphatidique est généré par l’hydrolyse du groupement de tête de phospholipides

membranaires par des PLD1/2 ou par l’ajout du groupement phosphate à un diacylglycérol

par une diacylglycérol kinase. La formation à partir du glycérol-3-phosphate se fait par

l’ajout d’un acide gras par des glycérol-3-phosphate acyltransférases39. Enfin, la formation

de LPA à partir du monoacylglycérol se fait par ajout d’un groupement phosphate en sn-3

par une monoacylglycérol kinase40.

La fonction principale du LPA intracellulaire est de servir d’intermédiaire au métabolisme

des glycérophospholipides. Il peut donc être rapidement pris en charge par des

lysophosphatases et des acyl glycerol-3-phosphate acyltransférases41 pour former

respectivement du glycérol-3-phosphate et de l’acide phosphatidique.

La famille des glycérol-3-phosphate acyltransférases (1 à 4) et les acyl glycérol-3-phosphates

acyltransférases sont localisées sur la membrane des mitochondries29,39 et du réticulum

endoplasmique28,39. La production du LPA est donc localisée à ces compartiments.

Cependant, le LPA peut être transporté entre différents compartiments intracellulaires par

son association avec la protéine de liaison cytosolique du LPA (cytosolic LPA-binding

protein)39. La protéine de transport est capable d’inhiber, dans la mitochondrie, ou de

stimuler, dans le réticulum endoplasmique, la production de LPA39.

1.2.2 Synthèse extracellulaire

À ce jour, deux mécanismes de production de LPA extracellulaire ont été décrits, soit à partir

d’acide phosphatidique, soit de phospholipides comme la phosphatidyl-sérine, -choline ou -

éthanolamine (Figure 4, partie du centre).

Des PLA1/2 peuvent hydrolyser un acide gras de l’acide phosphatidique pour former le LPA.

Ce mécanisme a été mis en évidence, dans les follicules pileux avec la PLA1 membranaire

spécifique pour l’acide phosphatidique, PA-PLA1α également appelé lipase H42-44. Il a aussi

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été montré sur des vésicules extracellulaires (extracellular vesicles, EV) par des PLA2

secrétées45 et dans des lignées de cellules cancéreuses d’ovaire par des PLA2 membranaires46.

Le second mécanisme requiert l’hydrolyse d’un des acides gras de la phosphatidyl-sérine, -

choline ou -éthanolamine par des PLA1/2 pour former des lysophospholipides42,47. Le

groupement de tête des lysophospholipides est ensuite clivé par une PLD pour former le LPA.

L’activité de la PLD dans les milieux extracellulaires est assurée par l’autotaxine et constitue

la source majoritaire du LPA extracellulaire48,49.

Cette voie de synthèse utilise des sources diverses de substrats. Les phosphatidyl -sérines, -

cholines ou -éthanolamines peuvent être présentes dans les lipoprotéines50 et à la suite d’une

asymétrie de la membrane plasmique des cellules42,46,47 et de certaines EV45,51. Les EV

peuvent aussi être une source directe de lysophospholipides51. L’oxydation de lipoprotéines

génère également des lysophospholipides qui peuvent être transformés en LPA par

l’autotaxine52-54. Enfin, le milieu extracellulaire peut contenir des quantités importantes de

lysophospholipides comme c’est le cas dans le sang où la concentration en

lysophosphatidylcholine est de l’ordre de 140 µM chez l’humain55-57.

1.3 L’autotaxine

L’autotaxine (abrégée en ATX dans les figures et manuscrit) est une enzyme secrétée de la

famille des ectonucléotides pyrophosphatase/phosphodiestérase (ENPP) et peut être notée

ENPP2. Les modèles d’invalidation génique de l’autotaxine sont léthaux à cause de son rôle

essentiel dans le maintien des vaisseaux lors de la vasculogenèse embryonnaire58-60 et lors du

développement du système nerveux59,61. Un défaut d’autotaxine ou de son activité chez des

souris adultes n’induit aucune létalité ni phénotype visible62. En revanche, elle est impliquée

dans le développement de nombreuses pathologies dont l’obésité63,64, des cancers65,66, des

maladies pulmonaires67, cardiovasculaires68,69 et inflammatoires70. À ce jour, l’ensemble des

effets biologiques de l’autotaxine ont été associés à la production de LPA58-60.

L’autotaxine présente une double activité catalytique. D’une part elle peut hydrolyser des

groupements phosphates sur des nucléotides par son activité

pyrophosphatase/phosphodiestérase71. D’autre part, elle peut former du LPA et de la

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sphingosine-1-phosphate à partir de lysophospholipide48,49 et de

sphingosylphosphorylcholine72, respectivement, par son activité de PLD (Figure 2).

L’affinité de l’autotaxine pour la lysophosphatidylcholine, un des principaux substrats pour

la formation du LPA, est trois fois plus forte que pour la sphingosylphosphorylcholine72 et

dix fois plus forte que pour les nucléotides49. L’activité PLD est donc son activité principale

et son affinité pour les lysophospholipides expliquent que les effets biologiques de

l’autotaxine sont associés avec la formation de LPA.

L’expression de l’autotaxine est détectée dans la quasi-totalité des tissus testés, à l’exception

des cellules musculaires lisses et des cellules endothéliales aortiques73-76. Les tissus adipeux

et lymphoïdes présentent une expression importante de l’autotaxine75,76. En effet, depuis qu’il

a été montré que l’expression de l’autotaxine dans le tissu adipeux affecte ses quantités

plasmatiques, le tissu adipeux est considéré comme une source majeure de l’autotaxine du

milieu extracellulaire76.

1.3.1 Isoformes

Le gène de l’autotaxine, noté ATX, est composé de 27 exons et 26 introns et est présent chez

l’humain sur la région chromosomiale 8q2473,77,78. La forme murine présente une homologie

de 93% et une structure similaire à la forme humaine73,79. De nombreux épissages alternatifs

du gène sont possibles et cinq isoformes ont été détectées chez l’humain, notées ATXα, β, γ,

δ et ε (Figure 3)73,74. Le gène ATX est fortement conservé dans l’évolution80 et les isoformes

α, β, γ sont présentes chez d’autres mammifères comme la souris et le rat73. L’ensemble de

ces isoformes ont des activités PLD et de pyrophosphatase/phosphodiestérase avec des

affinités similaires pour leurs substrats73,74. Les différentes isoformes se distinguent par leur

proportion et la localisation tissulaire de leur expression73. L’ATXβ est la plus représentée et

est considérée comme la forme canonique. Elle représente la forme la plus abondante dans

les tissus testés à l’exception du cerveau. C’est l’isoforme qui est la plus utilisée pour l’étude

de l’ATX73,74,81. ATXδ est la deuxième isoforme la plus fréquente et présente une distribution

similaire à ATXβ. ATXγ est l’isoforme majoritaire dans le cerveau, mais elle est peu détectée

dans les autres tissus73. ATXα et ATXε sont faiblement détectées dans les tissus73,74. En

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revanche, ATXα est la seule isoforme capable de lier les héparanes sulfates ce qui lui permet

de localiser la production de LPA à la membrane plasmique81.82

1.3.2 Structure et expression

Toutes les isoformes de l’autotaxine ont la même structure et le même mécanisme de

sécrétion (Figure 4, partie supérieur droite)73,74. L’autotaxine est d’abord sous forme de

pré-pro-enzyme et partage la structure des ENPP1-3 soit en N-terminal un peptide signal,

avec domaine transmembranaire, suivi de 2 domaines somatomedin B, puis le domaine

catalytique, et se termine avec un domaine de type nucléase en C-terminal. Contrairement

aux ENPP1 et 3 dont le peptide signal est conservé et permet l’ancrage du domaine

transmembranaire à la membrane plasmique, le peptide signal de l’autotaxine contient un site

de clivage83. Une fois le peptide signal clivé, la forme pro-enzyme de l’autotaxine entre dans

la voie de sécrétion classique, soit dépendante du Golgi83,84. Les domaines somatomedin B

et de type nucléase sont nécessaires à cette sécrétion. Le domaine de type nucléase permet

son transport au Golgi85, où une glycosylation dans les domaines somatomedin B permet sa

sécrétion et son activité catalytique86. Avant d’être libérée dans le milieu extracellulaire, la

forme pro-enzyme de l’autotaxine est clivée davantage en N-terminal par une pro-protéine

convertase de type furine et perd son domaine transmembranaire83. La forme extracellulaire

active de l’autotaxine consiste en N-terminal de 2 domaines somatomedin B avec une

glycosylation suivie du domaine catalytique et enfin du domaine de type nucléase83,86,87.

Le domaine catalytique de l’autotaxine assure l’activité nucléotide

pyrophosphatase/phosphodiestérase et PLD sur un même site actif88,89. Bien que le domaine

de type nucléase ne porte pas d’activité catalytique, il est essentiel pour assurer la liaison des

Figure 3: Structure des isoformes d’autotaxine. ATX autotaxine, SP peptide signal, SMB domaines

somatomedin B, PDE domaine central catalytique phosphodiestérase, NUC domaine C-terminal similaire à

nucléase. (Adaptée de Perrakis et Moolenaar, 2014(82))

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substrats et est nécessaire à l’activité du domaine catalytique90,91. Les produits de l’activité

catalytique de l’autotaxine, que sont le LPA et la sphingosine-1-phosphate, inhibent son

activité enzymatique83,87. Cette inhibition est de type mixte, c’est-à-dire que le LPA et la

sphingosine ne sont pas en compétition avec le substrat pour le site actif, mais se lient à un

deuxième site sur les domaines somatomedin B83,87.

Enfin, ce sont les domaines somatomedin B qui permettent la liaison de l’autotaxine aux

intégrines β1 et β387,92 et aux héparanes sulfates pour l’isoforme α81. Cela permet de localiser

la production de LPA à proximité des récepteurs aux LPA à la surface des cellules81,87,92.

1.3.3 Régulation de l’expression

L’expression du gène ATX est régulée au niveau épigénétique93,94, transcriptionnelle et post-

transcriptionnelle95. Sous le contrôle de nombreux facteurs de transcription, sa transcription

est stimulée par la famille de la protéine activatrice 1 (activating protein-1, AP-1) qui met en

jeu c-Jun96-98, la protéine de spécificité 1 (Specific protein 1, SP1)98, le facteur HOX1399, le

facteur nucléaire κ B (Nuclear factor κ B, NFκB)100-102, le facteur nucléaire des lymphocytes

T activés 1 (Nuclear factor of activated T cells 1, NFTA1)103, le signal de transduction et

d’activation de transcription 3 (Signal transducer and activator of transcription 3,

STAT3)104 ainsi que la β-caténine105. SP3 est le seul facteur de transcription identifié comme

un répresseur98. Enfin sa régulation post-transcriptionnelle est sous le contrôle de deux

protéines de liaison à l’ARN. L’antigène humain R (Human antigen R) permet la stabilisation

de l’ARN messager et stimule l’expression de l’ATX tandis que le facteur de liaison et

dégradation ARE/poly(U) 1 (ARE/poly(U)-binding/degradation factor 1) l’inhibe95.

Ces régulations de l’expression d’ATX sont mises en jeux par différents médiateurs

extracellulaires comme des facteurs de croissance ou encore des médiateurs pro-

inflammatoires. Les facteurs de croissance épidermique (Epidermal Growth Factor, EGF),

des fibroblastes (fibroblast growth factor, FGF)106 ainsi que le facteur de nécrose tumorale

(tumor necrosis factor, TNF)70,106,107 et l’interleukine (IL-) 6108 stimulent directement

l’expression d’ATX. L’activation des récepteurs de type Toll (Toll-like receptors, TLR) 3, 4

et 9 par, respectivement, des ARN doubles brins, des lipopolysaccharides et l’ADN,

stimulent également son expression de manière indirecte105,109. Les TLR induisent la

11

production d’interféron α et β qui activent le récepteur à l’interféron α/β 1 et stimulent

l’expression d’ATX109. Enfin, l’IL-1β a été rapportée comme un activateur106 et un

inhibiteur110,111 de son expression. Outre l’IL-1, le facteur de croissance transformant β106,

l’IL-4106 ainsi que les produits de l’autotaxine, soit le LPA et la sphingosine-1-phosphate107,

inhibent son expression.

1.4 L’acide lysophosphatidique vasculaire

Dans le compartiment vasculaire, qui est le compartiment d’intérêt des travaux de cette thèse,

le LPA est de forme acyl-LPA que ce soit dans le plasma ou dans le sérum humain, bien que

la présence d’alkyl-LPA ait été proposée dans certains contextes35,112-114. Les espèces acyl-

LPA 16:0, 18:0, 18:1, 18:2 et 20:4 sont parmi les espèces les plus représentées14. Cependant,

la proportion de chaque espèce varie du plasma au sérum. Dans le plasma, l’espèce la plus

représentée est l’acyl LPA 18:2 suivie du 18:1, 18:0, 16:0 et 20:414. Dans le sérum, l’acyl

LPA 20:4 et 18:2 sont trouvés en quantité similaire suivis de l’acyl LPA 18:1, 16:0 et 18:014.

Dans la vasculature, le LPA peut être associé avec l’albumine23,115 ou avec la gelsoline116,117.

L’association avec ces protéines protègent le LPA de la dégradation et peut moduler

positivement et négativement ses effets.

Le LPA vasculaire est formé directement dans le milieu extracellulaire par l’autotaxine qui

explique l’augmentation observée dans le sérum14,47,118. L’accumulation du LPA lors de la

préparation du sérum est associée avec la libération de l’autotaxine contenue dans les

granules des plaquettes119. L’activation plaquettaire est une importante source de manière

locale d’autotaxine119. L’autotaxine peut être associée avec différents acteurs vasculaires :

d’une part les cellules, notamment les plaquettes activées92,120 et les EV, et d’autre part avec

les lipoprotéines, où elle utilise les phospholipides oxydés comme substrat54,68. Le tissu

adipeux serait une source importante de l’autotaxine75,121. En effet, la perte d’expression de

l’autotaxine dans les adipocytes entraine la diminution de 40% des quantités de LPA

plasmatique dans des modèles murins75.

La quantité plasmatique de LPA chez les personnes saines est encore sujette à débat. En

fonction des études, elle varie en quantités de l’ordre de 0,1 µM122,123 jusqu’à atteindre

1 µM124,125 alors qu’un dernier groupe d’études détecte des quantités de l’ordre de

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0,7 µM14,126,127. L’ensemble des études établissent que la concentration de LPA plasmatique

est plus élevées chez la femme que chez l’homme14,122,123,126. Enfin, une étude a associé

positivement les quantités de LPA plasmatique avec l’indice de masse corporelle126. En

situation pathologique, les quantités de LPA plasmatique peuvent atteindre jusqu’à

12 µM124,125,127.

Figure 4: Synthèse, signalisation et dégradation du LPA. La partie supérieure gauche présente la synthèse

la synthèse intracellulaire du LPA par l’action des diacylglycerol kinases (DAGK); monoacylglycerol kinases

(MAGK); glycerophosphate acyltransférases (GPAT) et les phospholipases de type A et D (PLA1/2 et PLD).

Le LPA intracellulaire est dégradé par des phosphatases, des lipide-phosphate phosphatases (LPP) et par

l’acylglycerophosphate acyltransférase (AGPTA). La partie supérieure gauche présente la synthèse et la

sécrétion d’ATX. Le centre présente la synthèse extracellulaire du LPA par l’ATX et les PLA1/2 et sa

dégradation par les LPP. L’oxydation des lipoprotéines à faible densité peut également produire du LPA intra

et extracellulaire. La partie inférieure présente la signalisation induite du LPA sur les 7 récepteurs aux protéines

G (LPA1 à 6 et GPR87), le récepteur intracellulaire PPARγ et les canaux ioniques TRPV1, TREK1 et 2. La

figure a été créée à l’aide de BioRender.com.

14

1.5 Signalisation dépendante de l’acide lysophosphatidique

L’étude de la signalisation dépendante du LPA a débuté en 1996 avec la découverte du

premier récepteur au LPA et s’est étoffé au fil des ans7. Depuis, six récepteurs aux protéines

G sont considérés comme les cibles principales du LPA et ont été nommés récepteurs au LPA

1 à 6, notés LPA1-6 selon l’ordre de leur découverte7,128-132. Les LPA1-6 sont subdivisés en

deux familles, ceux dont l’expression est reliée aux gènes de différentiation endothéliales

(endothelial differentiation gene, Edg) et ceux qui sont proches des récepteurs purinergiques,

P2Y. En plus de ces récepteurs, le LPA peut activer le récepteur couplé aux protéines G 87

(G Protein-Coupled Receptor 87, GPR87)133,134. Enfin, il a été rapporté que le LPA active le

récepteur couplé aux protéines G P2Y10135. Cependant, une étude subséquente a identifié

non pas le LPA, mais la lysophosphatidylsérine (Lyso-PS) comme ligand de P2Y10136. Il n’y

a pas eu de publication additionnelle rapportant le LPA comme activateur du P2Y10.

Outre les récepteurs aux protéines G, le LPA est un ligand du récepteur intracellulaire activé

par les proliférateurs de peroxysomes (peroxisome proliferator-activated receptor gamma,

PPARγ)30, ainsi que de plusieurs canaux ioniques comme le récepteur transitoire à potentiel

vanilloïde 1 (transient receptor potential vanilloid 1, TRPV1)137 et les canaux à ion de

potassium apparenté TWIK-1 et -2 (TWIK related K+ channel-1 and -2, TREK-1/-2)138.

La signalisation du LPA a plusieurs niveaux de complexité. D’abord, il y a le patron

d’expression de chaque récepteur qui varie en fonction du tissu et du développement128,139.

Chaque récepteur peut s’associer avec différents médiateurs intracellulaires. Les récepteurs

aux protéines G activés par le LPA peuvent interagir avec quatre protéines G différentes

(Gα12/13, Gαq/11, Gαs et Gαi). Ensuite, les effets médiés par les récepteurs au LPA peuvent leur

être propres, partagés ou encore opposés à ceux des autres récepteurs au LPA. Enfin, les

récepteurs ont des affinités différentes pour les espèces moléculaires de LPA. Il en résulte

que les effets du LPA sur l’environnement cellulaire ne s’explique pas seulement en fonction

des récepteurs présents et de leur médiateurs intracellulaires associés mais également en

fonction des espèces moléculaires de LPA mis en jeux dans l’environnement extracellulaire.

La signalisation dépendante des récepteurs est récapitulée dans la partie inférieure de la

Figure 4.

15

1.5.1 La famille EDG des récepteurs couplés aux protéines G : LPA1, 2 et 3

1.5.1.1 LPA1/Edg2

En 1996, le LPA est identifié comme ligand du récepteur codé par le gène de la zone

ventriculaire 1 (ventricular zone gene-1)7. Le récepteur est nommé par la suite LPA1. LPA1

est codé par le gène LPAR1 dans la région chromosomique 9q31.3140. Il est le deuxième

membre de la famille des récepteurs couplés aux protéines G de type EDG. De ce fait, il

partage une homologie importante avec LPA2 et 3 qui sont également des récepteurs de la

famille EDG128,141. LPA1 est fortement exprimé dans le cerveau, le cœur et les intestins, mais

son expression est communément détectée dans d’autres tissus128,139. LPA1 est le récepteur

au LPA le plus étudié. Un de ses principaux rôles est le développement et le fonctionnement

du système nerveux142. Cependant, il régule aussi de nombreux mécanismes biologiques

comme la tumorigenèse143-145, l’ostéogenèse146,147, l’inflammation et l’immunité148,149 ou

encore le système vasculaire150-152.

Activation et signalisation

La position de l’acide gras sur le squelette de glycérol n’affecte pas l’interaction du LPA

avec LPA1, cependant les formes acyl-LPA présentent une affinité plus forte comparée aux

alkyl- ou alkeny-LPA8. Les espèces insaturées de LPA présentent une affinité plus forte avec

LPA1. Les espèces de LPA qui contiennent les acides gras 16:1, 18:1, 18:2, 18:3 et 20:4

présentent la plus grande affinité pour LPA1, viennent ensuite les formes saturées, d’abord

le LPA 16:0 et ensuite 18:08. LPA 12:0 et 14:0 sont capables d’activer le LPA1 mais

nécessitent de très forte concentrations8.

La signalisation de LPA1 repose sur son interaction avec les 3 protéines G, Gα12/13, Gαq/11, et

Gαi/o 8,153,154. Son association avec Gα12/13 permet l’activation de la signalisation dépendante

de RhoA/ROCK principalement impliquée dans le réarrangement du cytosquelette155. LPA1

active la voie de signalisation dépendante de la PLC et des PKC par son interaction avec

Gαq/11154. Cette voie permet notamment à LPA1 de réguler l’entrée d’ions à la membrane

cellulaire par l’activation du canal calcique TRPV1156,157 ou l’inhibition du canal potassium

TREK-1158. Enfin sous contrôle de LPA1, Gαi/o induit les signalisations dépendantes des

16

MAP kinases et de la PI3K153 ainsi que l’inhibition de l’adénylyl cyclase154. L’association de

LPA1 à Gαi/o permet notamment la transactivation du récepteur à l’EGFR159.

Le LPA1 présente des domaines PDZ en C-terminal qui est l’acronyme des trois premières

protéines où ce domaine a été mis en évidence160. Les domaines PDZ permettent l’interaction

de l’extrémité C-terminale avec des protéines spécifiques161. Seules deux protéines

interagissent avec LPA1 par les domaines PDZ, soit la protéine d’interaction en C-terminus

de type Gα (Galpha-interacting protein C-terminus, GIPC)162 et le facteur d’échange de

nucléotide guanine spécifique à RhoA (Rho-specific guanine nucleotide exchange factors,

RhoGEF)163. L’association avec GIPC induit la dégradation de LPA1 dans les endosomes et

ainsi inhibe sa signalisation du LPA1162,164. L’interaction avec RhoGEF stimule la

signalisation dépendante de RhoA163.

Principales fonctions

Dans le système nerveux, LPA1 est exprimé par les neurones centraux et périphériques ainsi

que par les cellules gliales comme les astrocytes, les cellules de Schwann ou les

oligodendrocytes165. LPA1 promeut leur migration, leur prolifération166 et leur différentiation

ainsi que la survie cellulaire pour les cellules de Schwann167 et les neurones168. Cependant,

LPA1 peut également induire l’apoptose des neurones par l’initiation de dysfonctionnements

mitochondriaux169. De plus, le LPA1 module les flux calciques neuronaux170,171, la

production de neurotransmetteurs170,172 ainsi que l’expression des gènes associés avec la

balance d’excitation et d’inhibition dans l’hippocampe173. Enfin, LPA1 est impliqué dans le

changement de morphologie154,165,174 et dans la formation de myéline167,175.

Il résulte que le modèle d’invalidation génique de LPA1 chez la souris entraine, entre autres,

une malformation de plusieurs régions du cerveau ainsi qu’une mortalité néonatale de 50 %

à cause d’un défaut dans le comportement d’allaitement142. Les études d’invalidations

géniques conditionnelles subséquentes ont mis en évidence un lien entre LPA1 avec

différents comportements comme l’anxiété173,176,177, la régulation des émotions173, la

consommation d’alcool173 ou de nourriture178 ou encore la mémoire spatiale176,177. Enfin, son

absence induit des symptômes similaires à ceux induits lors de la schizophrénie170,172,179.

Outre le développement et le comportement, LPA1 est impliqué dans la médiation de la

17

douleur. Dans la douleur neuropathique, qui est la douleur due à une blessure ou une maladie

du système de somesthésie, LPA1 participe à l’initiation et à l’amplification de la douleur

centrale et périphérique155,180,181. Plus récemment, LPA1 a été associé à la médiation de la

douleur d’origine inflammatoire182 et aux réponses anormales de douleurs dans le contexte

du diabète183. Enfin des études récentes s’intéressent à son rôle dans la progression de la

sclérose en plaque et de la sclérose latérale amyotrophique184,185.

En dehors de ses effets spécifiques au système nerveux, le LPA1 module la minéralisation

osseuse en stimulant sa formation186 et sa résorption146. Sa signalisation stimule la formation

osseuse par la différenciation des ostéoblastes en ostéocytes187-189. Elle induit aussi le

bourgeonnement de la membrane des ostéoblastes et par conséquent la libération d’EV190.

Bien qu’il augmente l’expression de médiateur anti-inflammatoire comme le suppresseur de

tumorigénicité 2 (suppression of tumorigenity 2)191, LPA1 induit également l’expression de

cytokines pro-inflammatoires par les ostéoblastes comme IL-6 et IL-8 qui promeuvent la

différenciation en ostéoclaste147,187,192. De plus, LPA1 stimule l’activité de résorption146.

LPA1 semble davantage favoriser la minéralisation de l’os en situation physiologique186,189.

Cependant dans des modèles pathologiques, son rôle dans la résorption est mis en avant146,147.

Similairement, LPA1 stimule la formation du cartilage en stimulant la prolifération des

chondrocytes et l’assemblage de fibronectine193,194, mais il semble lié à sa dégradation dans

l’arthrite rhumatoïde147,195. Cette différence peut venir de son implication dans

l’inflammation qui promeut l’activité des ostéoclastes et donc la dégradation osseuse comme

dans un modèle d’arthrite rhumatoïde147,192.

En effet LPA1 promeut l’inflammation par la production de nombreuses cytokines pro-

inflammatoires comme IL-1, IL-6, IL-8 et IL-17 par différents types cellulaires147-

149,187,192,196. Il favorise l’adoption d’un phénotype inflammatoire par les macrophages185 et

la différenciation des lymphocytes T en lymphocytes T auxiliaires149. Il induit également

l’expression des protéines d’adhésion des cellules endothéliales148,149 et le recrutement de

neutrophiles et de macrophages au site inflammatoire148,185,197,198. LPA1 a été associé avec

l’inflammation neuronale185,199, pulmonaire198, abdominale196, systémique196 ainsi que celle

dans la membrane synoviale qui contribue à la progression de l’arthrite

rhumatoïde147,149,195,200.

18

LPA1 promeut le développement de fibroses par son effet pro-inflammatoire201-203 et en

participant au recrutement et à la prolifération des fibroblastes150,204 ainsi qu’à la formation

de collagène198,203,205. Le LPA1 est essentielle pour le développement de la fibrose dans le

modèle de sclérodermie induit par la bléomycine206.

L’effet de LPA1 dans le recrutement de cellules immunitaires et dans la fibrose, est

partiellement médié par l’augmentation de la perméabilité des parois intestinales et

vasculaires150,207. Outre son action sur la perméabilité de l’endothélium vasculaire, LPA1

stimule le recrutement et la prolifération des cellules du muscle lisse vasculaire208,209 ce qui

lui permet d’induire la formation de néo-intima à la suite de dommages aux vaisseaux

sanguins209. LPA1 module le tonus vasculaire. L’activation du LPA1 des cellules

endothéliales induit la vasodilatation151,210 alors que son activation sur les cellules

musculaires lisses induit une vasoconstriction152.

Enfin, le LPA1 présente un rôle mixte dans la tumorigenèse. LPA1 peut stimuler la survie211,

la motilité212,213, l’invasion212,213, la prolifération cellulaire214 ou encore la formation de

métastases par différentes lignées cancéreuses213. Il peut aussi stimuler l’expression

d’oncogènes143,144, de facteurs de croissance215-217 et de cytokines215. De même, plusieurs

études impliquent LPA1 dans les mécanismes de résistance aux traitements anti-

cancéreux143,144. Cependant, de nombreuses lignées cancéreuses présentent une mutation

rendant LPA1 inactif ou réprimant son expression145. Il inhibe la progression de certaines

tumeurs par la répression de la motilité218,219 ou de l’expression de facteurs de croissance220.

1.5.1.2 LPA2/Edg4

LPA2 est codé par le gène LPAR2 situé dans la région chromosomique 19p13.11 chez

l’humain et présente une forte homologie avec les deux autres récepteurs de type EDG, LPA1

et LPA3128,141. L’expression de LPA2 est détectée dans de nombreux tissus mais à des

niveaux souvent plus faibles que LPA1128,139. Une forte expression de LPA2 est présente chez

les leucocytes et dans le tissu testiculaire128 et LPA2 est également retrouvé dans l’intestin et

le cerveau139,221. L’étude de LPA2 est principalement axée sur la protection de l’endothélium

intestinal, l’organisation du système nerveux et vasculaire ainsi que sur l’immunité. Enfin,

19

LPA2 stimule également la tumorigenèse ce qui explique pourquoi il est présent dans de

nombreuses lignées cancéreuses128,222-225.


L’affinité des espèces de LPA pour LPA2 n’est pas affectée par la position de l’acide gras

sur le squelette de glycérol8. En revanche, les formes acyl-LPA sont privilégiées par rapport

aux formes alkyl- et alkenyl-LPA8. Les formes de LPA avec les acides gras 16:0, 16:1, 18:1,

18:2, 18:3 et 20:4 ont l’affinité la plus forte avec LPA2, vient ensuite LPA 14:0 puis LPA

18:0 et enfin LPA 12:0 qui active très faiblement le récepteur8.

LPA2 est capable de s’associer avec 3 protéines G, Gα12/13, Gαq/11, et Gαi/o154,226,227. Bien que

LPA2 puisse s’associer à Gα12/13 pour activer RhoA/ROCK227, LPA2 peut aussi activer cette

voie par son association avec Gαq/11 201,228,229. L’activation de la voie RhoA/ROCK par Gαq/11

permet à LPA2 d’induire l’expression de l’intégrine β6 et la transactivation de la signalisation

au TGF-β 201,228,229. Gαq/11 permet également de promouvoir l’activation de la PLC et

l’accumulation de DAG et d’IP3. L’accumulation d’IP3 permet à LPA2 d’induire la

mobilisation de calcium, pas seulement par son association avec Gαq/11, mais également avec

Gαi/o230,231. L’association de LPA2 avec Gαi/o permet également l’activation des voies des

MAP kinases et de PI3 kinase/AKT ainsi que la transactivation d’EGFR232-234.

LPA2 se différencie des autres récepteurs au LPA par un niveau de régulation supplémentaire

de son activité. Son extrémité C-terminale présente des domaines de liaison de CXXC235,236

et PDZ235,237. À l’aide des domaines PDZ, LPA2 interagit avec le facteur 2 de régulation des

échanges sodium hydrogène (sodium/hydrogen exchanger regulatory factor 2, NHERF2)236-

238, la guanylate kinase associée à la membrane avec une orientation inversée 3 (membrane-

associated guanylate kinase with inverted orientation-3, MAGI-3)238,239 et RhoGEF163. Son

interaction avec RhoGEF promeut l’activation de RhoA163. L’interaction de LPA2 avec

MAGI-3 promeut son association avec Gα12/13 tandis que NHERF2 stimule celle avec

Gαq/11238. MAGI-3 et NHERF2 sont en compétition pour LPA2 et donc ont un impact sur la

voie de signalisation induite suite à l’activation de LPA2238.

20

Outre les protéines qui interagissent avec ses domaines PDZ, LPA2 présente également des

motifs CXXC qui peuvent lier la protéine d’interaction au récepteur de la thyroïde 6 (thyroid

receptor-interacting protein 6, TRIP6)235,236,240 et le facteur inducteur d’apoptose Siva-

1236,241. Le recrutement de TRIP6 au LPA2 amplifie l’activation de NFκB dépendante de

LPA2240. Contrairement à TRIP6, Siva-1 réprime la signalisation dépendante de LPA2236,241.

Une fois lié à Siva-1, LPA2 est ubiquitinylé, ce qui conduit à la dégradation de LPA2 et de

Siva-1241.

Les interactions de LPA2 avec NHEFR2 and TRIP6 permettent également de localiser ses

effets dans l’environnement intracellulaire235,242,243. Cela permet, par exemple, d’orienter la

migration cellulaire en fonction d’un gradient de LPA242, de localiser l’effet de LPA2 au

cytosquelette235 ou proche d’effecteurs membranaires243.


Similairement à LPA1, LPA2 protège les progéniteurs de neurones contre l’apoptose244, mais

semble induire l’apoptose des neurones169. LPA2 participe aussi à la transduction neuronale

par la libération de glutamate et la mobilisation de calcium245. Enfin, LPA2 stimule la perte

de myéline après des blessures du système nerveux246.

LPA2 est impliqué dans le maintien de l’intégrité vasculaire et intestinale247-249. Il régule les

échanges de liquide par l’activation d’échangeur d’anion qui lui donne un effet anti-

diarrhéique234,250. Il induit la production de prostaglandines E2 qui sont impliquées dans la

protection des cellules gastriques contre l’environnement délétère de l’estomac248. Enfin, le

LPA2 stimule plusieurs mécanismes cellulaires impliqués dans la survie cellulaire et la

résistance à la radiation251-255. D’une part, LPA2 promeut la réparation de l’ADN252-254.

D’autre part, il diminue différents signaux apoptotiques. LPA2 inhibe la translocation de

BAX à la mitochondrie ainsi que la production de médiateurs pro-apoptotiques solubles254.

Enfin, l’activation de LPA2 induit la dégradation du facteur pro-apoptotique Siva-2 qui lie le

LPA2 activé241.

Au niveau vasculaire, LPA2 stimule la lymphangiogenèse et l’angiogenèse par l’induction

d’IL-8256,257. Il participe également au recrutement des cellules du muscle lisse en partenariat

21

avec LPA1 en réponse à une blessure de la paroi vasculaire258. Enfin, LPA2 inhibe la

différenciation des progéniteurs myéloïdes vers les voies de mégacaryocytes et érythrocytes

dans les étapes précoces de différenciation et uniquement vers le destin d’érythrocytes dans

les étages tardives259-261.

LPA2 impacte le milieu vasculaire et intestinal également de par son rôle dans l’immunité et

l’inflammation. Chez les macrophages, il stimule leur recrutement262, la production de

purine246, de cytokines pro-inflammatoires263 et des métalloprotéases matricielles264. La

signalisation du LPA2 chez les macrophages est associée à de l’inflammation dans le système

nerveux central ainsi qu’à de l’inflammation musculaire, intestinale et vasculaire262-264.

L’importance de LPA2 dans l’immunité a été la plus étudiée dans le contexte allergique. En

effet, LPA2 est impliqué dans l’allergie en réponse à une stimulation des muqueuses,

systémique ou des voies respiratoires265. Au niveau pulmonaire, LPA2 stimule le recrutement

des éosinophiles, des lymphocytes TH2 au poumon266,267. Il induit l’activation des cellules

dendritiques, lymphocytes T et la production de cytokines pro-inflammatoires265-269.

Outre ses effets pro-inflammatoires, LPA2 induit la production de cytokines pro-fibrosantes

et la transactivation du récepteur au TGFβ201,228,229,270. De plus, il induit le recrutement et la

différenciation de fibroblastes en myofibroblastes ainsi que l’accumulation de fibronectine,

d’actine et de collagène270. Ses effets promeuvent le développement de fibroses, notamment

pulmonaire228,270.

Enfin, LPA2 est fréquemment exprimé dans les cancers et supporte leur développement.

C’est notamment le cas pour les cancers de l’intestin222,271,272, du sein273,274 et des

ovaires233,275-277. Il stimule la transformation tumorale278,279, la prolifération219,240,273,280,281,

migration219,273,276,277 et l’invasion des cellules tumorales222,273,276,277. Récemment,

l’expression de LPA2 par les cellules cancéreuses est un facteur de

chimiorésistance223,224,227,282.

1.5.1.3 LPA3/Edg7

LPA3 est codé par le gène LPAR3 situé dans la région chromosomique 1p22.3 chez l’humain.

Il présente une forte homologie avec les récepteurs autres récepteurs de type EDG, LPA1 et

22

2129,141. Bien que trouvé dans de nombreux tissus, LPA3 est fortement exprimé au cerveau,

au cœur, aux testicules, à l’utérus, et aux poumons129,283-285. Les principaux domaines où le

rôle de LPA3 est étudié, sont la grossesse286,287, le maintien du système vasculaire259,288,289,

l’immunité148,290 ainsi que dans le cancer215,291.


Le LPA3 a une affinité plus forte pour les espèces de LPA avec l’acide gras en position sn-2

et pour les formes insaturées8,129. LPA3 lie plus facilement d’abord les formes acyl-LPA puis

les alkyl- et enfin les alkeny-LPA8. Les formes de LPA avec les acides gras insaturées 18:1,

18:2 et 18:3 présentent la plus forte affinité avec LPA3 suivi du LPA 20:4 puis des formes

14:0, 16:0, 16:1 et 18:08. La forme LPA 12:0 est également capable d’activer LPA3, mais

seulement à de très fortes concentrations8.

LPA3 s’associe avec les protéines G, Gα12/13292,293, Gαq/11

283, et Gαi/o294. Quand LPA3 interagit

avec Gα12/13, il active la signalisation dépendante de RhoA, ROCK et YAP292,293. Par son

association avec Gαq/11, LPA3 peut activer la signalisation dépendante de la PLC154,219,295

ainsi que la mobilisation de calcium par l’activation de la PLC et la production d’IP3154,296.

Par l’activation de Gαi/o, LPA3 induit, d’une part, la signalisation dépendante de PI3K,

d’AKT et de NFκB219,256,297, qui permet notamment l’activation de la β-caténine297 et la voie

des MAP kinases105. D’autre part, l’interaction de LPA3 avec Gαi/o permet également

d’activer la voie de signalisation dépendante de la PLC, du DAG et des PKC qui est commune

avec Gαq/11297. Enfin, son association avec Gαi/o lui permet d’inhiber l’accumulation d’AMP

cyclique154. Les trois voies, dépendantes de Gα12/13, Gαq/11 et de Gαi/o, sont capables d’induire

la transactivation du récepteur à l’EGFR293,294,298. Enfin la signalisation dépendante de RhoA

et ROCK peut être activée par LPA3 de manière indépendante de Gα12/13219,296.


Similairement à LPA1, LPA3 est impliqué au cerveau dans la réponse aux douleurs

neuropathiques et anormales180,183,299,300. Les effets cellulaires qui ont été rapportés sont en

revanche bien plus limités que pour LPA1. LPA3 induit la formation chez les neurones de

neurites, soit un prolongement de celui-ci qui peut être un axone ou une dendrite154,300. Il peut

23

également induire la libération d’ATP par les macrophages du système nerveux, les

microglies301.

LPA3 a différentes fonctions au niveau vasculaire. D’une part, de manière opposée à LPA2,

LPA3 stimule l’érythropoïèse259,261,297. Il inhibe également la différenciation vers les

mégacaryocytes302. D’autre part, similairement à LPA2, LPA3 stimule l’angiogenèse et la

lymphangiogenèse par la production d’IL-8 et de VEGF256,288,298,303,304. Il est également

impliqué dans la réparation de la paroi vasculaire et du cœur après blessure par le recrutement

et la prolifération de cellules musculaires lisses et de cardiomyocytes209,289,293. Cependant les

effets de LPA3 sur les cardiomyocytes peuvent mener à une hypertrophie cardiaque305-307.

LPA3 permet le recrutement aux sites inflammatoires et l’activation de neutrophiles et de

monocytes ainsi que de ses formes différenciées que sont les macrophages et les cellules

dendritiques 105,197,290,308. Leur activation par LPA3 conduit à la libération de nombreux

médiateurs pro-inflammatoires comme le leucotriène B4, la prostaglandine E2 ou

CCL8105,197,290. Le recrutement des monocytes par LPA3 se fait par l’expression de cytokines

dont l’IL-1, CXCL8 (IL-8) et CCL2 ainsi que par l’expression de facteur d’adhésion par les

cellules endothéliales148. Enfin, LPA3 induit l’internalisation de lipoprotéines oxydées de

basse densité (oxidized low density lipoprotein, oxLDL) par les macrophages et donc leur

transformation en cellules spumeuses309,310. La motilité des cellules spumeuses est inhibée

par LPA3311.

Les modèles d’invalidation génique ont mis évidence que LPA3 est un acteur important dans

l’implantation des embryons dans l’endomètre utérin286,287. De même, chez l’humain, la

diminution de son expression dans les endomètres utérins est associée avec une baisse de la

fertilité à cause de soucis d’implantation des embryons312,313. LPA3 active la cyclooxygénase

2 et l’oxide nitrique synthase inductible314-316. Cela lui permet de stimuler la production des

prostaglandines E2 et I2286,315-317 et de transformer les cellules stromales en cellules

sécrétrices315. Cela permet à LPA3 de promouvoir l’implantation de l’embryon par le

développement de la vascularisation314,315 et la décidualisation, soit une modification de

l’endomètre permettant l’implantation de l’embryon306,314-316. Enfin, LPA3 est impliqué dans

les contractions utérines318,319.

24

LPA3 est fréquemment exprimé par les cellules cancéreuses215,275,320-323. LPA3 stimule la

migration et l’invasion de ces cellules144,215,219,292,321,323. De plus, il stimule leur survie,

notamment en réponse à des traitements, et leur prolifération cellulaire par l’inhibition de la

sénescence et l’expression de facteurs de croissance144,219,324,325. Cependant, l’absence

d’expression de LPA3 est associée à une mortalité plus forte dans le cancer du sein et lors de

métastases pulmonaires ou au cerveau291. De plus, des études mettent en évidence une

inhibition de la migration et de la survie ainsi que de l’angiogenèse282,326-328. Il en résulte que

l’implication de LPA3 dans la tumorigenèse est ambigüe.

1.5.2 Récepteurs couplés aux protéines G de type non-EDG

1.5.2.1 LPA4/P2Y9

LPA4 est le premier récepteur de type non-EDG identifié qui est codé par le gène LPAR4

présent dans la région chromosomique Xq21.1 chez l’humain. Il présente moins de 20 %

d’homologie avec les LPA1 à 3 mais est proche des LPA5 et 6132,141. LPA4 est fortement

exprimé dans les ovaires bien qu’une faible expression de LPA4 soit retrouvée dans de

nombreux tissus comme le cœur ou le thymus130,329. La signalisation de LPA4 a

principalement été étudiée dans le cancer330-332, l’ostéogenèse333,334, ainsi que la perméabilité

vasculaire335,336 et l’angiogenèse335,337-339.


L’affinité des espèces de LPA pour LPA4 a été étudiée de manière limitée. Le LPA4 préfère

les LPA sous forme acyl- puis alkyl- et enfin alkenyl-LPA130. Parmi les espèces testées, le

LPA avec l’acide gras 18:1 présente l’affinité la plus forte suivie du LPA 18:0, puis de 16:0

et enfin 14:0130.

Une fois activé, le LPA4 est le seul récepteur au LPA capable de s’associer aux quatre

protéines G soit Gα12/13, Gαq/11, Gαs et Gαi/o 130,329. L’activation de LPA4 peut donc conduire à

l’accumulation d’AMP cyclique par son association avec Gαs 130,340. Son association avec

Gα12/13 active la signalisation RhoA/ROCK et YAP/TAZ 339,341. Quand LPA4 est associé à

Gαi/o, il met en jeu la signalisation ERK et PI3K278,330 et peut induire l’entrée de calcium dans

la cellule par l’activation de canaux cationiques329. Enfin LPA4 peut aussi induire l’activation

25

de la PLC et la mobilisation du calcium intracellulaire par son association avec Gαq/11329.

Étonnamment, LPA4 peut interagir avec Gαi/o et Gαs, alors que Gαi/o peut inhiber

l’accumulation d’AMP cyclique dépendante de Gαs. Cependant LPA4 privilégie l’interaction

avec Gαs à celle avec Gαi/o329. Enfin, la signalisation dépendante de LPA4 peut inhiber

l’activité de PPARγ342.


Bien que LPA4 inhibe la motilité cellulaire330-332 et promeut l’infiltration des leucocytes dans

les tumeurs341, son implication dans la tumorigenèse est contreversée. LPA4 stimule

également la formation d’invadopode343, et la transformation des cellules en cellules

cancéreuses278. LPA4 réprime la formation de tissu osseux en inhibant la différenciation des

ostéoblastes333,334.

Dans l’angiogenèse, LPA4 est impliqué dans le bourgeonnement de nouveaux vaisseaux par

le réarrangement du cytosquelette d’actine des cellules endothéliales335,338,339,341. De plus,

LPA4 renforce les jonctions adhérentes des cellules endothéliales vasculaires335 et stimule le

recrutement des cellules de la paroi vasculaire comme les cellules du muscle lisse et les

péricytes337. Il permet donc d’assurer le maintien et de moduler la perméabilité de la paroi

vasculaire335,336,341. Il est notamment impliqué dans la transmigration des leucocytes336,341.

Son action dans le maintien et le développement du système vasculaire est partiellement

médiée en partenariat avec LPA6339.

1.5.2.2 LPA5

LPA5 est codé par le gène LPAR5 présent dans la région chromosomique 12p13.31 chez

l’humain. Le LPA5 présente une homologie plus importante avec les LPA4 et 6 qu’avec les

récepteurs LPA1 à 3132,141. Son expression est principalement détectée dans le cœur, le

placenta, le cerveau et les intestins131 et dans certaines cellules immunitaires344,345. LPA5 est

impliqué dans les accidents ischémiques transitoires346,347, le comportement comme

l’anxiété348, la nociception348, l’immunité349-352 la régulation de la progression tumorale353.

26


LPA5 présente une affinité plus forte avec des espèces de LPA sous ses formes alkyl- plutôt

qu’acyl-LPA112,354. Les espèces de LPA avec un acide gras 16:0, 18:1, 18:2, 18:3 et 20:4

présentent des affinités similaires pour LPA5. Seules les formes LPA 18:0 et 20:0 ont des

affinités plus faibles354. La farnesyl pyrophosphate et l’arachidonoyl-glycine sont également

des ligands de LPA5 même s’ils présentent des affinités plus faibles que le LPA354,355.

Les effets du LPA5 sont induits par son association avec deux protéines G. L’association à

la protéine Gα12/13 permet d’activer la signalisation RhoA/ROCK131. LPA5 interagit

également avec la protéine Gαq/11, ce qui induitl’activation de la PLC et des PKC131,356. Les

voies de signalisation en aval de LPA5 permettent l’activation de canaux ioniques 357,358 et la

transactivation du récepteur à l’EGFR357. Enfin la signalisation dépendante de LPA5 permet

l’accumulation d’AMP cyclique bien que LPA5 n’interagisse pas avec la protéine Gαs131.


La signalisation de LPA5 dans l’intestin et les reins permet l’absorption de liquides par

l’activation de l’échangeur d’ion sodium et potassium 3 356,357,359. Au cerveau, l’implication

de LPA5 dans la nociception et le comportement s’explique d’abord par son activation du

canal ionique TRPV1358. D’autre part, l’activation de LPA5 promeut un environnement

inflammatoire par l’activation des macrophages346,360, la différentiation de la microglie en

macrophage de type M346,361 et la production de cytokines346,352.

En dehors du cerveau, son action est plus ambiguë. LPA5 induit la sécrétion de cytokines

pro-inflammatoires chez les mastocytes362 et contrôle la voie d’activation des plaquettes

dépendante du LPA112,354,363. Cependant, LPA5 est décrit comme un activateur et un

inhibiteur de l’activation des macrophages351,360. Ses effets répresseurs de l’inflammation et

de l’immunité sont supportés également par son inhibition de la signalisation des récepteurs

des lymphocytes B349 et T350 et la libération de cytokines anti-inflammatoires351.

Enfin, dans le cancer, LPA5 diminue la chimiorésistance de différentes lignées

cancéreuses223,353,364 par l’inhibition de la migration353,365-368, de l’invasion332,369 et de la

survie cellulaire332,353. Bien que le LPA5 est majoritairement décrit comme un inhibiteur de

27

la tumorigenèse, plusieurs études ont rapporté que le LPA promeut la prolifération et la

motilité de certaine cellules cancéreuses et inhibe l’activité cytotoxique des lymphocytes T

CD8+ 350,370-372.

1.5.2.3 LPA6/P2Y5

LPA6, codé par le gène LPAR6 présent dans la région chromosomique 13q14 chez l’humain,

est le dernier ajout aux récepteurs au LPA. Il est un récepteur de la classe P2Y qui est

phylogénétiquement proche des récepteurs LPA4 et LPA5132,141. Bien que son étude soit

encore limitée, LPA6 est étudié dans les domaines classiques du LPA comme le

cancer331,332,373, l’immunité et l’homéostasie vasculaire336,351,374,375, mais également dans la

pousse de cheveux44,132.


Le LPA6 a besoin de concentration de LPA plus élevée que les LPA1 à 5 pour être activé376.

Les formes alkyl- et acyl-LPA partagent des affinités similaires pour le LPA6376. De plus, il

est, avec LPA3, le seul récepteur au LPA à préférer les formes de LPA avec l’acide gras en

position sn-2 sur le squelette de glycérol376. LPA6 utilise du LPA formé au niveau des

follicules pileux, non par l’autotaxine, mais par la PA-PLA1α qui forme des 2-acyl-LPA43,44.

Enfin, l’espèce de LPA avec un acide gras 18:2 présente l’affinité la plus forte pour LPA6,

puis vient le LPA 18:1, ensuite 20:4 puis 16:0 après 18:0 et en dernier le LPA 14:0376.

LPA6 active la signalisation RhoA/ROCK par son association avec la protéine Gα12/13375,377

ainsi que les signalisations des MAPK et de la PI3K par la protéine Gαi377. Enfin,

l’accumulation d’AMP cyclique, qui est dépendante de la protéine Gαs, mise en évidence par

une première étude132, n’a pas été observé par deux autres équipes132,376. L’association de

LPA6 avec la protéine Gαs est donc incertaine. L’activation de LPA6 induit la transactivation

du récepteur au EGF44, module l’expression de cytokines351, la motilité331,332,373 et l’adhésion

cellulaire336,374,375,378.

28


LPA6 est un acteur majeur dans la différentiation et la maturation des follicules pileux43,44,379.

Sa mutation est notamment associée à la perte de cheveux chez l’humain44,132. Outre le

domaine capillaire, LPA6 participe la formation de nouveaux vaisseaux vasculaires339 et la

transmigration des lymphocytes336 bien que ce soit LPA4 qui soit déterminant dans ces

processus.

1.5.2.4 GPR87

Le LPA a été identifié comme le ligand du récepteur orphelin couplé aux protéines G

GPR87133. Les rares études sur le GPR87 se concentrent sur son rôle pro-tumorale dans le

cancer380-382.

Bien que l’activation de GPR87 par le LPA ait été mise en évidence, aucune étude décrivant

en détail son interaction avec les espèces de LPA ou ses voies de signalisation n’a été publiée.

Une seule étude a lié les effets du LPA induits par GPR87 à l’activation de la voie AKT134.

1.5.3 Récepteurs non couplés à des protéines G

1.5.3.1 TRPV1

TRPV1 est un canal calcique exprimé fortement dans les neurones383,384. Il est également

présent dans de nombreux autres tissus dont les tissus nerveux, vasculaires et certaines

cellules immunitaires384. TRPV1 est impliqué dans la thermoception, la nociception et

l’inflammation385,386. L’activation de TRPV1 par le LPA a été mis en évidence dans le

système nerveux pour la nociception chronique et aigue137,156,157,387,388 et pour les

démangeaisons358. L’activation de TRPV1 est également impliquée dans la vasoconstriction

dépendante au LPA389.

Le LPA active TRPV1 de manière indirecte dans les neurones du ganglion spinal par sa

signalisation dépendante de LPA1156,157 ou de LPA5358. Le LPA peut interagir directement

avec TRPV1 pour l’activer137,358,387. Parmi les espèces testées, seules les formes de acyl-,

alkyl-LPA 18:1 et leur analogue l’acide phosphatidique 18:1 cyclique peuvent interagir

directement avec TRPV1387. Le site de liaison du LPA est situé sur la partie intracellulaire

29

de TRPV1358,387. En revanche, le mécanisme par lequel le LPA intracellulaire active TREK-

1 et -2 est encore inconnu.

1.5.3.2 TREK-1/-2

TREK-1 et -2 sont des canaux à ion potassium de la famille des canaux potassium à 2 pores.

Ces canaux sont fortement exprimés dans les neurones et les cellules cardiaques. Ils sont

également détectés dans les poumons, le tractus intestinal, les reins et les testicules390. Cette

famille de récepteurs est impliquée dans la conduction de potassium de la membrane. Ces

récepteurs jouent un rôle neuroprotecteur390 et sont impliqués dans la nociception155.

Le LPA inhibent l’activité de TREK-1 et -2 de manière indirecte par la signalisation

dépendante du LPA1/3155,158. Cependant, de manière similaire à TRPV1, le LPA

intracellulaire peut activer TREK-1 et -2138.

1.5.3.3 PPARγ

Le PPARγ est un facteur de transcription de la famille des récepteurs nucléaires. Fortement

exprimé dans les tissus vasculaires et adipeux, PPARγ a été associé avec le métabolisme des

lipides, la régulation de la fonction endocrine et l’inflammation391,392. L’activation du PPARγ

par le LPA promeut, entre autres, le remodelage de la paroi vasculaire35,393, ainsi que la

transition de monocyte en macrophage394,395.

La signalisation du LPA dépendante de LPA1 et 3 est capable de moduler positivement et

négativement l’activité du PPARγ33,394,396-399. Outre cette action indirecte, le LPA active

également le PPARγ par interaction directe30,399. L’activation du PPARγ peut être

indépendante de l’ajout de LPA extracellulaire31,393,398, mais au contraire dépendre de la

synthèse de LPA intracellulaire31.

1.5.3.4 Activation des cibles intracellulaires par le LPA

Le mécanisme d’action du LPA sur ses cibles intracellulaires n’est pas encore complétement

décrit. Bien que le LPA peut moduler ces récepteurs par sa signalisation dépendante de ses

récepteurs membranaires, il reste à élucider les mécanismes d’interaction directe. Le LPA

active le PPARγ chez des levures transfectées en absence d’hormones et des récepteurs au

30

LPA30. Il pourrait donc être internalisé par un mécanisme encore inconnu et avoir une action

transcellulaire. En revanche, l’activation de TRPV1 par le LPA5 est abolie par l’inhibition

de la PLD intracellulaire nécessaire à la production de LPA intracellulaire358. Par ailleurs,

stimuler la voie de synthèse de LPA dépendant d’une glycerol-3-phosphate acyltransferase 1

permet l’activation du PPARγ31 et promeut la migration cellulaire32. Ces études suggèrent un

rôle de second messager pour le LPA intracellulaire.

1.6 Régulation de l’activité du LPA : les lipide-phosphate phosphatases

L’activité du LPA est principalement régulée par sa dégradation en monoacyl-glycérol à la

suite de l’hydrolyse du groupement phosphate par des lipides phosphate-phosphatases400,401.

Les monoacyl-glycérols n’ont pas d’activité biologique propre à l’exception du 2-

arachidonylglycérol qui peut être impliqué dans la signalisation des cannabinoïdes402.

Différentes lipides phosphate-phosphatases existent chez l’humain en fonction des tissus et

des compartiments cellulaires considérés, comme la LPA phosphatase dans les

mitochondries403,404 ou la phosphatase prostatique acide dans le liquide séminal405.

Cependant, les régulateurs majeurs des quantités de LPA extracellulaires sont les lipide-

phosphate phosphatases (LPP)400.

Les LPP, anciennement nommées phosphatases d’acide phosphatidique, sont des protéines

transmembranaires qui peuvent hydrolyser le groupement phosphate de certains lipides

phosphorylés que sont le LPA, la shingosine-1-phosphate, les acides phosphatidiques et les

céramides-1-phosphate. L’hydrolyse du groupement phosphate peut être fait par les LPP

quand ces lipides sont associés à de l’albumine ou sous forme de micelle401. Il existe 3 classes

distinctes de LPP, soit les LPP1, LPP2 et LPP3. Toutes les LPP sont des protéines avec 6

domaines transmembranaires et qui présentent 3 sites catalytiques406. Les 3 classes de LPP

partagent la même orientation avec les extrémités N- et C-terminales cytosoliques et leurs

sites catalytiques sont situées dans le milieu extracellulaire ou du bord de la lumière des

membrane intracellulaires406,407. Les 3 classes de LPP sont présentes à la membrane

plasmique et ont une activité catalytique dans le milieu extracellulaire400,408,409, même si elles

sont associées à des sous-domaines distincts de la membrane plasmique410,411. Elles peuvent

également être localisées sur des membranes intracellulaires. Les LPP3 sont détectées au

31

niveau du réticulum endoplasmique412, de vésicules cytoplasmiques et dans le compartiment

périnucléaire413 alors que les LPP2 sont détectées au Golgi414 et sur des vésicules

cytoplasmiques413. Les LPP1 ont été décrites à ce jour uniquement à la membrane

plasmique410,411, même si elle a des effets intracellulaires409. Les LPP peuvent former des

homo ou hétérodimères ce qui n’affecte pas leur activité, mais affecte leur localisation407.

Outre leur localisation cellulaire, les LPP varient dans leur patron d’expression tissulaire. Les

LPP1 et 3 sont détectées dans de nombreux tissu au contraire des LPP2.

À l’inverse de l’invalidation génique de LPP2 qui ne présente pas de phénotype415,

l’invalidation de LPP3 est létale au stade embryonnaire à cause d’un défaut de

vasculogenèse416, et celle de LPP1 à une incidence sur la morphologie, la reproduction et le

pelage des souris417. De plus, la LPP2 promeut l’avancée du cycle cellulaire418 alors qu’au

contraire les LPP1 et 3 inhibent la prolifération et la survie cellulaire419,420. Même si les LPP

partagent une même activité enzymatique, structure et orientation, elles ont, par leur

distribution tissulaire et cellulaire, des fonctions distinctes bien qu’il existe une certaine

redondance entre LPP1 et LPP3.

Des études d’invalidations géniques conditionnelles ont montré que LPP1 et 3 inhibent

l’action du LPA421,422 et régulent les quantités plasmatiques de LPA400. L’action des LPP sur

la signalisation du LPA se fait, d’une part, par la dégradation du LPA extracellulaire dont le

LPA plasmatique400,423, et d’autre part, en agissant sur la signalisation intracellulaire en aval

des récepteurs au LPA409,424. La localisation des LPP à des domaines membranaires

spécifiques permet de localiser la production du LPA à certains sites378. Les mécanismes

d’action des LPP sur la signalisation intracellulaire des récepteurs au LPA ne sont pas encore

décrits en détail. Cependant, une étude a associé l’inhibition intracellulaire de la signalisation

du LPA par les LPP avec la réduction des quantités intracellulaires d’acide phosphatidique

409. Cela est supporté par le fait que les LPP modulent les quantités d’acide phosphatidique

intracellulaire413 qui sont nécessaires au recrutement de certains médiateurs importants de la

signalisation dépendante des protéines G telles les protéines ras425 ou Raf-1426.

32

2 Vésicules extracellulaires

J’utilise le terme EV selon la définition de la Société internationale pour les vésicules

extracellulaires (ISEV) soit « terme générique pour les particules naturellement libérées par

des cellules, qui sont délimitées par une bicouche lipidique et qui ne peuvent pas se répliquer,

i.e. ne contiennent pas un noyau fonctionnel »427. Les EV sont conservées dans l’ensemble

des trois domaines du vivant soit archée, procaryote et eucaryote y compris chez les

végétaux428. Chez l’humain, elles sont trouvées dans l’ensemble des fluides biologiques

comme le plasma, la lymphe ou les liquides synoviaux429-431. Les principaux rôles associés

aux EV sont la communication intercellulaire, l’élimination de composé cellulaire, la

coagulation, la modulation de l’inflammation et de la réponse immune432,433. Les EV qui sont

impliquées dans la progression de différentes pathologies inflammatoires, auto-immunes et

dans le cancer, peuvent servir de biomarqueurs434-436. Enfin leur utilisation comme des

plateformes médicamenteuses ou vaccinales est également étudiée435-437.

2.1 Diversité et formation des EV

2.1.1 Les classes : exosomes, microvésicules, corps apoptotiques

Les EV sont libérées soit de manière passive ou active par les cellules. Le feuillet externe de

leur membrane présente des protéines comme des récepteurs, des molécules d’adhésion, ainsi

que des lipides, notamment la PS438-441 (Figure 5). Leur cytosol contient des protéines et des

acides nucléiques soit de l’ADN, des ARN messagers ou encore des microARN438,439,442

(Figure 5). Les EV peuvent également contenir des organelles comme les mitochondries ou

le protéasome439,442,443. 438

La composition de la membrane et le contenu du cytosol des EV dépend d’abord de la cellule

qui les produit, puis du stimulus et enfin du mécanisme qui conduit à leur formation. En effet

bien que la classification des EV soit encore débattue, leur subdivision en trois catégories

selon leur mécanisme de formation est fréquemment trouvée dans la littérature. Ces trois

subdivisions sont les corps apoptotiques, les microvésicules et les exosomes432(Figure 6).

33

Tout d’abord, les corps apoptotiques sont des fragments de cellules entrées en apoptose. Ce

sont des EV de grande taille, entre 500 et 5 000 nm qui peuvent contenir de l’ADN et des

organelles. Les corps apoptotiques transfèrent des protéines et de l’ADN, dont des oncogènes

à des cellules hôtes444,445 et sont également une source d’auto-antigènes446.

Les microvésicules, aussi appelées microparticules ou ectosomes, sont les EV produites par

un bourgeonnement de la membrane plasmique447. Les microvésicules ont une forme de

sphère ou de tubule448 et une taille comprise entre 100 et 1 000 nm, similaire aux bactéries et

à différents agrégats protéiques dont les complexes immuns449. Les plus grandes peuvent

contenir différentes organelles442. Le bourgeonnement des microvésicules a été associé avec

la perte de l’asymétrie de la membrane plasmique par la mobilisation de calcium et l’action

des flippases, floppases et scramblases450-452. Bien que l’exposition de la phosphatidylsérine

(PS) à la suite de l’asymétrie ne soit pas ubiquitaire, la PS reste fréquemment utilisée comme

un marqueur des microvésicules448,453. Leur formation peut faire intervenir la protéine

ARF6454 ainsi que des protéines de la famille du complexe de tri endosomal nécessaire au

Figure 5: Récapitulatif du contenu trouvé dans les EV à l’exception des organelles. (Colombo et al.,

2014(434))

34

transport (i.e. endosomal sorting complex required for transport ou ESCRT) bien qu’on

eût initialement restreint leurs fonctions à la production d’exosomes455-458.

Enfin les exosomes sont des EV formées dans les corps multivésiculaires puis libérées dans

le milieu extracellulaire par la fusion des corps multivésiculaires à la membrane plasmique459.

Les exosomes ont une taille similaire au virus entre 50 à 150 nm460. La principale voie de

formation des exosomes dans les corps multivésiculaires repose sur les protéines

ESCRT459,461. Des voies indépendantes des ESCRT existent aussi et font intervenir soit des

céramides, des tétraspanines ou les protéines dites SIMPLE (i.e. small integral membrane

protein of lysosomes and late endosomes)459,462-464. La fusion les corps multivésiculaires

avec la membrane plasmique est sous le contrôle de protéines de la famille RAB (i.e. RAS-

related protein in brain)459,465.

L’origine cellulaire et les mécanismes de production des EV permet d’obtenir une grande

hétérogénéité dans la composition des EV détectées dans les fluides biologiques429. Cette

hétérogénéité est accrue par la nature des stimuli à l’origine de la formation des EV qui

modifient le contenu des EV en cours de formation et donc leur fonction432. Il en résulte

qu’une même cellule peut, par exemple, libérer des EV pro- et anti-inflammatoires en

fonction du contexte cellulaire466-469. 470

Figure 6 : Libération des différentes vésicules extracellulaires. Les cellules non-apoptotiques peuvent libérer

des microvésicules par l’invagination de la membrane plasmique et former des exosomes dans les corps

multivésiculaires qui sont libérés après fusion des corps multivésiculaires à la membrane plasmique. Les

cellules apoptotiques subissent une fragmentation de la cellule pour former des corps apoptotiques. (D’après

Bian et al., 2019(462))

35

Les EV peuvent interagir avec les composants du milieu extracellulaire et avec des cellules

par l’intermédiaire des protéines et des lipides présents à leur surface. L’interaction des EV

avec une cellule peut activer différentes réponses cellulaires dont leur absorption.

L’absorption des EV est faite soit par endocytose, soit par fusion de la membrane de l’EV

avec celle de la cellule432,459. L’absorption de l’EV permet le transfert de son contenu à la

cellule hôte et provoque des réponses cellulaires variées. Ce transfert peut être une source de

métabolites, mais également de modification de la cellule hôte. Par exemple, la fusion des

membranes peut apporter de nouvelles protéines membranaires ou encore l’apport d’ARNm

ou interférent peut modifier l’expression protéique de la cellule hôte432,459.

2.1.2 Isolation et étude

Les EV partagent des caractéristiques physiques similaires à différents composés présents

dans les fluides biologiques que ce soient les complexes immuns, les lipoprotéines, des

agrégats protéiques ou encore des virus471. Cela, ajouté à l’absence de consensus pour les

différentes classes d’EV que ce soit pour leurs caractéristiques physiques ou des marqueurs

spécifiques, rend difficile l’isolement et l’étude des EV472.

Les approches d’isolation d’EV reposent sur des critères distincts qui sont la densité, la taille

et la composition de la surface des EV473(Figure 7). Il est également possible d’isoler les EV

par leur précipitation dans un polymère473(Figure 7). Bien que la combinaison de plusieurs

Figure 7 : Les différentes méthodes d’isolation des EV. L’isolation des EV peut être faite en fonction de la

densité, la taille, la composition de leur surface ou par précipitation de polymère. Les avancées dans les

techniques microfluidiques ont permis le développement de puce d’isolement d’EV. DLD, Deterministic lateral

displacement (D’après Wang et al., 2018(469))

36

approches soit généralement privilégiée, deux méthodes sont plus communément utilisées,

soit l’ultracentrifugation, avec ou sans un gradient de densité, et la chromatographie

d’exclusion de taille473. Considérant qu’aucune technique ne permette l’isolation spécifique

ou complète d’une population d’EV, que ce soit en fonction du type, (exosomes ou

microvésicules) ou d’une origine cellulaire par exemple, il reste possible d’enrichir une

population ciblée.

Leur composition est étudiée de manière ciblée par des puces ARN/ADN ou par PCR pour

les acides nucléiques et par immunobuvardages de type western et des méthodes immuno-

enzymatiques de type ELISA (i.e enzyme-linked immunosorbent assay) pour les

protéines474,475. Les études non ciblées se font par séquençage et par spectrométrie de

masse474,476,477.

Il est aussi possible d’étudier les EV de manière individuelle. L’observation de la structure

et de la taille des EV a d’abord été faite à l’aide de la microscopie électronique et

atomique448,478. La microscopie optique est limitée aux plus grandes EV puisque la résolution

est limitée à 400 nm478. Cependant, la microscopie optique à fluorescence reste utilisée pour

détecter ou suivre des EV dans des tissus ou des modèles animaux, ce qui peut être fait par

vidéomicroscopie454,479. La taille des EV peut aussi être mesurée par des techniques de

diffusion dynamique de la lumière478 ou par suivi individuel de particule478,480. Ces approches

présentent cependant des inconvénients, notamment le temps et la complexité d’analyse d’un

grand nombre d’EV ou de plusieurs marqueurs simultanément. L’utilisation d’un cytomètre

en flux, dit de haute sensibilité d’une résolution pour les évènements jusqu’à une taille de

100 nm permet de combler ces limitations par l’analyse rapide d’un grand nombre d’EV. Ces

cytomètres en flux permettent la distinction de différentes populations d’EV en fonction de

leur taille, mais aussi selon différents marqueurs de surface ou intracellulaires tel que la

présence d’organelle429,442,478. Cette approche s’est imposée dans l’étude des EV477 et sera

centrale dans les travaux présentés dans cette thèse.

Les EV trouvées dans de la circulation sanguine sont pour la majorité issues soit des

plaquettes, dites PEV, soit des GR, dites REV448,481,482. Puisque les travaux de cette thèse

s’intéressent plus spécifiquement à ces EV, elles seront décrites plus en détail.

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2.2 Vésicules extracellulaires de plaquettes

2.2.1 Présentation de la plaquette

Les plaquettes sont les cellules les plus représentées avec les GR. Les plaquettes sont des

éléments du sang de l’ordre de 2 µm délimités par une membrane plasmique qui sont

produites par les mégacaryocytes. Elles ne contiennent pas de noyau et ne sont donc pas

capable de réplication483,484. Par conséquent, les plaquettes seraient conformes avec la

définition des EV faite par l’ISEV, citée précédemment. Cependant, par souci de clarté et

pour éviter un débat sur la nature des plaquettes, je vais, dans ce document, assimiler les

plaquettes à des cellules vasculaires. Les PEV correspondent donc aux vésicules libérées par

les plaquettes soit des exosomes ou des microvésicules de plaquettes.

Par le réarrangement des microtubules, les mégacaryocytes forment des protubérances qui

peuvent se subdiviser pour former plusieurs pro-plaquettes. Les mégacaryocytes étant situés

dans la moelle osseuse, il est nécessaire pour les protubérances qui contiennent les pro-

plaquettes de s’allonger à travers l’endothélium vasculaire pour atteindre la circulation

sanguine485,486. Une fois dans le flux sanguin, les pro-plaquettes sont scindées pour libérer

les plaquettes487,488. Au cours de l’élongation et de la formation des pro-plaquettes, le

mégacaryocyte fait la synthèse d’une grande quantité de protéines plaquettaires. Dans le

même temps, des protéines de membranes et des composants intracellulaires, dont des

organelles, vont être incorporés aux pro-plaquettes489. De fait, le contenu et la composition

membranaire des plaquettes reflète celui des mégacaryocytes. Elles présentent des organelles

incluant les mitochondries, les granules denses et α, le protéasome et les ribosomes.

D’ailleurs, les plaquettes sont capables de faire de la synthèse protéique grâce aux ARN

hérités des mégacaryocytes. Par l’intermédiaire de leurs granules, les plaquettes ont une

quantité importante de cytokines et chimiokines ainsi que de protéines membranaires. Une

partie des médiateurs présents dans les granules n’est pas transférée par le mégacaryocyte,

mais est acquise par internalisation d’EV490,491.

Le rôle premier des plaquettes est la coagulation. La présence de collagène ou du facteur de

von Willebrand induit l’activation des plaquettes492-495. Leur activation libère les granules α,

ce qui induit la présentation de protéines membranaires comme des intégrines à la surface

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des plaquettes et la libération de protéines, comme l’autotaxine, le fibrinogène ou encore le

facteur de von Willebrand dans le milieu extracellulaire. Parmi les protéines membranaires

des plaquettes activées, des intégrines permettent l’agrégation des plaquettes entre elles ainsi

qu’avec d’autres cellules vasculaires comme les GR pour former un caillot496. En parallèle,

l’activation induit l’exposition de la PS à la surface des plaquettes, ce qui permet le

recrutement de cofacteurs de la coagulation497. Cela aboutit à la production de thrombine qui

stabilise le caillot par l’assemblage de filaments d’actine498. La thrombine et les médiateurs

libérés par les granules, amplifient l’activation plaquettaire et la synthèse de thrombine. Cela

permet de stabiliser le caillot498,499.

Le rôle des plaquettes n’est pas limité à l’initiation et à la promotion de la coagulation. Elles

présentent un nombre important de récepteurs qui peuvent induire leur activation.

L’ensemble des TLR exprimés chez l’humain, soit TLR1 à 10, y sont trouvés500. Les TLR

permettent la reconnaissance des motifs moléculaires associés soit aux dommages cellulaires,

des molécules d’origine endogène, soit aux pathogènes, alors des molécules d’origine

exogène. Le rôle de chaque TLR est dépendant de sa localisation et du motif qu’il reconnait.

Les TLR1, 2, 4, 5 et 6 sont présents à la membrane et reconnaissent différents types de

molécules de structures de micro-organismes501. Les TLR3, 7, 8 et 9 sont présents dans des

vésicules intracellulaires et reconnaissent des motifs d’acides nucléiques501. Également à leur

surface, plusieurs récepteurs aux fragments cristallisables des immunoglobulines (Ig) sont

présents et permettent de reconnaitre les IgA, E et G502-504. Elles présentent également des

récepteurs pour la thrombine505, l’ADP506, les prostaglandines506, différentes chimiokines507,

la sphingosine-1-phosphate508 et le LPA, en particulier LPA5 comme déjà présenté dans les

sections précédentes112,354,363. Il en résulte que les plaquettes peuvent être activées dans de

nombreux contextes inflammatoires et lors des réponses immunitaires innées et adaptatives.

En effet, les plaquettes activées sont capables de phagocytose, notamment de

pathogènes509,510, et elles libèrent des médiateurs pro-inflammatoires492,511,512. Ensuite, les

cytokines libérées et les intégrines présentes à la surface des plaquettes activées permettent

le recrutement et l’activation des cellules vasculaires dont les neutrophiles513,514, les

monocytes515, les cellules dendritiques515 et les lymphocytes516-519. L’activation des

lymphocytes T CD8+ par les plaquettes se fait lors de la présentation antigénique, par

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l’intermédiaire du complexe majeur d’histocompatibilité de classe I (CMH I)520. Les

plaquettes contiennent un protéasome fonctionnel qui permet la production d’antigène pour

l’apprêtement du CMH I520-522. L’activation des plaquettes ne se limite pas uniquement à

stimuler la réponse immunitaire. Elles libèrent également des modulateurs de l’immunité

adaptative telle la sérotonine ou encore le facteur plaquettaire 4 qui inhibent respectivement

l’activation des cellules dendritiques523 ou des lymphocytes T524.

2.2.2 Description générale

Les PEV constituent la première population d’EV dans le sang et représentent plus de la

moitié des EV trouvées dans le sang448,481,482. Elles sont libérées de manière passive en

réponse aux pressions physiques exercées par le flot sanguin sur les plaquettes, dites

contrainte de cisaillement (i.e. shear stress)525 ou lors de leur entreposage suite à un

prélèvement sanguin526,527. Les EV peuvent également être produites de manière active par

l’activation des plaquettes en réponse à une multitude de signaux qui ont été brièvement

présentés dans la section précédente, dont la thrombine528 ou le LPA112,354,363. La libération

de PEV en réponse à la thrombine ou au collagène est associée à l’activation d’une

scramblase dépendante du calcium et à la perte d’asymétrie de la membrane plasmique452,529.

Les plaquettes présentent différentes protéines de surface dont des intégrines, la PS et le

CD41439. Ce dernier est souvent utilisé comme marqueur de l’origine plaquettaire des EV.

Les PEV contiennent une grande diversité de protéines intracellulaires dont des récepteurs,

des facteurs de transcription, de coagulation et des cytokines439. En plus des différentes

protéines, les PEV peuvent aussi contenir des microARN530,531. Enfin, elles peuvent aussi

contenir des organelles comme le protéasome ou les mitochondries442. Ces dernières peuvent

également être libérées par les plaquettes activées sans nécessairement être contenues dans

une vésicule, elles sont appelées alors mitochondries libres442,527. Les PEV présentent une

grande hétérogénéité de contenu429. D’une part, celles de grandes tailles ont une plus grande

probabilité d’avoir des mitochondries fonctionnelles et d’autre part les stimuli à l’origine de

la libération des PEV modulent leur contenu et donc leur activité429,468,532.

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Les PEV sont éliminées de la circulation par phagocytose par les macrophages533 ou par

internalisation par les cellules endothéliales vasculaires si elles présentent de la PS534. Les

PEV négatives pour la PS seraient également drainées dans les vaisseaux lymphatiques535.

2.2.3 Fonctions

Les fonctions des PEV sont le reflet de celles des plaquettes, soit la coagulation466,467 et la

modulation de l’immunité. Les PEV peuvent adhérer à l’endothélium vasculaire endommagé

par leur fixation au collagène, au facteur de von Willebrand, au fibrinogène et aux

plaquettes466,536. De plus, certaines PEV peuvent exposer du facteur tissulaire et de la PS ce

qui permet le recrutement de différents facteurs de la coagulation et la production de

thrombine526,536,537. Cependant dans certaines conditions, les plaquettes libèrent également

des PEV avec des propriétés anticoagulantes468,469.

Dans l’immunité, les PEV ne sont pas seulement une source de médiateurs pro-

inflammatoires et de motifs de dommages associés aux dégâts cellulaires, mais peuvent

moduler l’activité de certaines cellules immunitaires439,442,538-540. Elles peuvent notamment

induire la différenciation de lymphocytes T régulateurs et aussi inhiber leur activité541,542.

Les PEV induisent également la différenciation et l’activation de lymphocyte B543,544.

Certaines sous-populations de PEV sont capables de présenter des antigènes et d’activer la

lymphoprolifération443,544. Les PEV ont donc un rôle dans la stimulation, mais également

dans l’inhibition de la réponse immune. Conséquence de leur petite taille, les PEV peuvent

diffuser dans les tissus et propager l’inflammation, comme c’est notamment le cas dans le

compartiment synovial de patients souffrant d’arthrite rhumatoïde447,545.

2.3 Vésicules extracellulaires de globules rouges

2.3.1 Présentation des globules rouges

Les GR sont des cellules sanguines les plus représentées avec des concentrations autour de

5x109 cellules/mL chez l’humain et représentent approximativement 45% du volume

sanguin. Leur fonction principale est d’assurer les échanges gazeux d’oxygène et de dioxyde

de carbone entre les poumons et les tissus.

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Les globules rouges sont constitués de 1 à 3% de réticulocytes546. Les réticulocytes sont

formés dans la moelle osseuse par l’énucléation des érythroblastes, avant de rejoindre la

circulation sanguine. Ils ont une taille de 10 à 13 µm. Bien qu’ils ne présentent plus de noyau,

leur contenu faible en ARN messagers permet encore la synthèse de protéines nécessaires à

leur maturation547. Lors de la maturation, les réticulocytes produisent une importante quantité

d’hémoglobine, dont la synthèse débute plus tôt dans l’érythropoïèse. Cependant la

production d’hémoglobine est cette fois combinée à une dégradation des organelles, comme

les mitochondries, et également celles nécessaires à la synthèse protéique comme les

ribosomes548-551. Les réticulocytes présentent aussi une forte activité de RNase551,552 et il y a

également une dégradation ou une expulsion de protéines membranaires et intracellulaires,

soit par des autophagosomes553 ou bien par la production d’EV554-557. La maturation permet

d’une part la perte de nombreuses fonctions cellulaires des réticulocytes, mais aussi un

remodelage important de la membrane avec la perte de 20% de sa surface558-560. Ce

remodelage permet l’obtention de la forme biconcave qui leur confère une déformabilité

importante560,561. L’issue du processus de maturation aboutit aux érythrocytes.

Les érythrocytes constituent 97 à 99% des globules rouges546. Ce sont des cellules d’une

taille de 6 à 8 µm d’une forme biconcave qui contiennent de grande quantité d’hémoglobine

(plus de 90% de leur masse sèche) pour le transport d’oxygène et de dioxyde de carbone.

L’étape de maturation fait en sorte que les érythrocytes ne présentent ni noyau, ni organelles.

Il n’y a donc pas de synthèse de protéine chez les érythrocytes même s’ils contiennent encore

des ARN, notamment de petites tailles et des ARN interférents562-564. L’absence de

mitochondrie les empêche de consommer l’oxygène qu’ils transportent. Ils utilisent donc la

glycolyse comme source d’ATP560. Ils ont d’ailleurs une réserve importante d’ATP qui leur

permet de moduler leur forme ainsi que le tonus vasculaire par sa libération565. Enfin, leur

élimination de la circulation sanguine est faite après environ 120 jours par leur phagocytose

par des macrophages. Plusieurs mécanismes sont proposés (Figure 8). Le premier est que

BAND-3, la protéine transmembranaire la plus représentée à la membrane des érythrocytes,

soit ciblée par des auto-anticorps naturels (i.e. natural occurring antibodies)566. Le

deuxième reposerait sur la présence de CD47 à la membrane. CD47 interagit avec SIRPα

pour inhiber la phagocytose. Le vieillissement des érythrocytes est associé à une diminution

du CD47 membranaire567,568. Également, chez des érythrocytes vieillissants, l’interaction de

42

CD47 avec SIRPα permet sa liaison au macrophage et sa subséquente phagocytose569. Le

troisième est que les érythrocytes peuvent présenter à leur surface un signal de phagocytose

qui est la PS570-572. La PS qui est normalement dans le feuillet interne de la membrane peut

être exposé à la surface de cellules lors du vieillissement des érythrocytes573,574 ou lorsqu’ils

sont stimulés par du LPA575-577. La perte de l’asymétrie de la PS à la membrane est

partiellement expliquée par à une baisse de l’activité des flippases lors du vieillissement des

érythrocytes578. 579

Bien que le rôle des GR ait longtemps été confiné aux transports et aux échanges gazeux, de

nouvelles fonctions vasculaires émergent. Les GR seraient, d’une part, impliqués dans la

coagulation et la thrombose580. En effet, les GR peuvent s’agréger entre eux581 et également

avec les plaquettes par l’interaction du ligand Fas, côté plaquette, au récepteur Fas, côté

GR496 ou par sa liaison avec la fibrine582,583. L’association du ligand Fas à son récepteur

permet l’exposition de la PS par les GR et leur interaction avec les cellules

Figure 8: Les globules rouges et leur élimination par les macrophages. Les GR présentent de la PS à leur

surface suite à leur activation ou à leur vieillissement. La PS peut lier directement la stabilin-2 et le Tim-4 des

macrophages ou indirectement à ceux-ci par l’intermédiaire de Gas-6 et des lactadhérines ou la

trhombospondine-1. La présence du CD47 à la surface des GR permet d’inhiber la phagocytose. La protéine

Band-3 peut être liée à des auto-anticorps naturels (Nabs) ce qui induit la phagocytose des GR par

l’intermédiaire des récepteurs aux fragments cristallisables (Fc receptor). (D’après de Back et al., 2014(575))

43

endothéliales496,584. L’exposition de la PS permet également la génération de

thrombine496,584,585. Enfin, la présence de GR au sein du thrombus le stabilise et permet

d’accroitre la résistance de la fibrine à la lyse565,586,587.

D’autre part, les GR seraient un modulateur de l’inflammation vasculaire. Ils servent

d’entreposage vasculaire pour plusieurs cytokines et chimiokines588-590. Les cytokines

présentes chez les GR proviennent soit d’un stade plus précoce de l’érythropoïèse590, soit du

captage de cytokines circulantes par le récepteur aux antigènes et chimiokines Duffy (i.e.

Duffy antigen/chemokine receptor)588,591-593. Les cytokines et chimiokines stockées par les

GR peuvent être sécrétées par ces derniers et stimuler différentes cellules immunitaires dont

les neutrophiles et les lymphocytes T594,595. Outre les cytokines, les globules rouges

présentent le TLR9 qui permet la liaison de l’ADN CpG notamment l’ADN

mitochondriale596. Enfin, le CD235a et le récepteur Duffy permettent la liaison des GR à

plusieurs pathogènes et leur élimination lors de la phagocytose des GR par les

macrophages596-601.

2.3.2 Description générale

Les REV sont la deuxième population d’EV la plus abondante dans le système vasculaire

après celle d’origine plaquettaire448. Les REV peuvent présenter à leur surface le canal d’ion

Band 3, la PS, des tétraspanines et des glycoprotéines comme le CD235a, ou la glycophorine

A602. Cette dernière est notamment utilisée comme un marqueur spécifique des EV de GR.

Les REV contiennent notamment de l’hémoglobine et des ARN de petites tailles602.

La libération d’EV par les GR est souvent considérée comme un mécanisme passif en réponse

à des contraintes de cisaillement603,604, à leur vieillissement605,606 ou à leur stockage557.

Cependant, les GR libèrent également des EV de manière active au cours de leur maturation,

lors de l’hémolyse et en réponse à certains stimuli comme le LPA575,576. Le LPA est le seul

médiateur endogène connu qui peut activer la production de REV, mais des composés comme

le calcium ionophore ou le phorbol-12-myristate-13-acétate peuvent également

l’induire575,576. La production d’EV par les GR peut être faite de manière dépendante et

indépendante de la mobilisation intracellulaire de calcium575,576.

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2.3.3 Fonctions

La production d’EV par les GR à plusieurs fonctions. Lors de la maturation, la libération

d’EV par la voie des exosomes ou par bourgeonnement, permet le remodelage de la

membrane du GR ou l’élimination de médiateurs intracellulaires tel le récepteur au facteur

tissulaire554-557,575. Ce mécanisme est également présent lors du vieillissement des GR

matures et des érythrocytes, qui perdent notamment de l’hémoglobine par la libération de

REV607.

Les REV agissent dans la continuité des fonctions des GR. Grâce à leur cargo en

hémoglobine, ils participent à la régulation des échanges gazeux par le captage de l’oxyde

d’azote circulant608-610 ou encore à la libération d’espèces réactives de l’oxygène611. Le

captage de l’oxyde d’azote par les REV inhibe la vasodilatation dépendante des cellules

endothéliales vasculaires608,609. La présentation de PS à leur surface permet également de lier

l’hème du milieu extracellulaire612.

Également, les REV sont capables d’initier et de promouvoir la coagulation441,613,614. Elles

sont d’une part une source mobilisable du facteur de von Willebrand615 et induisent

l’expression de protéines d’adhésion par cellules endothéliales615. Et d’autre part, elles

participent à la production de thrombine par l’activation du facteur XII441 ou par la cascade

de kallicréine616. De manière similaire aux GR et aux plaquettes, la production de thrombine

est initiée par l’exposition de la PS à la surface des REV, ce qui permet la liaison de cofacteur

de la coagulation ainsi que des inhibiteurs de la coagulation comme la protéine C

activée440,441.

Outre leur rôle dans la coagulation, elles participent à la modulation de l’immunité et de

l’inflammation. La génération de thrombine par les REV permet d‘activer le système du

complément617. Ils permettent également les interactions entre les plaquettes, les

neutrophiles, les lymphocytes T et les monocytes/macrophages614,618. Ces interactions

résultent en une production de nombreuses cytokines et chimiokines pro-

inflammatoires614,615,618 et également à l’activation des cellules endothéliales vasculaires615.

Sur les neutrophiles, les REV favorisent leur recrutement et leur « amorçage » (i.e.

priming)619,620. En parallèle à ces effets d’activation de l’immunité, les REV ont un potentiel

45

d’inhibition. En effet, leur internalisation inhibe l’activation et la survie des lymphocytes T621

et diminue la production de cytokines pro-inflammatoires pour les macrophages622.

3 L’acide lysophosphatidique et les vésicules extracellulaires

dans les maladies rhumatismales auto-immunes systémiques

3.1 Polyarthrite rhumatoïde

La polyarthrite rhumatoïde (PAR) est une maladie rhumatismales auto-immune systémique

avec une prévalence de 0,5 à 1% de la population et touche principalement les femmes qui

représente de 60 à 80% des cas623. La susceptibilité à développer l’PAR dépend en grande

partie de facteurs génétiques mais il existe de nombreux facteurs environnementaux comme

le tabagisme624. Le développement de la PAR se fait par l’activation et la propagation de la

réponse immunitaire adaptative contre des protéines modifiées du soi, notamment les

protéines citrullinées, ou d’anticorps appelés « facteur rhumatoïde »625. Cette première

réponse immunitaire ne suffit pas au déclenchement d’une PAR625. Un deuxième stress (i.e.

additional hit), comme la formation de complexes immuns ou l’activation du complément,

est nécessaire pour déclencher l’inflammation et le recrutement des cellules immunitaires au

niveau du tissu synovial de manière chronique. L’inflammation chronique du tissu synovial

conduit au cours du temps à la destruction du cartilage et de l’os. Les effets de la PAR ne se

limitent pas aux articulations et présentent des manifestations systémiques. Il existe

également une forme d’PAR dite séronégative où les patients ne présentent pas d’anticorps

dirigés contre des protéines du soi modifiées. Les patients atteint par une PAR sont

notamment plus à risque de développer des maladies cardiovasculaires, qui sont la première

cause de surmortalité des patients PAR par rapport à la population générale626-628. Les

patients PAR sont notamment sujet à un risque accru d’infarctus du myocarde et

d’évènements cérébrovasculaires plus élevés627,629. Ce risque a été associé à un

développement plus rapide de l’athérosclérose et à une instabilité accrue des plaques

d’athéroscléroses qui lors de leur rupture, entraine la formation d’un thrombus et le blocage

du flux sanguin627,629,630.

Le LPA a été montré comme un promoteur important de la pathophysiologie de la PAR dans

plusieurs modèles murins de PAR70,147,192. Chez l’humain, l’autotaxine est détectée dans le

46

liquide synoviale et son expression ainsi que celles de certains récepteurs au LPA, comme

LPA1, sont augmentés dans les tissus synoviaux, notamment les fibroblastes synoviaux de

type B70,149,192,631. Les fibroblastes synoviaux de type B contribuent à l’inflammation et à

l’hyperplasie du tissu synovial. Le LPA stimule la prolifération des synoviocytes de types B

et induit la libération de médiateurs pro-inflammatoires comme l’IL-6 et l’IL-8149,631,632 et de

métalloprotéases matricielles350. L’action du LPA ne se limite pas aux fibroblastes

synoviaux. Il stimule également le recrutement et l’activation de lymphocytes et de

macrophages dans le tissu synovial192,633. Enfin, il participe à la dégradation du cartilage et

de l’os par la formation et l’activation des ostéoclastes147,192.

L’inflammation dans les tissus synoviaux de patients PAR met également en jeux les EV.

Les patients présentent des quantités importantes d’EV d’origines diverses comme de

fibroblastes, de lymphocytes et de macrophages634-637. Le liquide synovial présente

également une infiltration d’EV de la circulation sanguine sous forme de PEV447,545,637,638.

Les PEV stimulent l’inflammation par l’apport d’IL-1α et β, ainsi que par l’induction de l’IL-

6 et l’IL-8 par les fibroblastes synoviaux447. Les PEV sont une source d’auto-antigènes, tel

que des protéines citrullinées ou de complexes immuns, et elles activent la réponse

inflammatoire lorsqu’elles sont internalisées par les neutrophiles545,639. Outre les PEV, les

EV du liquide synovial stimulent l’inflammation synoviale par la diffusion de microARN et

par l’apport de protéines citrullinées dans le tissu synoviale640-643. Les EV du liquide synovial

augmentent l’activation et la survie des lymphocytes636,641. Enfin, les EV des articulations

inflammées de patients PAR ont été proposées comme étant un facteur dans la coagulation

locale élevée et seraient impliquées dans les dépôts de fibrines visibles chez les patients

PAR644.

3.2 Lupus érythémateux disséminé

Le lupus érythémateux disséminé (LED) est une maladie rhumatismale auto-immune

systémique avec une prévalence qui varie de 19 à 159 par 100 000 personnes en fonction de

la zone géographique645. Le LED affecte principalement les femmes et, bien que le biais varie

fortement parmi les études, le ratio est considéré comme étant de 9 femmes pour 1

homme623,645.

47

Le LED est une pathologie complexe qui se caractérise par des atteintes à de multiples

organes et par un grand nombre de symptômes possibles. Parmi les manifestations les plus

fréquentes sont retrouvés les atteintes rénales, dite néprhite lupique, articulaires, cutanées ou

encore neurologiques645. Le fait que le LED puisse se présenter sous des formes très variées,

que ce soit sur des critères cliniques ou sérologiques, rend difficile son diagnostique. De plus,

le retard dans le diagnostique du LED augmente les risques dommages irréversibles aux

organes646.

Plusieurs systèmes de classification du LED ont été créés pour faciliter la conduite d’étude

clinique. Le premier a été fait par l’American College of Rheumatology (ACR) sur la base

de 11 critères cliniques et sérologiques647,648. Un patient doit présenter au moins 4 critères

pour être considéré comme atteint du LED. Un deuxième est fait par le Systemic Lupus

International Collaborating Clinics (SLICC) sur la base de 17 critères cliniques et

sérologiques648,649. Pour être considéré comme atteint du LED selon cette classification, un

patient a besoin de 4 critères, dont au minimum un clinique et un sérologique ou avoir une

atteinte rénale, la néphrite lupique, avec simultanément une mesure d’anticorps antinucléaire

ou contre l’ADN. Enfin, en 2019, l’ACR et l’European Alliance of Associations for

Rheumatology (EULAR) ont proposé une nouvelle classification basée sur 22 critères

cliniques et sérologiques648,650. Un patient est classifié comme atteint du LED s’il est positif

pour des anticorps antinucléaires et présente un score égal ou supérieur à 10. Cette nouvelle

classification présente une meilleure sensibilité, soit la probabilité que le test détecte la

maladie, que les classifications sur les critères ACR ou SLICC. Bien qu’elles puissent être

utilisées dans le cadre d’un diagnostique, ces classifications restent des outils pour aider la

recherche et n’ont pas une vocation à être couramment utilisé pour poser un diagnostic ou

pour décider d’un traitement.

La progression dans le temps du LED peut se faire de trois manières. D’abord la forme

quiescente, qui ne présente pas de symptôme clinique, mais qui est actif selon les critères

sérologiques, ensuite une forme active de manière chronique, et enfin une dernière forme

cyclique, qui alterne des poussées de la maladie avec des phases de rémission651. De plus, les

patients ont besoin d’un suivie important pour réduire, d’une part les risques de complication

liés à la pathologie et à son traitement, et d’autre part les dommages permanents aux organes.

48

Il est donc important d’évaluer l’activité de la maladie chez les patients645,651. Bien qu’il

existe plusieurs index, le plus utilisé est l’index SLEDAI pour systemic lupus erythematosus

disease index. Le SLEDAI est basé sur la présence de 24 critères qui inclus des atteintes de

9 organes différents lors des 10 jours précédents le test. Le SLEDAI permet l’obtention d’un

score qui augmente avec l’activité de la maladie652.

L’étiologie du LED reste encore floue mais implique des composantes génétiques et

environnementales645. Le LED est causé par une réponse de l’immunité innée et adaptative

contre des acides nucléiques ou des complexes protéiques contenant des acides nucléiques

originaires du soi, des phospholipides et des protéines mitochondriales653,654. La réponse

innée repose en grande partie sur la production d’interféron de type I par les cellules

dendritiques, les granulocytes et les neutrophiles655-657. La production d’interféron se fait

principalement en réponse à l’activation des TLR 7 et 9, respectivement par l’ARN simple

brin et par l’ADN CpG non méthylé657. Tandis que l’activation des lymphocytes B et T

conduit à la production de nombreux auto-anticorps et à la libération de cytokines658-661. Les

patients LED présentent des dommages aux organes en fonction de la nature locale de la

réponse immunitaire excessive aux auto-antigènes dans ces organes662-666. Similairement aux

patients RA, les patients affectés par le LED sont plus à risque que la population générale de

développer des maladies cardiovasculaires665,667-669. Les premières causes de mortalité chez

patients LED sont les maladies cardiovasculaires dont l’athérosclérose665,669.

Les patients lupiques présentent des quantités élevées d’EV circulantes. Similairement à leur

rôle dans la PAR, les EV stimulent l’inflammation principalement par la diffusion de

microARN670-672. Cela résulte à la libération augmentée de cytokines et chimiokines pro-

inflammatoires importantes dans la progression de la LED comme l’interféron-α, un

interféron de type I671-673. Elles sont également une source d’auto-antigènes674-676. Enfin, chez

les patients LED, les quantités de PEV sont associées avec l’épaississement de la paroi

vasculaire, un facteur de risque des accidents vasculaires674.

3.3 Comorbidité : athérosclérose

Les maladies cardiovasculaires représentent la première cause de mortalité au monde677-679.

L’athérosclérose est la principale cause des maladies cardiovasculaires678,679. L’inflammation

49

joue un rôle moteur dans la progression de l’athérosclérose ce qui explique de la trouver

comme facteur de comorbidité dans la PAR et le LED qui sont toutes deux des pathologies

associées à une forte inflammation systémique vasculaire627,629,630,665,669.

L’athérosclérose consiste en un épaississement de la paroi vasculaire par la formation d’un

noyaux fibreux et lipidique qui conduit à une réduction du flux sanguin. La progression de

l’athérosclérose se fait en trois phases (Figure 9). Les vaisseaux sanguins sont composés de

trois couches tissulaires, l’intima, la média et l’adventice, dans l’ordre d’éloignement à la

lumière du vaisseau. Dans la phase d’initiation, des lipoprotéines de la circulation

s’accumulent dans l’intima de la paroi vasculaire680. Cette accumulation permet la création

d’une plaque d’athérosclérose680. Au cours de la phase de progression, la plaque

d’athérosclérose se développe par la poursuite de l’accumulation de lipides et par le

recrutement de cellules. D’une part les cellules musculaires lisses de la média voient leur

prolifération et leur migration stimulées681. D’autre part, il y a également une infiltration par

des leucocytes, majoritairement des macrophages, mais également des lymphocytes T et des

neutrophiles682,683. Une fois présent dans la plaque d’athérosclérose les macrophages y

prolifèrent683. L’accumulation de lipides ne dépend plus uniquement des lipoprotéines

circulantes, mais également de l’intégration de cellules spumeuse dans la plaque. Les cellules

spumeuses sont des macrophages ou des cellules musculaires lisses gorgées de lipides684,685.

L’accumulation des lipides et le recrutement des cellules immunitaires dans la plaque

d’athérosclérose est facilité par l’activation de l’endothélium qui le rend plus perméable686.

Au fil des années les cellules présentes dans la plaque induisent la production de matrice

extracellulaire et accentuent la capture et la rétention des lipides678,681. Cette production

permet aussi la mise en place d’une chape fibreuse qui permet de stabiliser la plaque681,687.

Au cœur de la plaque se forme une accumulation de cellules mortes, de macrophages et de

cellules du muscle lisse, ainsi que de lipides qui est appelé noyau nécrotique688,689.

50

La progression de la plaque peut déboucher sur plusieurs complications, qui constituent la

dernière phase. D’une part, la plaque peut prendre trop d’espace dans la lumière du vaisseau

sanguin, ce qui va gêner le flux sanguin et peut mener à des ischémies lorsque l’apport en

oxygène devient insuffisant. D’autre part, si l’instabilité de la plaque est trop grande, une

rupture de la plaque peut se produire690-692. À la suite de cette rupture, le noyau nécrotique

va au contact de la circulation déclencher la coagulation et la formation d’un thrombus. La

formation de ce dernier peut bloquer le passage du sang686,690.

Figure 9: Progression d’une plaque d’athérosclérose. L’activation de l’endothélium augmente sa

perméabilité et induit la libération de cytokines pro-inflammatoires et l’expression de molécules d’adhésion.

La perméabilité de l’endothélium facilite l’accumulation de lipides tandis que les cytokines et les molécules

d’adhésion facilitent le recrutement des cellules immunitaires, principalement les macrophages.

L’inflammation au sein de la paroi vasculaire cause d’une part, le recrutement et la prolifération des cellules du

muscle lisse et d’autre part la formation de cellules spumeuses par internalisation de lipides par les macrophages

et les cellules du muscle lisse. Il en résulte un épaississement localisé de la paroi vasculaire. Le stress oxydatif

et l’hypoxie à l’intérieur de la plaque induit l’apoptose cellulaire et la formation d’un cœur nécrotique riche en

lipide. La plaque d’athérosclérose est protégée par la formation d’une chape fibreuse. Le développement de la

plaque résulte en une obstruction partielle de la lumière du vaisseau. La plaque peut rompre à cause d’un

amincissement de le chape fibreuse ou à cause d’une instabilité de la plaque. La rupture de celle-ci expose le

cœur nécrotique riche en lipides et en protéines matricielles aux plaquettes sanguines, ce qui induit la formation

d’un thrombus. (D’après Skeoch et Bruce, 2015(675))

51

Des modèles murins d’athérosclérose ont mis en évidence l’implication de l’autotaxine et de

certains récepteurs du LPA dans la progression de l’athérosclérose693,694. L’autotaxine

transportée par les lipoprotéines peut former du LPA lors de leur oxydation et favoriser

l’accumulation du LPA dans les plaques d’athérosclérose34,68,113. Le LPA participe à

l’inflammation et à l’hyperplasie de l’endothélium vasculaire par la libération de cytokines

pro-inflammatoires311,695-697. Il est également impliqué dans la formation de néo-intima par

la stimulation de la migration et de la prolifération des cellules endothéliales et du muscle

lisse311,698-700. Il augmente l’absorption de lipides par les macrophages ce qui permet leur

transformation en cellules spumeuses309,395,701. Le LPA inhibe la migration des cellules

spumeuses ce qui pourrait contribuer à leur rétention dans la plaque d’athérosclérose311.

Enfin, lors de la rupture de la plaque, le LPA et les fibres de collagène activent les plaquettes

enclenchant ainsi le processus de coagulation34,112,113.

Les plaques d’athérosclérose présentent différentes populations d’EV dont les REV702-705.

L’injection de certaines EV accélèrent la progression de l’athérosclérose dans un modèle

murin706. Les EV sont capables de moduler plusieurs mécanismes mis en jeu dans le

développement de l’athérosclérose. Tout d’abord, elles stimulent la migration et le

recrutement des lymphocytes et des monocytes/macrophages dans le la paroi vasculaire704,706-

709. Elles induisent également la production et la libération de cytokines et chimiokines pro-

inflammatoires702-704,706,710. D’autres EV sont impliquées dans le recrutement et la migration

de leucocytes. Les EV sont des apports de lipides aux macrophages et stimulent leur rétention

dans la plaque710-713. Cette modulation du contenu lipidique des macrophages favorise leur

transformation en cellules spumeuses711. Les EV sont également impliquées dans la

formation de néovascularisations et de microcalcifications au sein de la plaque

d’athérosclérose, ce qui la déstabilise714,715. Enfin le processus de coagulation qui se produit

lors d’une rupture de plaque implique les EV, comme cela a été décrit dans la section sur les

PEV et les REV.

Cependant, certaines populations d’EV ont des effets protecteurs contre l’athérosclérose.

Certaines EV protègent l’intégrité des cellules endothéliales716,717. D’autres peuvent inhiber

le recrutement et l’activation des monocytes et macrophages718-720. Enfin des EV peuvent

avoir des effets positifs et négatifs sur la progression des plaques comme c’est le cas pour les

52

EV de monocytes qui stimulent la formation de cellules spumeuses, mais inhibent

l’activation de lymphocytes T et la production de cytokines pro-inflammatoires711.

4 Objectif

Le LPA promeut le développement de plusieurs pathologies inflammatoires dont la PAR et

l’athérosclérose. Il est par ailleurs le seul médiateur endogène connu capable d’induire

l’activation des GR ce qui entraine notamment la libération de REV. De plus, un rôle des GR

dans la modulation de l’inflammation est proposé. Outre la capacité de stocker et libérer des

cytokines par les GR, leurs REV ont des activités pro-inflammatoires et stimulent la

coagulation. L’hypothèse au centre de cette thèse est que le LPA via l’activation des GR peut

promouvoir l’inflammation vasculaire et participer aux dommages vasculaires, comme

l’athérosclérose, associés aux maladies rhumatismales auto-immunes systémiques.

Notre premier objectif était d’évaluer le potentiel activateur de différentes espèces de LPA

sur les GR et d’examiner les voies de signalisation impliquées.

Un deuxième objectif était d’évaluer au sein de cohortes de patients atteints de maladies

rhumatismales auto-immunes systémiques si l’autotaxine et des niveaux élevés d’EV

sanguines étaient associés à un risque accru de thrombose et au développement de plaques

athéromateuses.

Ce projet permettrait donc d’approfondir l’implication du LPA et des EV sur les dysfonctions

du système vasculaire dans le contexte de maladies systémiques et auto-immunes telle l’PAR

et le LED.

53

Chapitre 1 : Interplay between LPA2 and LPA3 in LPA-

mediated phosphatidylserine cell surface exposure and

extracellular vesicles release by erythrocytes 1 Résumé

Un rôle plus large des globules rouges (GR) dans l’homéostasie vasculaire émerge,

notamment dans les évènements thrombotiques et dans l’inflammation. L’acide

lysophosphatidique (LPA) est le seul activateur connu des GR et induit l’exposition de

phosphatidylsérine et la libération de vésicules extracellulaires de GR (REV). Par des

approches de cytométrie en flux à haute sensibilité, nous avons étudié l’effet d’espèces

majeures de LPA plasmatique sur les GR. Trois de ces espèces induisent la présentation de

la PS et la libération de petites REV PS- et de grandes REV PS+ par l’activation du récepteur

LPA3. L’activation des GR est possible dans le plasma et libère des REV similaires à celles

trouvées dans le plasma de patients. Une quatrième espèce inhibe l’activation des GR par le

récepteur LPA2. Nos résultats suggèrent que les espèces de LPA présentent des activités

biologiques différentes chez les GR en fonction de l’activation des récepteurs LPA2 et/ou

LPA3.

54

Interplay between LPA2 and LPA3 in LPA-mediated phosphatidylserine cell surface

exposure and extracellular vesicles release by erythrocytes

Stephan Hasse1, Anne-Claire Duchez2, Paul Fortin2, Eric Boilard1, Sylvain G. Bourgoin1

1Centre de recherche du CHU de Québec-Université Laval, Centre ARThrite de l'Université

Laval, Département de microbiologie-infectiologie et d’immunologie, Université Laval,

Québec, QC, Canada G1V 4G2.


Laval, Département de médecine, Faculté de médecine, Université Laval, QC, Canada G1V

4G2.

Corresponding author:

Sylvain G. Bourgoin, PhD

Centre de Recherche du Centre Hospitalier Universitaire de Québec

Faculté de Médecine de l’Université Laval

2705 Boul. Laurier, Québec, QC, Canada G1V 4G2

[email protected]

Phone: +1 (418) 525-4444, ext. 46136 Fax: (418) 654-2765

Key words: LPA, Erythrocytes, LPA receptors, Phosphatidylserine, Autoimmunity,

Extracellular Vesicles

55

2 Abstract

Evidence is growing for the role of red blood cells (RBCs) in vascular homeostasis, including

thrombogenic events and inflammation. Lysophosphatidic acid (LPA) is known to induce

phosphatidylserine (PS) exposure and the release of RBC Extracellular Vesicles (REVs).

Using high sensitivity flow cytometry, we examined the effects and the mechanisms by

which the LPA species commonly found in human plasma could activate RBCs. We report

that LPA 16:0, 18:0 and 18:1, but not LPA 20:4, induced PS exposure and the release of

small PS- and large PS+ REVs through LPA3 receptor signalling in RBCs. The release of

large PS+ REVs required higher concentrations of LPA. RBCs were not activated by LPA

20:4. Interestingly, blockade of LPA2 enhanced LPA-mediated PS- REV release in RBCs.

Furthermore, LPA receptor agonists and antagonists highlighted that LPA 20:4 inhibited

LPA3-dependent PS exposure and, through the LPA2 receptor, inhibited PS- REV

production. Activation of RBCs with LPA 18:1 in normal plasma stimulated the release of

PS- and PS+ REVs. REVs released in response to LPA were similar to those found in the

plasma of systemic lupus erythematosus patients. Our results suggest that LPA species

exhibit different biological activities in RBCs through targeting LPA2 and/or LPA3

receptors.

56

3 Introduction

Red blood cells (RBCs) have long been limited to their role as vehicles for gas exchanges

between blood and tissues and considered passive for other vascular processes such as

inflammation or coagulation. However, a lower concentration of RBCs in polycythemia vera

patients is associated with a lower number of thrombotic events and a lower death rate from

cardiovascular causes [1], thereby suggesting a role for RBCs in coagulation and thrombosis.

Furthermore, inhibition of RBC - platelet interaction protects from arterial thrombosis in

vivo[2]. In vitro experiment shows that interaction between RBCs and platelets contributes

to thrombin generation and thrombus formation[2]. A small subset of RBCs exposing

phosphatidylserine (PS) at their surface could contribute up to 40 % to the thrombin

generated in blood. PS exposition by RBCs promoted their binding to endothelial cells[3]

and could contribute to retinal vein occlusion[3]. In addition to the coagulation process,

RBCs constitute a storage site of vascular cytokines[4, 5] and are potential modulators of

blood cytokines and chemokines. Some such as IL-33 come from an earlier stage of RBC

development[6]. Others such as IL-8 are actively captured by RBCs through the Duffy

antigen receptor for chemokines[4]. Furthermore, RBCs can release cytokines in the milieu

without undergoing hemolysis[7]. Such RBC conditioned media could promote neutrophil

transmigration in vitro[8] and lymphocyte T survival and growth[7].

The release of extracellular vesicles (EVs) by RBCs also contributes to coagulation and

vascular inflammation. EVs are small vesicles from 30 nm up to 1 µm. EVs can originate

from the endosomal compartment before being released (exosomes) or through plasma

membrane budding of activated cells (microvesicles, ectosomes or microparticles). The

release of EVs containing membrane receptors by RBCs (REVs) is a mechanism associated

with a loss of functional responses during maturation of erythrocytes[9-11]. REVs are

capable of initiating and sustaining coagulation in vitro[12]. The increase of

hypercoagulation following REV injection in a mouse model of transfusion highlights their

role in coagulation processes[13]. REVs procoagulant activity is in part explained by the

upregulation of endothelial cell adhesion markers[14] and as a source of thrombin[15]

through the activation of factor XII or of the kallikrein cascade[12]. REVs could also have a

role in immunity, as suggested by the exacerbation of pro-inflammatory cytokine production

57

by REVs in lipopolysaccharide-induced inflammation[16]. REVs promote inflammation

further through thrombin generation[15, 16] and activation of the complement cascade[16].

With platelet EVs, REVs are the main EV population in the bloodstream[17]. Even if REV

release has long been considered passive in response to shear stress[18], aging[19, 20] or

storage[21], REV release can be induced in vitro by a mediator present in the plasma, the

lysophosphatidic acid (LPA)[22].

LPA is a bioactive lipid formed by a phosphate group on a glycerol backbone attached to a

fatty acid. LPA activity is mediated by six widely expressed LPA receptors (LPA1-6) and

atypical receptors such as the peroxisome proliferator-activated receptor γ[23]. The

functional responses to LPA depend on the pattern of LPA receptor expression and LPA

species affinity for the receptors[24, 25]. LPA is a critical mediator of the vascular system

homeostasis[26, 27]. LPA contributes to pro-thrombotic events and inflammatory diseases

such as atherosclerosis[28, 29]. LPA pro-inflammatory and pro-thrombotic functions are in

part explained by the activation of vascular cells such as platelets[30] and endothelial

cells[29]. It promotes platelet aggregation[30], cytokine production[29], and immune cell

recruitment[31]. However, knowledge on LPA impact on RBCs is limited. LPA is known to

have a dual role in the early steps of hematopoiesis. LPA3 promotes erythropoiesis at the

expense of megakaryopoiesis[32, 33], while LPA2 inhibits erythropoiesis[34]. LPA induces

the aggregation of mature RBCs[35], as well as the presentation of surface PS and the release

of pro-thrombotic REVs[22, 36].

Since RBCs and REVs have an active role in vascular physiology and LPA-mediated RBC

activation contributes to inflammation and coagulation, we studied the effect of common

blood LPA species on RBC PS exposure and EV release.

4 Material and methods

4.1 Products

LPA species (16:0, 18:0, 18:1, and 20:4), LPA3 agonist 2S-OMPT, and LPA1/3 antagonist

VPC 32183 were from Avanti Polar Lipids (Alabaster, Alabama, USA). LPA2 agonist GRI-

977143 and LPA2 antagonist H2L5186303 were from Tocris Bioscience (Bristol, UK).

58

4.2 Human plasma samples

Platelet-free plasma (PFP) samples, from systemic lupus erythematosus (SLE) patients and

age- and gender-matched healthy controls, were obtained from the Biobank and Repository

Data for Systemic Autoimmune Rheumatic Diseases of the CHU de Québec-Université

Laval Research Center. PFPs were prepared from blood samples using previously described

standardized procedures[37], which are presented in the section Platelet and EV-free plasma

preparation. All blood samples were obtained after informed consent. The study was

reviewed and approved by the ethics review board of the CHU de Québec-Université Laval

(Project # 2016-2558).

4.3 RBC isolation and activation

Whole blood was collected with sodium citrate as an anticoagulant from healthy anonymous

donors after obtaining informed consent. The blood was centrifuged at 282 g for 10 min to

separate the RBC pellet from plasma and leukocytes. The RBC pellet was collected and

washed thrice in phosphate-buffered saline (PBS: 1 mM KH2PO4, 154 mM NaCl, 3 mM

Na2HPO4; pH 7.4) unless otherwise stated. Washed RBCs were used right away at 108

cells/mL in PBS, when not stated otherwise, and were stimulated up to 2 h at 37 °C under

continuous agitation. In experiments looking at RBC activation in the presence of calcium or

albumin, washed RBCs in HEPES buffered physiological solution (HPS: 145 mM NaCl, 7.5

mM KCl, 10 mM glucose, 10 mM HEPES, pH 7.4 as previously described[36]) containing

2 mM CaCl2 (Sigma Aldrich, Oakville, ON, Canada) or 1 % lipid-free bovine serum albumin

(BSA, Sigma Aldrich, Oakville, ON, Canada) were stimulated with LPA 18:1 for 1 hour at

37 °C. Washed RBCs in EV-free and PFP (V/V), prepared as described in the section below,

were used to mimic physiological conditions. RBCs were centrifuged at 282 g for 10 min,

resuspended one last time in EV-free PFP (V/V), and stimulated with LPA 18:1 for 1 h and

24 h under rotating agitation at 37°C. PBS and HPS were prepared with chemical of

analytical grade obtain from Sigma Aldrich (Oakville, ON, Canada) and HEPES from Wisent

(St-Bruno, Québec, Canada).

59

4.4 Platelet and EV-free plasma preparation

PFP were prepared as previously described[37]. Blood from healthy donors was centrifuged

at 282 g for 10 min at room temperature (RT). The plasmatic fraction was centrifuged at 2

500 g for 20 min at RT. The supernatant was further centrifuged 3 times at 3 500 g for 5 min

at RT to obtain PFP. Finally, PFP was centrifuged at 100 000 g for 90 min at 18 °C to remove

all EVs. The plasma free of both platelets and EVs of 3 healthy donors were pooled and

stored at -20 °C before use.

4.5 RBC and REV labeling for flow cytometry

Following stimulation, 15 µl of stimulated RBCs at 108 cells/mL or 2 µl RBCs in PFP (V/V)

were collected and labeled with 3 µl anti-CD235a-PECy7 (BD Bioscience Canada,

Mississauga, ON, Canada), a marker of RBC, and 3 µl Annexin V FITC (BD Bioscience

Canada, Mississauga, ON, Canada) for PS detection, in 100 µl final of Annexin V binding

buffer (BD Bioscience Canada, Mississauga, ON, Canada) for 30 min in the dark at RT.

Labeling was stopped by adding 200 µl of Annexin V binding buffer and samples were

analyzed within 90 min to prevent labeling loss. PS exposure on RBCs and REVs were

analyzed using a high sensitivity flow cytometer BD Canto II Special Order Research Product

with a small particle option as previously described[38]. Performance tracking of high

sensitivity flow cytometry was done the day of use with BD cytometer setup and tracking

beads (BD Bioscience Canada, Mississauga, ON, Canada). REVs and PS+ REVs absolute

concentrations were determined using 2 µm APC polystyrene beads (BD Bioscience Canada,

Mississauga, ON, Canada) or 3 µm polystyrene beads (Polysciences, PA, USA) of known

concentration in each sample. The gating strategy for RBC activation and REV detection are

presented in Figure 1 A and B. The gate to discriminate the event of a size between 100 nm

and 1,000 nm was set using silica particles of known dimensions of 100, 500 and 1,000 nm

(Kisker Biotech GmbH & Co. Steinfurt, Germany) (Figure 1C). Voltages for the detection

of RBCs were set as follows: FSC at 160 Volt (V), SSC at 300 V, PECy7 at 450 V and FITC

at 500 V. A diode was used to optimize RBC detection. REVs were detected with the

following settings: FSC-PMT at 390 volts (V), SSC at 460 V, PECy7 at 500 V, FITC at 400

V and APC at 550 V. Detection of REVs in the plasma of SLE patients were done with the

60

following settings: 300 V, SSC at 300 V, PECy7 at 500 V, FITC at 500 V and APC at 500

V.

4.6 Control for REV detection by flow cytometry

To evaluate the sensitivity of REV detection, we performed serial dilution, triton treatment

and ultracentrifugation of REV samples as previously described[38]. RBCs were first

removed by centrifugation at 1 300 g for 10 min at RT, and the supernatants were then

centrifuged two more times at 3 500 g for 10 min at RT. EVs were destroyed using 0.05 %

Triton X-100 (Sigma Aldrich, Oakville, ON, Canada) for 1 h at 37 °C or removed by

centrifugation at 100 000 g for 1 h and 30 min at 18 °C. EV depletion data are express as a

percentage of the total number of EVs in untreated samples. Labeling in PBS with 50 µM

EDTA (Wisent, St-Bruno, Québec, Canada), instead of Annexin V buffer, verified the

specificity of Annexin V labeling. We also performed a coincidence test for the detection of

REVs to verify that our measurements were quantitative. Two-fold serial dilution of REV

samples were prepared and measured by high sensitivity flow cytometry (Figure 1F).

4.7 Analysis and statistics

Flow cytometry data were analyzed using FlowJo V10 software (FlowJo, LLC, OR, USA),

and statistical analyses performed using GraphPad Prism 7.0 software (GraphPad Software,

San Diego, USA). Since the number of repetitions for each experience is under 15, we only

used nonparametric tests for our statistical analysis. The strength of activation by LPA

species or LPA receptor agonists showed substantial variations between blood donors.

Therefore, when looking for increase or inhibition of REV production by RBCs, we mitigated

the variations by normalizing the data to the positive control. The Mann-Whitney test

assessed the difference between two experimental conditions. Multiple comparisons used a

two-way ANOVA with Dunnett's multiple comparison post-test to analyze RBC activation

in response to increasing concentrations of LPA and stimulation length. Statistical analyzes

used Kruskal-Wallis tests with Dunn’s multiple comparison post-test or Friedman's test with

Dunn's multiple comparison post-test. Post-test comparisons used the positive or negative

controls, and results were expressed as the mean ± standard error of the mean.

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5 Results

5.1 Detection of activated RBCs and REVs by flow cytometry

We monitored PS exposure at the surface of RBCs as a marker of RBC activation. Events

positive for the RBC marker CD235a and Annexin V to detect PS were considered (Figure

1A). The gating for PS+ RBCs was validated using RBCs stimulated with calcium ionophore

as a positive control (Figure 1A). We also used the RBC marker CD235a to detect REVs.

Events of a size comprised between 100 nm and 1 000 nm and positive for the RBC marker

CD235a were considered as REVs (Figure 1B). We further discriminated the PS+ REVs

using Annexin V-FITC labeling (Figure 1B). The gate to discriminate the events between

100 nm and 1 000 nm were set with 100 nm, 500 nm and 1 000 nm beads (Figure 1C). To

validate the specificity of REV detection, we monitored the loss of signal for REVs in

supernatants of RBCs activated with LPA 18:1 after treatment with Triton X 100 or

ultracentrifugation at 100 000 g, which destroys the EV lipid bilayer and pellets REVs,

respectively. Triton X 100 treatment or ultracentrifugation reduced the REV populations by

at least 95% compared to untreated supernatants (Figure 1D). The data confirmed the

specificity of REV detection using our experimental approaches. Calcium-free medium

supplemented with EDTA abolished Annexin V labeling of REVs and validated the

specificity of PS detection by Annexin V at the surface of REVs (Figure 1E). Finally, serial

dilution of REV samples resulted in similar reduction of REV detection (Figure 1F).

Although, we detected fewer event, it did not affect the mean nor the median fluorescence of

the RBC marker, CD235a-PECy7, which confirmed that each event we measured is a single

EV and not a cluster of EVs (Figure 1F-G).

5.2 LPA species differentially activate RBCs.

Though LPA is known to induce PS exposure and EV release by RBCs, there is no report on

individual LPA species. We choose to evaluate 4 LPA species, i.e. LPA 16:0, 18:0, 18:1 and

LPA 20:4. It allowed us to have LPA with a long or short fatty acid, either unsaturated,

monounsaturated, or polyunsaturated. Furthermore, all 4 species are commonly found in the

plasma[39, 40] and they are also produced in large quantities following platelet activation[40,

62

41] and during lipoprotein oxidation[40]. We used a concentration of 10 µM of LPA species

to stimulate RBCs, as was done in a previous study of RBC activation by LPA[22].

LPA 16:0, 18:0, and 18:1 induced PS exposure by RBCs (Figure 2A) and REV release

(Figure 2B and C). Surprisingly, LPA 20:4 did not induce PS exposure (Figure 2A) nor

REV release (Figure 2B and C). Our results show a functional discrepancy between LPA

species. Further analysis of LPA-mediated RBC activation used LPA 18:1, since it is the

most potent species that we tested.

5.3 Characterization of RBC activation by LPA 18:1.

We assessed the kinetics of externalization of PS and REV release by RBCs incubated with

various concentrations of LPA 18:1. High concentrations of LPA, 10 µM, and 20 µM resulted

in rapid and transient PS exposure by RBCs (Figure 3A). Furthermore, LPA induced

significant REV accumulation for all concentrations tested except the lowest (Figure 3B).

REV release in the media is seen as soon as 2 min after stimulation and reaches a maximum

after 1 h (Figure 3B). Interestingly, low concentrations of LPA (2.5 µM) induced significant

REV accumulation (Figure 3B) but not PS exposure by RBCs (Figure 3A). Altogether the

data indicate that low concentrations of LPA 18:1 can induce the production of REVs by

RBCs in the absence of PS cell surface exposure.

Interestingly, PS- and PS+ REVs have distinct sizes. PS- REVs show relative size of about

100 nm. On the other hand, PS+ REVs have a size between 500 nm and 1 000 nm (Figure

3C). Using the same gating strategy, we detected similar smaller PS- and larger PS+ REV

subpopulations in PFPs of SLE patients (Figure 3D).

LPA 18:1 induced the release of PS+ REVs in a dose-dependent manner (Figure 3E). In

opposition, the production of PS- REVs reached a plateau with a concentration of 5 µM

(Figure 3F). RBCs released mainly PS- REVs when stimulated with 2.5 µM (87.3 % ± 5.5)

and 5 µM (85.7% ± 3.4) while PS+ REVs were produced at concentration of 10 µM (53.9 %

± 12.5) and 20 µM (90.2 % ± 3.4) (Figure 3G). Thus LPA 18:1, depending on the

concentration, induces the release of 2 distinct populations of REVs, small PS- and large PS+

possibly through two different mechanisms. To further understand the mechanisms leading

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to RBC activation and the release of those two REV populations, we investigated whether

the production of those REVs was induced through the activation of LPA receptors.

5.4 LPA3 receptor induce RBC activation.

To our knowledge no published paper investigated the presence of LPA receptors on mature

RBCs and only two LPA receptors, LPA2 and 3, were reported on the precursors of RBCs[32,

33]. We first investigated the implication of the receptors LPA1 and LPA3 since both

receptors often mediate similar functions. Since no agonists for LPA1 are available, we

stimulated the RBCs with an agonist selective for LPA3, 2-OMPT. RBC stimulation with

2S-OMPT resulted in PS exposure and REV release (Figure 4A and B). As for RBC

activation with LPA 18:1, a low concentration of 2S-OMPT only induced the release of PS-

REVs (Figure 4B), and a high concentration of 2S OMPT led to significant accumulation of

both PS- and PS+ REVs (Figure 4C and D). To confirm that LPA activation of RBCs goes

through LPA3 (or LPA1), we stimulated RBCs with LPA 16:0, 18:0, and 18:1 in the presence

of VPC32183, an LPA1/3 receptor antagonist. VPC32183 reduced LPA 16:0-, 18:0-, and

18:1-induced cell surface exposure of PS and REV release by RBCs (Figure 4E and F).

Furthermore, VPC32183 strongly reduced the release of PS+ REVs in response to LPA 16:0,

18:0, and 18:1 (Figure 4G). VPC32183 strongly inhibited the release of PS- REVs in

response to LPA 16:0 and 18:0 or 18:1 to a lesser extent. Altogether these data suggest that

LPA-mediated RBC activation is dependent on LPA1/3 receptors.

5.5 LPA2 receptor inhibits PS- REV formation.

Knowing the complexity and redundancy of LPA signaling and the role played by LPA2 in

the regulation of erythropoiesis, we examined if LPA2 also contributes to LPA mediated

RBC activation and production of REVs. RBC incubation by GRI-977143, an LPA2 agonist,

did not induce RBC PS exposure (Figure 5A) nor the release of REVs (Figure 5B). We

further tested LPA2 implication in RBC activation by stimulating RBCs with LPA species

in the presence of H2L5186303, an LPA2 antagonist. H2L5186303 did not affect RBC PS

exposure induced by LPA 16:0, 18:0, and 18:1 (Figure 5C). Surprisingly, H2L5186303

enhanced the production of REVs induced by LPA 16:0 and 18:1 compared to the control

without H2L5186303, 199.2 % ± 88.0 (p=0.0350) and 157.3 % ± 59.5 (p=0.0450),

64

respectively (Figure 5D). Stimulation of RBCs with LPA 16:0 and 18:1 in the presence of

H2L5186303 only increased the release of PS- REVs (Figure 5F), but not that of PS+ REVs

(Figure 5E). To confirm the ability of LPA2 to inhibit PS- REV release, we induced the

release of REVs in the presence of 2S-OMPT, a selective LPA3 agonist, and the LPA2

agonist GRI-977143. As shown in Figure 5G, 10 µM of GRI-977143 reduced to 41.76 % ±

14.03 (p=0.0011) the production of PS- REVs. A higher concentration of GRI-977143 (20

µM) did not further inhibit the production of PS- REVs (Figure 5G), this might be due to

unknown off-target effects of high concentrations of the LPA2 antagonist. The data suggest

that LPA2 inhibits PS- REV release.

5.6 LPA 20:4 inhibits both RBC PS exposure and the production of PS-

REVs.

Since LPA2 has an inhibitory action, we assessed if LPA 20:4, which showed no activation

effect, could inhibit RBC activation. To address this point, we stimulated RBCs with 2S-

OMPT in the presence of LPA 20:4. Interestingly, 20µM LPA 20:4 reduced 2S-OMPT

mediated RBC PS exposure (11.30 % ± 8.81 versus 77.80 % ± 22.50; p=0.0107) (Figure 6A)

and strongly inhibited the production of REVs. Of note, LPA 20:4 had no impact on the

release of PS+ REVs (Figure 6B) but drastically reduced the production of PS- REVs induced

by 2S-OMPT (Figure 6C). Since PS+ REV accumulations were similar in the absence or

presence of LPA 20:4, we reasoned that LPA 20:4 mediated inhibition of PS- REV release

may be mediated by LPA2. To confirm this, we tried to rescue 2S-OMPT activation in the

presence of LPA 20:4 by adding the LPA2 antagonist, H2L5186303. The addition of

H2L5186303 enhanced in a dose-dependent manner LPA 20:4-mediated inhibition of PS cell

surface exposure induced by 2S-OMPT (Figure 6D). As expected, H2L5186303 at 30 µM

antagonized the inhibitory effect of LPA 20:4 on 2S-OMPT-mediated PS- REV release by

RBCs (Figure 6E). Together, these data support that LPA 20:4 inhibits the release of PS-

REVs through LPA2 signaling and inhibit PS exposure on RBCs through an LPA2

independent mechanism.

65

5.7 RBC activation by LPA in physiological condition.

All previous experiments used calcium- and albumin-free PBS. Given the presence of

albumin and millimolar concentration of calcium in the plasma, the vascular environment

likely impacts the ability of LPA to activate RBCs. Albumin binds numerous lipids including

lyso-phospholipids, thereby buffering the concentration of albumin-free LPA which prevents

the degradation of LPA by phosphatases and serves as a carrier to deliver this lipid to cells

and tissues [42, 43]. To evaluate the impact of albumin and calcium, two main components

of blood, on LPA-mediated effect on RBCs we used HEPES buffered physiological solution

(HPS) as described previously for RBC incubation in the presence of a high concentration of

calcium[36]. The strongest activator of RBCs LPA 18:1 was selected for this series of

experiments. LPA 18:1 induced PS externalization by RBCs and calcium enhanced LPA

18:1-mediated cell surface exposure of PS (Figure 7A). LPA 18:1 also stimulated the release

of PS+ (Figure 7B) and PS- REVs (Figure 7C). However, the release of PS+ REVs was lower

(Figure 7B), and that of PS- REVs strongly reduced in the presence of calcium (Figure 7C).

The addition of 1 % BSA to the medium supplemented with or without calcium abolished

LPA 18:1-mediated PS exposure by RBCs (Figure 7A) and the release of PS+ and PS- REVs

(Figure 7B and C).

Since major blood components impact LPA-mediated RBC activation, we investigated next

LPA effects in a closer physiological condition. We resuspended washed RBCs in an

equivalent volume of PFP- and EVs-free plasma to mimic the vascular compartment. In those

conditions, the stimulation with LPA 18:1 for 1 h or 24 h had no discernable impact on the

level of PS exposure by RBCs (Figure 7D). However, RBCs stimulated for 1 h with 20 µM

of LPA 18:1 showed a significant release of PS- REVs (Figure 7F) and higher quantities of

PS+ REVs albeit not significant (Figure 7E). After 24 h, the accumulation of PS+ REVs was

significantly higher for RBCs stimulated with 20 µM of LPA 18:1 (Figure 7E). REV

populations produced by RBCs stimulated with LPA 18:1 in EVs-free PFP, as characterized

by flow cytometry (Figure 7F), resemble those detected in the PFPs of SLE patients (Figure

3D). Interestingly, SLE patients showed higher plasmatic PS+ and PS- REV levels compared

to plasma of healthy controls (Figure 8A and B). Our data show that the REVs released by

RBCs in vitro can be observed in conditions that approximate to the vascular compartment.

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6 Discussion

In this study, we showed that three LPA species, 16:0, 18:0, and 18:1, activate RBCs by

inducing PS exposure and the release of REVs. The REVs produced in response to LPA are

mainly small PS- EVs, but another population of larger PS+ EVs was also released. LPA-

mediated PS exposure and REV release as well were through the LPA3 receptor and possibly

LPA1. Among the LPA species tested, LPA 20:4 inhibited PS exposure on RBCs and the

release of small PS- REVs. Inhibition by LPA 20:4 of the release of the PS- REVs is through

the LPA2 receptors (Figure 9). Our data do not exclude the possibility that LPA 16:0 and

18:1 bind to LPA2 and contribute to limiting RBC activation through LPA3. Finally, we were

capable of reproducing part of RBC activation by LPA, including PS exposure and the release

of PS+ and PS- REVs, using conditions that mirror the vascular environment.

Even if we validated that LPA3 activates RBCs by inducing both PS exposure and REV

release using an agonist for LPA3 and an antagonist for LPA1 and LPA3, a role for LPA1

remains unclear. LPA species affinity for the LPA receptors is dependent on the fatty acid

chain[24, 25]. LPA 20:4 binding affinity for LPA3 has been reported among the lowest for

LPA species, whereas LPA 20:4 binding affinity for LPA1 is similar to LPA 16:0, 18:0 and

18:1[25]. A role for LPA1 in RBC activation is not excluded but could not be addressed in-

depth due to the lack of LPA1 selective agonists. Furthermore, even if LPA1 and LPA3

usually mediate similar biological functions, a role for LPA in promoting erythropoiesis has

been associated solely with LPA3[32]. To this day, no study showed an implication of LPA1

in RBC functions. An unambiguous approach to determine if LPA1 modulates the production

of REV and PS exposure by RBCs would require mouse knockout for LPA1.

LPA2 is known to inhibit to erythropoiesis[34]. In this study we provide evidence that

inhibition of small PS- REV release by LPA 20:4-activated RBCs depends on LPA2. Among

the species we tested, the one with the highest affinity for LPA2 is LPA 20:4[25], which is

in line with the effects of 20:4 reported in this study. However, LPA 16:0 and 18:1 could also

activate LPA2 but with a lower affinity than LPA 20:4[25]. Our data suggest that LPA2 can

mitigate LPA 16:0 or 18:1-mediated production of EVs by RBCs, as evidenced by the

increased production of EVs in the presence of an LPA2 antagonist. Other methods such as

67

silencing of LPA receptors are not amenable in mature RBCs. However, the observation that

LPA2 antagonist H2L5186303 can abolish the inhibitory effect of LPA 20:4 on LPA3

agonist-mediated production of EVs reinforces the hypothesis that signaling through LPA2

inhibits the release of small PS- EVs (Figure 9).

Increased inhibition of LPA 20:4-mediated PS exposure with H2L5186303 was unexpected

in experiments where RBCs were activated by the LPA2 agonist 2-S-OMPT. H2L518630 is

a competitive antagonist with a 50% inhibitory concentration (IC50) one hundred times

inferior for LPA2 than for LPA3. Competition between H2L518630 and 2-S-OMPT from

binding to LPA3 is unlikely since LPA 20:4-mediated inhibition of PS- REV release by 2-S-

OMPT is abolished by the LPA2 receptor antagonist. The inhibition of PS exposure by RBCs

seen with increasing concentration of H2L5186303 could be due to an unknown off-target

effect in the presence of LPA 20:4. Further, studies needed to unravel the mechanisms by

which LPA 20:4 modulates RBC PS exposure. Signaling through LPA5 is a pathway that

contributes to activation platelets, which share the same progenitor cell as RBCs[44].

Moreover, activation of LPA5 by 20:4 was reported[44]. The absence of available selective

agonists/antagonists for LPA5, LPA4, and LPA6 precludes further analysis of signaling

pathways involved in LPA 20:4 mediated inhibition of PS exposure in mature RBCs.

Depending on the study, the concentration of LPA in human plasma ranges from 50 nM to

1 µM[45-47], and up to 14 distinct LPA species are detected[45]. Most studies focus on 6

species: LPA 16:0, 18:0, 18:1, 18:2, 20:4 and 22:6; and with the following relative

abundance: LPA 18:2 > LPA 20:4 > LPA 16:0 ≥ LPA 18:1 = LPA 18:0 = LPA 22:6[39, 45-

47]. Platelet activation, a major driving force behind LPA increase in plasma, leads to LPA

16:0, 18:0, 18:1 and 20:4 accumulation with no significant changes in LPA 18:2 and 22:6

levels[40, 48, 49]. In the present study, we selected four LPA species (i.e. LPA 16:0, 18:0,

18:1 and 20:4) that are consistently detected in human plasma and produced upon platelet

activation or lipoprotein oxidation[40]. Since LPA 18:2 is the most prevalent species in

human plasma and the levels of LPA 22:6 and 18:2 are increased in acute coronary syndrome

patient[40], a future study of those LPA species on RBCs would be of interest.

An analysis of EVs present in human PFP by cryo-electronic microscopy reported that 95 %

of EVs in PFP were under 1 µm with an average diameter of 275 ± 150 nm[17]. Furthermore,

68

the same study found that only a fraction of PFP REVs were PS+[17]. Similarly, Zetasizer

analysis reported average size of 200 nm for REVs produced in vitro[36]. These findings

support our observations that RBCs release mainly small PS- EVs. However, confocal

microscopy and classic flow cytometry studies also highlighted the production of PS+

REVs[22, 36]. The previous study using classic flow cytometry reported that 98 % of REVs

produced in response to LPA were PS+[22]. Less than 1 % of EVs are detected using classic

flow cytometry as compared to cryo-electronic microscopy[17]. The main limitation of

classic flow cytometry is the detection of the small size vesicles released by cells. Our

findings suggest that the PS+ REVs detected in classic flow cytometry could apply only to

the largest REVs that only account for a small fraction of total REVs[17, 36].

We showed that plasma from healthy donors deprived of cells and EVs, LPA 18:1 was able

to induce the release of PS+ and PS- REVs. Since plasma components can alter the LPA

effects on RBCs, plasma composition may play a crucial role in inhibiting or promoting RBC

activation by LPA. Vascular inflammation is associated with chronic rheumatic

inflammatory diseases. PS+ and PS- REV levels are higher in pathologies with vascular

inflammation such as SLE. Further studies should aim at determining whether RBCs from

SLE and rheumatoid arthritis patients are more prone to activation by LPA or whether the

plasma of patients enhances LPA-mediate PS externalization and EV production by RBCs

from healthy controls.

Patients with rheumatoid arthritis and SLE suffer from comorbidities, with cardiovascular

diseases being the most preponderant factor[50, 51]. Cardiovascular dysfunctions

(atherosclerosis, coronary heart disease, stroke, heart failure) and thromboembolic events

correlated to systemic inflammation in rheumatoid arthritis and SLE[52]. REVs and PS

exposing RBCs were associated with activation of the blood coagulation cascade[3, 13] and

vascular inflammation[16, 53, 54]. Vascular LPA levels are higher in patients with

atherosclerosis[55] and those suffering from acute coronary syndrome[56, 57]. Plasma from

rheumatoid arthritis patients shows higher levels of autotaxin, the enzyme responsible for

LPA extracellular production[58]. Activated platelets are a significant source of LPA

including LPA 16:0, 18:0, 18:1, and 20:4[49]. The present study shows that some of those

species induce PS exposure and REV release by RBCs. Since both PS exposure and REVs

69

can promote coagulation and inflammation[13, 15, 16], RBC activation by LPA could be a

noteworthy mediator for the amplification of inflammation and the coagulation cascade.

Therefore, LPA capacity to modulate RBC physiology is a pathway worth investigating to

understand how RBCs contribute to vascular physiology in atherosclerosis or rheumatoid

arthritis.

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74

8 Figures and legends

Figure 1. RBC activation and REV detection by high-sensitivity flow cytometry. (A)

RBCs were first gated according to size and granularity in SSC and FSC. Events were

considered for the RBC marker CD235a conjugated to PECy7 (upper left and right panels)

75

and for PS exposure, a marker of RBC activation, using FITC-conjugated Annexin V (upper

right panel). RBC stimulated with 5 µM of calcium ionophores served as positive control for

RBC activation. (B) REVs were defined as events with a size between 100 nm and 1 000 nm

(EV gate) and positive for the RBC marker CD235a (REV gate). Events in the FITC channel

were considered PS positive (PS+) REVs (REV PS+ gate). (C) The EV gate in SSC-H

(granularity) and FSC-PMT-H (relative size) were set with polystyrene beads of 100 nm,

500 nm, and 1 000 nm. (D) Specificity of REV detection was validated by the clearance of

REVs with 0.05% Triton X-100 (TX-100) treatment which destroys EV’s lipid bilayer and

by 100 000 g ultracentrifugation (ultra) which pellets EVs, (n=4). Results are the mean

percentage ± SD of the untreated condition (Ctrl). Statistical comparisons used the Kruskal-

Wallis test with the Dunnet post-test. (E) Calcium-free PBS supplemented with 50 µM of

EDTA (EDTA) was used for Annexin V labeling of REVs to validate the specificity of PS

detection at the surface of REVs. Data are the mean percentage ± SD of the labeling done in

Annexin V binding buffer (Ctrl). Statistical comparisons used the Mann-Whitney test. (F-H)

Two-fold serial dilutions of REV samples were quantified by high sensitivity flow cytometry

using polyester counting beads. REV concentrations and calculated dilution factors (F), the

mean (G) and the median (H) intensity of fluorescence for each dilution are presented (n=3).

Figure 2. RBC activation by LPA varies depending of the fatty acid. (A) Percentage of

RBCs exposing PS, (B) REVs and (C) PS+ REVs released in response to 10 µM LPA 16:0

(n=8), 18:0 (n=10), 18:1 (n=10) and 20:4 (n=4) after 1 h stimulation measured by high-

sensitivity flow cytometry. Data are the mean ± SEM, comparisons were done using Kruskal-

Wallis tests with Dunnet post-test, * p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

76

Figure 3. LPA induced RBC activation leads to two distinct REV populations. (A)

Percentage of RBCs exposing PS and (B) kinetics of REV released in response to increasing

concentration of LPA 18:1 measured by high-sensitivity flow cytometry (n=4). Statistical

comparisons used a two-way ANOVA with Dunnett's multiple comparison post-test. (C)

Representative dot plot for PS expression (left panel) and size (right panel) of REV released

77

by RBC in response to 10 µM of LPA 18:1 and (D) in PFP of SLE patients. (E) PS+ REV

and (F) PS- REV amount released in response to increasing concentration of LPA 18:1 after

0, 2, 15, 30, 60, and 120 min of stimulation measured by high-sensitivity flow cytometry.

(G) The relative percentage of PS- and PS+ REVs released in response to increasing LPA

18:1 concentration are the mean ± SEM. Statistical comparisons used a two-way ANOVA

with Dunnett's multiple comparison post-test, * p <0.05, **p <0.01, ***p <0.001, ****p

<0.0001.

Figure 4. LPA1/3 mediates RBC activation by LPA. (A) The percentage of RBCs exposing

PS, (B) REVs, (C) PS+ REVs, (D) PS- REVs released by RBCs stimulated with LPA3 agonist

2S-OMPT at 10 and 20 µM for 1 h measured by high-sensitivity flow cytometry (n=10). (E)

Percentage of RBCs exposing PS, (F) REVs, (G) PS+ REVs and (H) PS- REVs released in

response to LPA 16:0 (n=5), 18:0 (n=6) or 18:1 (n=7) in presence and in the absence of

LPA1/3 antagonist VPC32183 (VPC) at 5 or 15 µM measured by high-sensitivity flow

cytometry. REVs (total, PS+, PS-) are expressed as a percentage of total LPA-induced REVs.

78

Data are presented as mean ± SEM, Kruskal-Wallis test with Dunnet post-test, * p <0.05,

**p <0.01, ***p <0.001, ****p <0.0001.

Figure 5. LPA2 inhibits PS- REV production. (A) The percentage of RBCs exposing PS,

(B) REVs released by RBCs stimulated with LPA2 agonist GRI-977143 (GRI) at 10 and

20 µM for 1 h measured by high-sensitivity flow cytometry (n=10). (C) The percentage of

RBCs exposing PS, (D) REV, (E) PS+ REV and (F) PS- REVs released by RBCs stimulated

with 10 µM of LPA 16:0 (n=5), 18:0 (n=6) or 18:1 (n=7) in presence of LPA2 antagonist

H2L5186303 (H2L5) at 5 and 15 µM measured by high-sensitivity flow cytometry. REV

amounts (total, PS+, PS-) are expressed as a percentage of LPA induced REVs. (G) PS- REV

released by RBCs stimulated with 10 µM of 2S-OMPT in the presence of GRI measured by

high-sensitivity flow cytometry. PS- REV amounts are expressed as a percentage of LPA

induced REVs. All data are presented as mean ± SEM, Kruskal-Wallis test with Dunnet post-

test, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

79

Figure 6. LPA 20:4 inhibits PS- REV production through LPA2 and PS exposure by

RBCs. (A) The percentage of RBCs exposing PS, (B) PS+ REVs, and (C) PS- REVs released

by RBCs stimulated with 10 µM of 2S-OMPT in the presence of LPA 20:4 measured by

high-sensitivity flow cytometry. (D) Percentage of RBCs exposing PS and (E) PS- REVs

released by RBCs stimulated by 10 µM of 2S-OMPT in the presence of 5 µM of LPA 20:4

and H2L5186303 (H2L5) measured by high-sensitivity flow cytometry. PS- REVs are

expressed as a percentage of 2S-OMPT-induced REV release. Data are presented as mean ±

SEM, Kruskal-Wallis test with Dunnet post-test, * p <0.05, **p <0.01, ***p <0.001, ****p

<0.0001.

80

Figure 7. LPA 18:1 induces PS+ REVs in platelet-free and EV-free plasma from healthy

donors. (A) The percentage of RBCs exposing PS, (B) PS+ REVs, and (C) PS- REVs released

by RBCs stimulated for 1 h by LPA 18:1 in the absence or presence of 1 % BSA and calcium

2 mM (n=4) measured by high-sensitivity flow cytometry. Statistical comparisons used a

two-way ANOVA with Dunnett's multiple comparison post-test. For each incubation milieu,

we compared RBC stimulation to their respective control without LPA. (D) The percentage

of RBCs exposing PS, (E) PS+ REVs, and (F) PS- REVs released after 1 h or 24 h stimulation

by LPA 18:1 of RBCs in EV-free PFP (V/V) measured by high-sensitivity flow cytometry

(n=5). Statistical comparisons used the Friedman test with Dunn's multiple comparison post-

test (G). Representative dot plot for PS expression (left panel) and size (right panel) of REV

populations produced by stimulation of washed RBCs in EV free PFP (V/V) with LPA 18:1

at 20 µM. * p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

81

Figure 8. High plasmatic quantities of PS+ and PS- REV are present in SLE patients.

(A) PS+ REV and (B) PS- REV plasmatic amounts in SLE patients (n=102) and healthy

controls (n=30) measured by high-sensitivity flow cytometry. Statistical comparisons used

the Mann-Whitney test. * p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

82

Figure 9. LPA signaling in RBCs. LPA 16:0, 18:0 and 18:1 activate LPA3 on RBC which

induces PS exposure and the release of small PS- REVs and large PS+ REVs. LPA2 activation

by LPA 20:4 inhibits the release of small PS- REVs when RBCs are stimulated. LPA 16:0

and 18:1 can also activate LPA2. LPA 20:4 inhibit PS exposure of stimulated RBC through

an unknown mechanism. Created with BioRender.com.

83

Chapitre 2 : Plasma level of red blood cell-derived

phosphatidylserine positive extracellular vesicles are

associated with thrombosis in systemic erythematous

lupus patients

1 Résumé

Les cellules activées libèrent des vésicules extracellulaires (EV). Les EV promeuvent la

coagulation et l’inflammation notamment en étant une source d’antigènes du soi. Les patients

atteints par le lupus érythémateux disséminé (LED) présentent un inflammation vasculaire

importante et ont un risque accru de développer des maladies cardiovasculaires. C’est

pourquoi nous pensions que les EV de plaquettes et de globules rouges (REV) pouvaient être

corrélées à l’activité de la maladie et aux dommages cardiovasculaires qui sont associés au

LED. Bien que les PEV et les REV soient augmentées chez les patients LED, elles ne sont

pas associées avec l’activité de la maladie. Cependant, la stratification de la cohorte en

fonction des REV positives pour la phosphatidylsérine a mis en évidence une incidence plus

importante de thrombose chez les patients qui en présentent de grandes quantités.

84

Red blood cell-derived phosphatidylserine positive extracellular vesicles are

associated with thrombosis in systemic erythematous lupus patients

Stephan Hasse1, Anne-Sophie Julien2, Anne-Claire Duchez1, Chenqi Zhao1, Eric Boilard1,

Paul Fortin3, Sylvain G. Bourgoin1


Laval, Département de microbiologie-infectiologie et d’immunologie, Université Laval,

Québec, QC, Canada G1V 4G2.

2Département de mathématiques et statistique, Université Laval, QC, Canada G1V 4G2.


Laval, Département de médecine, Faculté de médecine, Université Laval, QC, Canada G1V

4G2.

Short title: Extracellular vesicles promote thrombosis in SLE

Corresponding author:

Sylvain G. Bourgoin, PhD

Centre de Recherche du Centre Hospitalier Universitaire de Québec

Faculté de Médecine de l’Université Laval

2705 Boul. Laurier, Québec, QC, Canada G1V 4G2

[email protected]

Phone: +1 (418) 525-4444, ext. 46136 Fax: (418) 654-2765

Key words: Extracellular vesicles, ATX, CD62P, phosphatidylserine, platelets, erythrocytes,

lupus, thrombocytopenia, thrombosis

85

2 Abstract

Background. Extracellular vesicles (EVs) released by blood cells have pro-inflammation

and pro-coagulant action. Systemic lupus erythematosus (SLE) patients present high vascular

inflammation and are prone to develop cardiovascular diseases. Therefore, we postulated that

the EV populations found in blood, platelet EVs (PEVs) and red blood cell EVs (REVs) are

associated with SLE disease activity and SLE-associated cardiovascular accidents.

Method. We assessed ATX plasma levels by ELISA, the platelet activation markers PAC1

and CD62P, ATX bound to platelets, and the amounts of plasma PEVs and REVs by flow

cytometry in a cohort of 102 SLE patients, including 29 incident cases of SLE and 30

controls. Correlation analyses explored the associations with the clinical parameters.

Result. Platelet activation markers were increased in SLE patients compared to control, with

the marker CD62P associated with the SLEDAI. The incident cases show additional

associations between platelet markers (CD62P/ATX and PAC1/CD62P) and the SLEDAI.

SLE patients presented higher levels of PEVs, phosphatidylserine positive (PS+) PEVs,

REVs, and PS+ REVs, but there is no association with disease activity. When stratified

according to the plasma level of PS+ REVs, the group of SLE patients with a high level of

PS+ REVs presented a higher number of past thrombosis events and higher ATX levels.

Conclusion. Incident and prevalent forms of SLE cases present similar levels of platelet

activation markers, with CD62P correlating with disease activity. Though EVs are not

associated with disease activity, the incidence of thrombosis is higher in patients with a high

level of PS+ REVs.

86

3 Introduction

Systemic lupus erythematosus (SLE) is a Systemic Autoimmune Rheumatic Disease

(SARD). SLE patients present a wide range of clinical phenotypes. One characteristic of SLE

is a high vascular inflammation associated with damages in multiple organs [1]. Patients with

SLE have a higher risk of dying from cardiovascular diseases [2]. The vascular inflammation

associated with SLE development relates to an immune response to autoantigens [3, 4] and

a production of type I interferon [5-7].

Platelet activation also induces the liberation of extracellular vesicles [8]. Extracellular

vesicles (EVs) are small vesicles liberated by activated cells. The EVs includes exosomes

and microvesicles. The fusion of multivesicular bodies with the plasma membrane releases

the exosomes. Plasma membrane budding generates the microvesicles, which often occurs

after cell loss of the membrane phospholipid asymmetry. Some EVs formed after the loss of

the plasma membrane asymmetry presents the phosphatidylserine (PS) at their surface. EVs

from platelets (PEVs) and red blood cells (REVs) can, through the exposition of PS, recruit

mediators of the coagulation cascade and initiate the formation of blood cloth [9-11]. SLE

patients present higher levels of platelet activation in SLE is a source of autoantigen, notably

by the release of free mitochondria [8, 12]. SLE patients have increased circulating EVs,

notably from platelet origin [13, 14] and serve as a source for interferon-α and bind sites for

immune complexes [15-17]. The exosomes are also associated with enhanced pro-

inflammatory cytokine and chemokine production, notably type I interferon in SLE [15, 16,

18].

PEVs are the largest EV population in the blood, which are found in higher amounts in the

plasma SLE patients. However, besides that PEVs are a source of autoantigens and bind

immune complexes, there is little knowledge on their role in SLE [14, 19]. In some studies,

there was an association between PEVs and the SLE disease index (SLEDAI) [14], but no

association was reported in others [20]. However, PEVs were associated with the progression

of atherosclerosis in SLE patients through the thickening of the vascular wall [19].

Platelet activation also releases autotaxin (ATX), a phospholipase with pro-inflammatory

properties. ATX is associated with the pathophysiology of rheumatoid arthritis and

87

cardiovascular diseases [21-23]. ATX catalyzes the production of lysophosphatidic acid

(LPA), one of the few known activators of red blood cells which induce the liberation of

REVs [24, 25]. SLE are more at risk to suffer from cardiovascular diseases that are the

leading cause of death for SLE patients. EVs and platelet activation are factors in the

development of cardiovascular diseases and other autoimmune diseases. PEVs and REVs

have pro-inflammatory and pro-coagulant activities [9, 10, 26-28]. Since SLE patients have

a high level of plasma EVs, we focused our study on PEVs and REVs, the two most abundant

EV populations found in the blood, and the associated vascular events.


4.1 SLE patients and healthy donors

SARD-BDB (Systemic Autoimmune Rheumatic Disease biobank and database repository of

the CHU de Québec-Université Laval) recruited prevalent SLE patients with a disease

duration superior to 15 months and incident SLE patients with a disease duration equal or

under 15 months. A control group formed by 30 healthy donors, under no medication and

without known illness, was recruited (mean age 50±8 years, female 63.33%).

4.2 SARD-BDB protocol

The SARD-BDB provided the plasma and platelet-free plasma (PFP) from SLE patients. The

PFP was processed using previously described standardized protocols [12]. Patients included

in the study gave informed written consent according to the declaration of Helsinki. The

ethics review board of the CHU de Québec-Université Laval reviewed and validated the

study (Project # 2016-2558). SLE patients had to meet the American College of

Rheumatology (ACR) classification criteria for SLE revised in 1997 [29, 30].

Antiphospholipid syndrome (APS) was diagnosed according to the 2006 revised Sapporo

criteria [31]. Variables were collected at the time of the first visit to the SARD-BDB

including, sociodemographic variables, diseases characteristics, clinical variables, common

hematology tests, cardiovascular and thrombosis risk factors, and current use medication.

88

4.3 Flow cytometry

4.3.1 Detection of platelet activation

Five µL of PRP were incubated 30 min at room temperature in the dark with 3 µL of anti-

CD41-V450 (BD Bioscience Canada, Mississauga, ON, Canada), a marker of platelets, 15

µL of anti-CD62P-APC and 15 µL of anti-PAC1-FITC (BD Bioscience Canada,

Mississauga, ON, Canada) and anti-ATX (BD Bioscience Canada, Mississauga, ON,

Canada) in 100 µL of PBS. The samples were mixed with 400 µL of PBS to stop labelling

and analyzed using a high sensitivity flow cytometer BD Canto II Special Order Research

Product with the gating strategy described in supplementary figure 1A. The flow cytometer

settings were as follows: FSC at 300 V, SSC at 335 V, Pacific blue at 500 V, FITC at 500 V,

PE at 500 V and APC at 500 V.

4.3.2 Detection of plasmatic EVs

Five µL of PFP were incubated for 30 min at room temperature and in the dark with 3 µl

anti-CD41-V450 (BD Bioscience Canada, Mississauga, ON, Canada), a marker for platelet-

derived EVs, and with 3 µl anti-CD235a-PECy7 (BD Bioscience Canada, Mississauga, ON,

Canada) to label the REVs. To detect PS exposed on the outer membrane leaflet, we added

3 µl Annexin V FITC (BD Bioscience Canada, Mississauga, ON, Canada) to the plasma

samples in 100 µl final Annexin V binding buffer (BD Bioscience Canada, Mississauga, ON,

Canada). The samples were mixed with Annexin V binding buffer (200 µL) to stop labelling

and processed under 90 min on a high sensitivity flow cytometer BD Canto II Special Order

Research Product with a small particle option as previously described [32]. The

supplementary figure 1B shows the gating strategy for the detection of plasmatic PEVs and

REVs. Silica particles of 100, 500 and 1,000 nm (Kisker Biotech GmbH & Co. Steinfurt,

Germany) allowed to set up a gate differentiating the events of size between 100 to 1 000 nm

(Supp. Fig. 1B). The flow cytometer settings were as follows: FSC at 300 V, SSC at 300 V,

PECy7 at 500 V, FITC at 500 V and APC at 500 V. To determine the absolute amounts of

REVs and PEVs in the samples, we added known concentrations of 2 µm APC polystyrene

beads (BD Bioscience Canada, Mississauga, ON, Canada) or 3 µm polystyrene beads

(Polysciences, PA, USA).

89

Specificity of EV detection was validated by destroying EVs from the sample by Triton X-

100 treatment or pelleting EVs by a 100 000 g ultracentrifugation (Supp. Fig. 1D). Labelling

in EDTA-supplemented buffer and absence of annexin V buffer was used to validate the

specificity of Annexin V labelling (Supp. Fig. 1E). Finally, a coincidence test validated that

our measurements of PEV and REVs were quantitative (Supp. Fig. 1F and G).

Every day before monitoring platelets activation markers and plasma EVs, a test of

performance tracking of high sensitivity flow cytometry was done using BD cytometer setup

and tracking beads (BD Bioscience Canada, Mississauga, ON, Canada).

4.4 Autotaxin measurement

Plasmatic concentrations of autotaxin (ng/mL) were quantified using a Human ENPP-

2/Autotaxin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and following

the manufacturer instructions.

4.5 Analysis and Statistics

Flow cytometry data analysis used the FlowJo V10 software (FlowJo, LLC, OR, USA) and

statistical analysis with GraphPad Prism 7.0 software (GraphPad Software, San Diego, USA)

and SAS version 9.4 (SAS Institute Inc, Cary, North Carolina, USA). Comparisons between

groups used the Kruskal-Wallis tests with Dunn’s multiple comparison post-test or the

Wilcoxon Mann Whitney test for continuous variables, depending on the number of groups.

The Exact Pearson Chi-Square Test was used to compare groups for discrete variables.

Spearman’s correlation coefficient (rs) determined the association between continuous

variables. Only variables monitored at the first visit (baseline) were considered for the

analyses.

5 Results

5.1 Patient’s characteristics

The characteristics at baseline of the 102 SLE patients included in the cohort are presented

in Table 1. For 29 of 102 patients, the SLE diagnosis was 15 months or less before their

90

inclusion in the cohort. These patients that started their treatments recently and had not their

SLE under control were considered incident cases. The SLE prevalent cases were the patients

treated for more than 15 months. Women represented 83% of all SLE cases. About a quarter

of the patients had thrombocytopenia (23.5%). SLE patients with renal disorders and

antiphospholipid syndrome represented 24.5% and 15.7% of the SLE cohort, respectively.

The prevalence of thrombophilia in the SLE cohort was 11.8%. Furthermore, 30 patients had

atherosclerosis plaques, and 42 had no plaques (Table 1).

5.2 SLE patients present higher platelet activation and plasma EV levels at

baseline

When all the prevalent and the incident cases were considered at baseline, SLE patients

present a higher percentage of platelet with the activation marker PAC1 and CD62P by

comparison to healthy controls (Table 2). In addition, a higher number of platelets were

positive for the phospholipase ATX (Table 2). ATX is known to be secreted by and to bind

the integrins of activated platelets [33-35]. However, the total ATX plasma level was not

different between the SLE and the healthy group (Table 2). The plasma of SLE patients

shows high levels of platelet- and RBC-derived EVs, including those exposing PS at their

surface (Table 2). Furthermore, 79.3% of the plasma PEVs and 23.6% of the plasma REVs

were PS+ in the control group, and this proportion increased respectively to 87.8% and 45.2%

in the SLE cohort (Table 2).

5.3 Prevalent and incident SLE patients show similar levels of plasma EVs

and platelet activation.

We investigated the difference between recently diagnosed and established cases of SLE by

comparing the incident and prevalent cases (n=29 and n=73, respectively). The levels of

platelet activation markers and plasma EVs of the prevalent SLE were not significantly

different from those of the incident SLE cases (Fig. 1A & 1B). ATX plasma concentration

in healthy controls, prevalent and incident SLE cases are similar (Fig. 1C). However, plasma

EV levels in prevalent and incident cases were significantly different from those of the

control group (Fig. 1C & 1D). The incident SLE cases show no significant difference for the

91

platelet activation marker PAC1 or CD62P compared to healthy control (Fig. 1A, upper &

middle panels). In comparison to healthy patients, we report a significantly higher

percentage of platelets exposing both the activation markers PAC1 and CD62P (Fig. 1A,

lower panel). The prevalent and the incident SLE cases did not show a significant difference

for the platelet activation marker PAC1 compared to healthy control (Fig. 1A, upper panel).

In contrast, the amounts of CD62P positive (Fig. 1A, middle panel) and PAC1-CD62P

double-positive platelets (Fig. 1A, lower panel) were significantly different from the control

group. Compared to healthy controls, the levels of double-positive PAC1-ATX (Fig. 1B,

upper panel), double-positive CD62P-ATX (Fig. 1B, middle panel), and triple-positive

PAC1-CD62P-ATX platelets (Fig. 1B, lower panel) were significantly different with those

of prevalent but not those the incident SLE cases.

5.4 Platelet activation is associated with the SLEDAI score in incident cases

of SLE.

Despite significant increases in the incident and prevalent SLE groups, there was no

association between the EV populations and the SLEDAI score (Table 3). Only CDP62P, a

marker of platelet activation, was consistently associated with a higher SLEDAI score for the

incident and prevalent cases, or when we considered the whole SLE cohort patients

(Table 3). However, incident SLE cases show additional associations with platelet activation

markers as the levels of platelets highly positive for both CD62P and ATX or CD62P and

PAC1 are significantly associated with the SLEDAI score (Table 3).

5.5 Higher PS+ REVs are associated with vascular damages in SLE patients.

Incident and prevalent SLE patients did not present differences in plasmatic levels of ATX,

platelet activation and for both RBC and platelet EVs (Fig. 1A-E). Therefore, we choose not

to distinguish between the incident and prevalent SLE cases while analyzing the potential

clinical implications of high plasma levels of PS+ REVs. Based on the levels of PS+ REVs,

we divided the SLE cohort into two distinct groups (Fig. 1E). One group of patients had a

concentration of PS+ REVs similar to that of healthy controls. The second group of patients

showed a high plasma concentration of PS+ REVs. We applied a cut off at 1000 EV/µL to

92

distinguish the patients with a level of plasma PS+ REVs within the range of the healthy

controls from those with EV concentrations superior to the cut-off. The latter were considered

high plasma levels of PS+ REVs. Fifty-two SLE patients (51%) had a plasma PS+ REV

number over the cut-off level. The 50 SLE patients with a concentration of plasma PS+ REVs

lower than the cut off were considered “normal” for these biomarkers.

As anticipated, the SLE patients with a higher level of PS+ REVs also show a higher number

of REVs and PEVs (total and PS+) compared to those with “normal” concentrations of PS+

REVs (Table 4). Furthermore, the high PS+ REVs levels are associated with a lower platelet

count and high plasma level of plasma ATX (Table 4). Compared to patients with a normal

PS+ REV level, the plasma ATX amount was significantly higher in patients with elevated

PS+ REVs. The high PS+ REV counts were not associated with the disease duration or recent

disease onset but correlated with thrombophilia (Table 4). Only two patients with low plasma

PS+ REV levels (4%) had a history of venous thrombosis. Among patients with high PS+

REVs, 5 (10%) presented a history of venous thrombosis, 4 (8%) of arterial thrombosis and

one (2%) of microcirculation thrombosis. In this group of patients, we also highlight an

association between the plasma levels of PS+ REVs and the presence of autoantibodies

(Table 4). Surprisingly, patients with high amounts of PS+ REVs tend to have a lower

SLEDAI score, and antiphospholipid syndrome incidence tended to be higher than for

patients with low PS+ REV levels (Table 4).

93

6 Discussion

In this study, we report on a high plasma level of REVs in SLE patients. Furthermore, the

percentage of PS+ REVs in the plasma of SLE patients almost doubled compared to the

healthy group. The patients with a high level of plasma REVs also have elevated plasma

PEVs. There was no correlation between the EV levels, including those that are PS+, and the

SLEDAI. About half of the SLE patients had PS+ REV levels like the healthy controls.

However, a high level of PS+ REVs was positively associated with thrombocytopenia and a

higher incidence of thrombotic events.

A high level of platelet activation markers and plasma PEVs were reported previously in SLE

patients [36]. CD62P (P-selectin) is only present on the activated platelet following the fusion

of the α-granules with the plasma membrane [37]. The platelet activation marker CD62P (P-

selectin) is elevated in SLE patients and is positively associated with the SLEDAI score [36].

This study confirms the positive association between the number of CD62P+ platelets and

disease activity. The stratification of the incident and prevalent cases of SLE did not highlight

differences regarding the levels of plasma EVs, PS+ EVs, and platelet activation markers. In

incident SLE cases, the percentages of platelets positive for CD62P, CD62P/ATX or

PAC1/ATX double-positive, and PAC1/CD32P/ATX triple-positive, were not statistically

different from the healthy group. It may be due to the small number of newly diagnosed SLE

patients included in the present study. However, in the incident and prevalent cases, the

SLEDAI score correlated with platelet activation as monitored by the cell surface exposure

of CD62P. In incident SLE cases, we found other associations between the platelet activation

markers and the SLEDAI score. Those include the CD62P/ATX and CD62P/PAC1 double-

positive platelets. PAC1 monitor the activation of αIIbβ3 integrin complex in platelets [38,

39]. Of note, the ATX stored in α-granules and released upon platelet activation can bind the

platelet αIIbβ3 integrin [33-35]. It would suggest that the liberation of α-granule and the

activated form of platelets integrins contributes to the early phase of SLE disease progression.

While, at later stages, when the disease becomes chronic, only the liberation of the content

of α-granule is of importance.

94

It was possible to stratify the SLE patients based on the levels of plasma PS+ REVs. About

half of the patients had plasma levels of PS+ REVs below the threshold of 1 000 REVs/µL

found in healthy controls. The other half were SLE patients with high to very high levels of

PS+ REVs. The results suggest that a high amount of plasma PS+ REVs is associated with a

higher incidence of cardiovascular events. A longitudinal study on incident cases with no

antecedent thrombosis or cardiovascular diseases would confirm if SLE patients with a high

level of plasma PS+ REVs are more at risk of thrombosis. REVs exposing PS+ can recruit

different actors of the coagulation cascade and be a source for the generation of large

quantities of thrombin [10, 40]. REVs are also a source of the von Willebrand factor [41].

SLE patients with a high plasma level of PS+ REVs also show high amounts of plasma ATX.

ATX is the enzyme that produces LPA [42]. The binding of ATX to αIIbβ3 integrin of

activated platelets enhance its catalytic activity [33, 34]. Though we did not monitor the

plasma LPA levels, a role for LPA in RBC activation and production of PS+ REVs cannot be

excluded [24, 43].

Recent studies highlighted a possible role for phosphatidylserine-specific phospholipase A1

in SLE physiopathogenesis [44, 45]. Serum levels of phosphatidylserine-specific

phospholipase A1 are significantly higher in SLE patients with high disease activity. Besides,

patient treatment with immunosuppressive therapies lowered the amount of serum

phosphatidylserine-specific phospholipase A1 [44]. Serum phosphatidylserine-specific

phospholipase A1 and ATX are also higher in patients with lupus nephritis [45]. However,

there was an inverse correlation between the levels of serum ATX and disease activity [45].

Of note, we observed that patients with a normal plasma PS+ REV level tend to have a higher

SLEDAI and lower levels of plasma ATX. The role of ATX and phosphatidylserine-specific

phospholipase A1 in SLE pathophysiology is not known. Increased expression of

phosphatidylserine-specific phospholipase A1 is associated with many pathological

conditions, including autoimmune and cardiovascular diseases [46]. Phosphatidylserine-

specific phospholipase A1 can hydrolyse PS into lysoPS, and ATX can hydrolyze the lysoPS

into LPA [46]. On one side, long-chain lysoPS may contribute to immune cell activation,

including macrophages [47]. On the other side, stimulation of red blood cells with LPA

induces the liberation of REVs [24, 25]. LPA induces the production of REVs by red blood

cells in a concentration- and LPA species-dependent manner and through activation of

95

LPAR3 [48]. Low concentrations of LPA induce of the production of PS- REVs while the

production of PS+ REVs by red blood cells requires concentrations of LPA ≥ 5 µM [48].

Further studies should determine if the SLE patients with high plasma of PS+ REVs and ATX

also show elevated amounts of phosphatidylserine-specific phospholipase A1.

A higher percentage of patients with elevated amounts of plasma PS+ REV presented

abnormal quantities of glycoprotein and cardiolipin auto-antibodies. In SLE patients, higher

disease activity and clinical manifestations such as thrombosis were associated previously

with glycoprotein and cardiolipin antibody levels [49-53]. During our study, a change in the

coding variables occurred for cardiolipin and glycoprotein auto-antibodies. The changes may

have affected the validity of the statistical analysis between the group, especially for auto-

antibody variables.

Atherosclerosis is a lead cause of cardiovascular incidents and is a known comorbidity factor

in SLE [54-58]. PEVs exposing PS were associated with accelerated thickening of the intima-

media in SLE patients [19]. In addition, several EV populations promote the development of

atherosclerosis through the recruitment of immune cells in the vascular wall and the

production of cytokines[59-61]. Besides, through the uptake of EVs, vascular wall infiltrated

macrophages accumulate lipids and transform into foam cells [62-64]. Macrophages

phagocyte the PS+ EVs at a higher rate [65]. Therefore, even if PS+ REVs are not associated

with SLE progression, they still could be implicated in the progression of atherosclerosis and

thrombotic events associated with SLE. We did not dispose of sufficient measurements to

investigate a potential link between the levels of PS+ REVs and the carotid intima-media

thickness test. The overtime impacts of PS+ REVs and other blood cell-derived PS+ EVs on

the thickening of carotid intima-media of SLE patients would be worth investigating in SLE

patients.

In summary, SLE patients show a high number of plasma PEVs and REVs and high surface

exposure of the platelet activation markers PAC1 and CD62P compared to age- and gender-

matched controls. There are no significant differences between the incident and prevalent

cases of SLE. The levels of EVs do not correlate with the SLEDAI score. However, the

analyses of CD62P exposure on the platelet surface show an association with SLEDAI,

consistent with the documented association between this platelet activation marker and

96

disease activity. The plasma PS+ REVs segregate the patients into groups with normal and

high PS+ REV levels, respectively. The analyses show a higher incidence of

thrombocytopenia and thrombotic events in SLE patients with a high level of PS+ REVs.

Further studies are required to determine if the plasma level of PS+ REVs is a potential

biomarker for managing the cardiovascular risk of individuals with SLE.

97

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8 Figures, legends and tables

Table 1: characteristics for SLE patients included in the study at baseline.

Demographics mean±SD or % (n)

Age, years, n=102 49.98 ± 14.58

Gender, Female, n=102 83.33 (85)

BMI, n=101 25.49 ± 4.66

Disease duration, years, n=98 10.63 ± 12.07

Onset, n=102

Incident 28.43 (29)

Prevalent 71.57 (73)

APS, n=102 15.69 (16)

ACR criteria n=99 % (n)

Arthritis 77.45 (79)

Thrombocytopenia 23.53 (24)

Malar rash 25.49 (26)

Discoid rash 17.65 (18)

Hemolytic anemia 3.92 (4)

Renal disorder 24.51 (25)

Medication n=102 % (n)

NSAID/ Cox-II Inhibitors 24.51 (25)

Antipalutic drugs 75.49 (77)

Immunomodulators 84.31 (86)

Methotrexate 15.69 (16)

Biologic agents 6.86 (7)

Steroids 21.57 (22)

Other DMARD 27.45 (28)

Prednisone 20.59 (21)

Clinical characteristics mean±SD or % (n)

SLEDAI, n=99 3.07 ± 3.70

Platelet, 109/L, n=101 227.20 ± 74.91

MPV, fL, n=101 8.92 ± 6.10

Hemoglobin, g/L, n=101 129.34 ± 12.29

CRP, mg/L, n=82 4.47 ± 7.25

ESR, mm/hour, n=94 13.55 ± 16.55

Lupus anticoagulant, n=97 10.78 (11)

Anti-cardiolipine IGG, n=97 equivocal 1.96 (2)

abnormal 14.71 (13)

Anti-cardiolipine IGM, n=97 equivocal 0.98 (1)

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abnormal 12.74 (12)

Glycoprotein IGG, n=97 abnormal 7.84 (8)

Glycoprotein IGM, n=97 abnormal 14.71 (15)

Cardiovascular damages mean±SD or % (n)

Thrombophilia, n=102 Venous 6.86 (7)

Arterial 3.92 (4)

Microcirculation 0.98 (1)

Plaque, n=72 29.41 (30)

CIMT, n=35 0.62 ± 0.12

ACR American college of rheumatology; APS antiphospholipid syndrome; BMI body mass index; CIMT carotid intima-media thickness; CRP C reactive protein; DMARD Disease Modifying Anti Rheumatic Drug; ESR erythrocyte sedimentation rate; MPV mean platelet volume; NSAID nonsteroidal anti-inflammatory drug; SLEDAI SLE disease activity index.

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Table 2: High platelet activation and EV quantities are found in SLE patients at baseline.

healthy SLE Pvalue

ATX, ng/mL 257.8 (219.9; 340.1) 251.4 (197.9; 334.6) 0.2428

Platelet, %

PAC1+ 3.65 (1.10; 8.50) 7.85 (2.88; 15.75) 0.0109

PAC1+ ATX+ 0.60 (0.20; 1.00) 1.90 (0.60; 4.30) 0.0026

CD62P+ 2.35 (1.70; 4.30) 5.55 (2.33; 9.53) 0.0044

CD62P+ ATX+ 0.70 (0.30; 0.80) 1.35 (0.60; 4.35) 0.0029

PAC1+ CD62P+ 0.70 (0.40; 1.40) 2.00 (0.90; 4.98) 0.0002

PAC1+ CD62P+ ATX+ 0.28 (0.15; 0.63) 0.85 (0.379; 3,24) 0.0011

Evs, EVs/µL

PEVs 478 (275; 1073) 3569 (1732; 7183) <0.0001

PS+ PEVs 377 (221; 816) 3228 (1581; 6299) <0.0001

REVs 423 (296; 602) 1936 (846; 4527) <0.0001

PS+ REVs 90 (55; 143) 1064 (165; 2304) <0.0001

Results are presented as median (Interquartile range), Pvalue based on Wilcoxon Mann Whitney Test

Table 3: Spearman correlation between our measurement and the total SLEDAI score for

SLE patients.

All SLE Prevalent SLE Incident SLE

n rs Pvalue n rs Pvalue n rs Pvalue

Plasmatic ATX 95 -0.17 0.0998 69 -0.17 0.1695 26 -0.18 0.3717

Platelet activation 39 22 17

PAC1+ 0.09 0.5859 -0.08 0.7141 0.46 0.0646

PAC1+ ATX+ 0.02 0.9032 -0.18 0.4273 0.41 0.1040

CD62P+ 0.48 0.0021 0.43 0.0479 0.59 0.0127

CD62P+ ATX+ 0.19 0.2492 0.03 0.8818 0.56 0.0207

PAC1+ CD62P+ 0.27 0.0927 0.01 0.9790 0.65 0.0045

PAC1+ CD62P+ ATX+ 0.12 0.4565 -0.04 0.8600 0.43 0.0817

PEVs 99 72 27

Total 0.06 0.5377 0.01 0.9093 0.28 0.1574

PS+ 0.05 0.6389 0.00 0.9842 0.25 0.2122

REVs 99 72 27

Total -0.07 0.4806 -0.09 0.4623 0.04 0.8275

PS+ -0.08 0.4274 -0.12 0.3166 0.16 0.4325

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Table 4: Comparison of SLE patients with low and high PS+ REVs.

PS+ REV

Demographic characteristics low high Pvalue

Gender, n Female 44 (88) 41 (79) 0.2897

Age, years 52.00 (41.00; 62.00) 50.50 (37.50; 59.00) 0.5592

Disease duration, years 7.93(0.76; 21.64) 3.58 (1.41; 15.87) 0.1426

Onset, n Incident 16 (32) 13 (25) 0.5124

Prevalent 34 (68) 39 (75)

APS 4 (8) 12 (23) 0.0551

Clinical characteristics

SLEDAI 2.00 (0.00; 6.00) 1.50 (0.00; 4.00) 0.0776

Platelet, 109/L 242.00 (195.00; 291.00) 202.00 (176.50; 246.00) 0.0262

MPV, fL 8.70 (8.00; 10.00) 8.90 (8.05; 9.55) 0.6992

Hemoglobin, g/L 129.00 (121.00; 137.00) 131.00 (124.00; 138.00) 0.3264

CRP, mg/L 2.90 (1.21; 3.42) 2.00 (1.00; 5.00) 0.7683

ESR, mm/hour 8.00 (4.00; 20.00) 6.00 (3.00; 14.00) 0.1674

Lupus anticoagulant presence 4/41 (10) 7/48 (15) 0.5374

Anti-cardiolipine IGG abnormal 3/37 (8) 10/50 (20) 0.0700

equivocal 2/37 (5) 0/50 (0)

Anti-cardiolipine IGM abnormal 3/37 (8) 9/50 (18) 0.1600

equivocal 1/37 (3) 0/50 (0)

Glycoprotein IGG abnormal 1/37 (3) 7/50 (14) 0.1300

Glycoprotein IGM abnormal 4/37 (11) 11/50 (22) 0.2500

Laboratory measurements

ATX, ng/mL 227.75 (183.10; 295.30) 274.50 (207.83; 408.73) 0.0318

PEVs, EVs/µL 2419 (1012; 4566) 4853 (2532; 8230) 0.0006

PS+ PEVs, EVs/µL 2042 (866; 4051) 4384 (2238; 7696) 0.0006

REVs, EVs/µL 842 (426; 1387) 4436 (3064; 7166) <.0001

ACR criteria

Arthritis 37 (76) 42 (84) 0.3262

Thrombocytopenia 9 (18) 15 (30) 0.2414

Malar rash 17 (35) 9 (18) 0.0705

Discoid rash 11 (22) 7 (14) 0.3080

Hemolytic anemia 3 (6) 1 (2) 0.3622

Renal disorder 12 (24) 13 (26) 1.0000

Cardiovascular damages

Thrombophilia 2 (4) 10 (19) 0.0283

Medication

NSAID/ Cox-II Inhibitors 14 (28) 11 (21) 0.4931

Prednisone 8 (16) 13 (25) 0.3299

Immunomodulators 42 (86) 44 (88) 0.7742

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Continuous variables are presented as median (Interquartile range), Pvalue based on Wilcoxon Mann Whitney Test. Categorical variables are presented as n (%), Pvalue based on Exact Pearson Chi Square Test.

Fig. 1: High platelet activation and EV levels are found in incident and prevalent SLE

patients at baseline. (A) Percentage of platelets expressing the activation marker PAC1

(upper panel), CD62P (middle panel) and the combination of PAC1 and CD62P (Lower

106

panel) for the healthy (n=30), prevalent (n=22) and incident SLE (n=18) group. (B)

Percentage of platelets expressing ATX and the activation marker PAC1 (upper panel),

CD62P (middle panel) or the combination of PAC1 and CD62P (Lower panel) for the healthy

(n=30), prevalent (n=22) and incident SLE (n=18) group. (C) Plasma concentration of ATX

for the healthy (n=30), prevalent (n=70) and incident SLE (n=28) (D) Number of plasma

PEVs (upper panel) and PS+ PEVs (lower panel) for the healthy (n=30), prevalent (n=73)

and incident SLE (n=29) group. (E) Plasma REV (upper panel) and PS+ REV levels (lower

panel) for the healthy (n=30), prevalent (n=73) and incident SLE (n=29) group. Data are

presented as median with interquartile range, Kruskal-Wallis test with Dunnett post-test,

*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Supplementary Figure 1: Platelet activation and EV detection by high-sensitivity flow

cytometry. (A) Plasma samples of SLE patients were assessed by high-sensitivity flow

cytometry (BD Canto II Special Order Research Product). Platelet were first gated according

to size (SSC) and granularity (FSC), and then for labeling with V450 fluorochrome-

conjugated antibodies directed against CD41a (CD41-V450-H), a platelet marker. Platelets

where then analyzed for the expression of several activation markers, firstly FITC

fluorochrome-conjugated antibodies directed against PAC1 (PAC1-FITC-H) alone or in

combination with ATX-PE-H, the APC fluorochrome-conjugated antibodies directed against

CD62P (CD62P-FITC-H) alone or in combination with ATX-PE-H, and lastly the

combination of PAC1-FITC-H and CD62P-FITC-H. Platelets positive for both PAC1 and

CD62P activation markers and labeled with PE fluorochrome-conjugated antibodies directed

against ATX (ATX-PE-H) were monitored. (B) PFP samples of SLE patients were assessed

by high-sensitivity flow cytometry (BD Canto II Special Order Research Product). Events

positive for the EV gate set for relative sizes comprised between 100 nm and 1 000 nm on

SSC and FSC-photomultiplier tube (PMT). EVs positive for the expression of CD41-V450-

H were considered PEVs and EVs positive for the expression of PECy7 fluorochrome-

conjugated antibodies directed against CD235a (CD235a-PECy7-H), a marker of RBC, were

considered REVs. Finally, we assessed the fluorescence labelling of PEVs and REVs using

FITC-conjugated Annexin V which binds PS (Annexin V-FTIC-H). (C) The EV gate in SSC-

H (granularity) and FSC-PMT-H (relative size) were set with polystyrene beads of 100 nm,

500 nm, and 1 000 nm. (D) Specificity of PEV and REV detection was validated by the

clearance of PEVs and REVs with 0.05% Triton X-100 (TX-100) treatment (n=6) which

destroys EV’s lipid bilayer and by 100, 000g ultracentrifugation (ultra) which pellets EVs,

(n=3) which are presented as the percentage of the untreated control (Ctrl). Data show the

mean percentage ± SD. Each condition was compared to its untreated control using the paired

t test. (E) Calcium-free PBS supplemented with 50 µM of EDTA (EDTA) was used for

Annexin V labeling of PEVs and REVs to validate the specificity of PS detection at their

surface (n=6) which is presented as the percentage of untreated (Ctrl). Data show the mean

percentage ± SD. Statistical comparisons used the paired t test. (F) Two-fold serial dilutions

of PEV samples were quantified by high sensitivity flow cytometry using polyester counting

beads. PEV concentrations and calculated dilution factors (left panel), and the median (right

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panel) intensity of fluorescence for each dilution are presented (n=3). Data are presented as

mean ± SD (G) Two-fold serial dilutions of REV samples were quantified by high sensitivity

flow cytometry using polyester counting beads. REV concentrations and calculated dilution

factors (left panel), and the median (right panel) intensity of fluorescence for each dilution

are presented (n=3). Data are presented as mean ± SD.

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Discussion

1 Mise en contexte

Le LPA est un lipide bioactif avec un rôle important dans la physiologie vasculaire. Le LPA

est un médiateur pro-inflammatoire qui est associé avec la progression de pathologies

inflammatoires comme l’arthrite rhumatoïde, une maladie rhumatismale auto-immune, ou

encore comme l’athérosclérose. Le LPA est notamment associé à l’activation des plaquettes

et à l’activation de la cascade de la coagulation lors de la rupture de plaques

d’athéroscléroses. Outre les plaquettes, le LPA est également connu comme le seul activateur

endogène des GR.

L’activation de cellules induit la libération d’EV. Les EV sont de petites vésicules

membranaires libérées dans le milieu extracellulaire par les cellules. Elles sont notamment

impliquées dans la communication intercellulaire et dans la régulation de l’environnement

vasculaire avec des effets pro-inflammatoires et sur le processus de la coagulation.

Les travaux présentés dans cette thèse ont examiné la modulation de l’activité des GR

par le LPA et leur implication possible dans la promotion des dommages vasculaires

associés aux MRAS.

2 Impact des limitations techniques dans l’analyse des vésicules

extracellulaires

La recherche sur les EV est un champ d’investigation récent qui ne présente pas encore

d’approches standardisées que ce soit dans la nomenclature, l’isolation, la caractérisation,

l’entreposage ou encore l’analyse des EV. Bien que les recommandations de l’ISEV

permettent d’avoir de plus en plus d’études qui respectent des normes communes, il reste

encore de nombreuses considérations méthodologiques et expérimentales à résoudre427,471,721.

Ma thèse étant centrée sur l’analyse d’EV par cytométrie en flux à haute sensibilité, je me

suis heurté à plusieurs limitations et problèmes posés par cette approche. Je vais revenir sur

les plus significatifs.

111

L’analyse d’EV dans des échantillons obtenus sur de grandes périodes, lors d’études

longitudinales par exemple, se heurte à plusieurs limitations. L’évolution de la sensibilité de

l’appareil lors d’un bris peut modifier grandement les limites de détection de l’appareil. Cela

peut rendre difficile, voire impossible, l’inclusion de mesure prise avant et après le bris dans

une même étude, et cela même avec l’utilisation d’un contrôle interne et une quantification

absolue. Même sans bris, la puissance des lasers diminue au fur et à mesure de leur utilisation.

Cette diminution réduit progressivement la fluorescence détectée pour un même réglage. Si

cela n’est pas vérifié et pris en compte régulièrement, il y a un risque de perdre les populations

de EV qui sont proches de la limite de détection.

De plus, le comportement des lots d’anticorps peut également varier au cours de l’étude

même pour des cibles bien établies. Cela peut donc conduire à l’abandon de marqueur

d’intérêt en cours d’étude à cause d’une perte de sensibilité de l’anticorps pour sa cible sur

les EV ou par l’apparition d’agrégats d’anticorps détectés par le cytomètre en flux à haute

sensibilité qui parasitent la détection des EV ciblées.

Ces soucis sont apparus au cours de mes travaux de thèse. Dans une première situation,

l’utilisation de tubes d’un même lot, mais commandés à des temps différents sur un même

échantillon produisait des résultats différents bien que l’analyse soit effectuée en parallèle

avec le même protocole. Cependant, le même test sur des cellules conduisait à des résultats

similaires. Dans la deuxième situation, un changement de lot d’anticorps a résulté dans

l’apparition d’agrégats non spécifiques qui se superposaient avec nos populations d’intérêt.

Le test des anticorps sur des populations d’EV de grande taille et sur des cellules a montré

une même spécificité de l’anticorps par rapport au lot précédent. Dans les deux cas, les

compagnies nous ont communiqué qu’elles n’avaient pas modifié le protocole de production

des anticorps et que la validation des anticorps avait été faite sur des cellules. L’absence de

validation des anticorps sur des EV pose plusieurs problèmes. D’une part, cela augmente la

variabilité des mesures déjà importante avec cette approche et d’autre part limite la

reproduction de résultats et donc limite leur validation722,723.

Réaliser les mesures une fois sur l’ensemble des échantillons dans un laps de temps limité

permet de réduire l’impact de l’évolution du matériel et des anticorps utilisés. Cependant, si

le projet requiert l’obtention d’échantillon sur de grandes périodes comme cela était notre

112

cas, il est nécessaire d’entreposer les échantillons à analyser ce qui peut avoir un impact sur

les résultats. En effet, la préparation et l’entreposage des EV peuvent induire la perte des EV

les plus fragiles ou de forte densité ainsi que modifier leur membrane724,725. Il en résulte qu’en

fonction de la solution d’entreposage des EV et des cycles de congélation / décongélation, le

contenu en EV de l’échantillon peut augmenter ou diminuer427,724,726. De plus au cours du

temps, malgré des conditions adéquates d’entreposage la quantité d’EV diminue dans les

échantillons727. Enfin, l’effet de l’entreposage n’affecte pas les populations d’EV de manière

homogène728.

La modification des populations d’EV peut mener à des analyses biaisées. Les

problématiques liées à l’entreposage des EV sont également importantes lorsque les

échantillons sont analysés au fur et à mesure du projet, mais peuvent être limitées notamment

par l’analyse des EV directement dans les échantillons frais, soit sans entreposage. Plusieurs

études conseillent cette approche pour les analyses dans les liquides biologiques427,724.

3 Résumé des travaux et discussion

3.1 L’acide lysophosphatidique et les vésicules extracellulaires de globules

rouges

Bien qu’il soit déjà connu, que le LPA active les GR par l’induction de la présentation de PS

à leur surface ainsi que par la libération d’EV positives pour la PS, les mécanismes ont été

peu étudiés575,576. L’externalisation de la PS par les GR en réponse au LPA est associée à la

mobilisation de calcium intracellulaire et à l’activation de la PKC. Les deux protéines G,

Gαq/11, et Gαi/o, peuvent être associées aux récepteurs au LPA et sont capables d’activer la

PKC. Par ailleurs, aucune étude n’avait étudié la possible hétérogénéité d’effet des

différentes espèces moléculaires de LPA sur l’activation des GR. Au vu de la littérature

limitée sur l’activation des GR par le LPA, nous avons voulu évaluer l’impact des espèces

moléculaires de LPA sur les GR, en particulier celles qui sont les plus abondantes dans la

circulation. Nous avons donc privilégié quatre espèces de LPA qui d’une part représentaient

la diversité des espèces moléculaires du LPA (saturé, mono-insaturé et poly-insaturé).

D’autre part, ces espèces étaient des formes majeures dans le plasma d’individu sain, mais

étaient produites lors de l’activation plaquettaire et lors de la préparation de sérum14,47,118,119.

113

Nous avons montré que l’activation des GR par le LPA varie en fonction de l’espèce

moléculaire avec notamment le LPA 20:4 qui ne présente aucun effet activateur. Et

contrairement aux études précédentes, nous avons montré que l’activation des GR par le LPA

ne débouche pas uniquement sur la libération de REV PS+, mais également sur la libération

de plus petites REV négatives pour la PS575,576. Cela s’explique en partie par la sensibilité de

la technique que nous avons utilisée qui permet de détecter des EV de petites tailles

(~ 100 nm). De plus, nous avons identifié un nouvel effet du LPA, il peut aussi inhiber

l’activation des GR. Enfin, nous avons été les premiers à associer l’effet du LPA sur les GR

matures à l’activation des récepteurs LPA2 et LPA3.

Notre étude de l’effet des espèces moléculaires de LPA sur les GR s’est heurtée à plusieurs

limitations. L’expression des récepteurs au LPA, a été mise en évidence dans les progéniteurs

myéloïdes communs729 et certains comme le LPA2 et les LPA3 sont impliqués dans la

régulation de l’érythropoïèse260,261,297. Cependant, leur présence chez les GR matures reste

encore inconnue. Des travaux antérieurs dans le laboratoire sur différentes cellules connues

pour exprimer les récepteurs au LPA ont montré que les anticorps disponibles contre les

récepteurs aux LPA ne permettaient pas la détection par immuno-buvardage de leur

expression basale. De plus, bien que les GR matures présentent des ARN messagers, la

maturation des globules rouges après l’énucléation présente une forte activité de dégradation

de l’ARN547,551,564 et l’hémoglobine contenue par les GR altère l’efficacité de la transcription

et de l’amplification des ARN par la technique de PCR730,731. Ces deux raisons pourraient

expliquer pourquoi nous n’avons pas pu détecter d’ARN messager pour les récepteurs au

LPA par des approches de RT-PCR. Il en résulte que nous n’avons pas pu valider la présence

des récepteurs au LPA autrement qu’avec des approches fonctionnelles soit par l’utilisation

d’agonistes et d’antagonistes. La disponibilité d’agonistes et d’antagonistes sélectifs pour les

récepteurs au LPA a limité le nombre de récepteurs que nous avons pu étudier. Il serait donc

intéressant d’approfondir les mécanismes d’action du LPA sur les GR à partir de modèles

murins ou par la différenciation in vitro de progéniteurs de GR. Ces modèles permettraient

notamment de supprimer ou de moduler l’expression de manière certaine des récepteurs au

LPA à des étapes précoces de l’érythropoïèse et ainsi s’assurer de leur absence sur les GR

matures.

114

Bien que nous ayons montré que l’activation des GR par le LPA est possible dans le plasma,

il n’y a pas encore d’estimations fiables des concentrations de LPA présente en circulation.

En effet, les études chez des sujets sains font état de concentrations allant de 50 nM à 1 µM

et jusqu’à 12 µM dans des situations pathologiques14,124,125,127,732. Il n’existe pas de protocole

standardisé pour le dosage du LPA plasmatique et il peut être synthétisé ou dégradé lors de

la manipulation d’échantillon sanguin. Les techniques d’extractions du LPA pour également

produire du LPA de façon artéfactuelle. Cela explique la grande variabilité entre les études

et empêche une estimation précise des concentrations du LPA plasmatique. De plus, la demi-

vie du LPA est courte et sa production peut être faite proche de son site d’action suite à la

liaison de l’autotaxine aux intégrines ou aux héparanes sulfates présents à la surface des

cellules. La concentration locale de LPA pourrait donc fortement varier de celle mesurée

dans la circulation générale, mais avoir un plus grand impact dans l’activation des cellules

vasculaires.

Nous avons aussi montré que les différentes espèces moléculaires de LPA n’ont pas toutes le

même potentiel activateur avec certaines qui montrent uniquement un effet inhibiteur pour

des concentrations physiologiques. L’activation des GR par la concentration de LPA

plasmatique pourrait donc dépendre de l’équilibre entre les espèces activatrices et inhibitrices

et pas seulement de la concentration totale de LPA.

De plus, nous avons montré que l’environnement module l’activation dépendante du LPA. Il

reste donc difficile à évaluer si l’activation par le LPA des GR est possible et est impliquée

dans des situations physiologiques ou pathologiques. Même si l’activation des GR n’a été

montrée que pour le LPA, ceux-ci peuvent être activés par d’autres voies signalétiques. En

effet, les GR expriment notamment le TLR9 qui peuvent lier de l’ADN mitochondrial596.

L’activation du TLR9 pourrait constituer un autre signal activateur que le LPA. Il serait donc

aussi intéressant de voir l’impact d’autres signaux activateurs des GR sur l’activation

dépendante du LPA. Stimuler les GR avec du PFP issu de patients souffrant de différentes

pathologies permettrait d’étudier plus en détail la potentiel implication du LPA plasmatique

dans les processus pathologiques.

Enfin, selon mes observations, le LPA induit deux populations de REV. Les REV de grande

taille PS+ ont déjà été associées dans des études antérieures à la coagulation et à

115

l’inflammation441,614. Cependant, l’études fonctionnelles des REV utilisent des REV

provenant de l’entreposage de GR441,613,614. Le stimulus à l’origine de la production des EV

est connu pour moduler leur composition et leur fonction429,468,532. Il serait donc important de

valider les effets biologiques des REV PS+ et PS- induit par le LPA dans l’inflammation et la

coagulation. De plus, les EV PS+ et PS- ont des demi-vies et une bio-distributions

différentes733. Les REV PS+ et les REV PS- pourraient donc jouer des rôles différents dans

l’environnement vasculaire.

3.2 Les vésicules extracellulaires de globules rouges dans le lupus

érythémateux disséminée

Contrairement à notre hypothèse, les quantités plasmatiques d’autotaxine chez les patients

LED étaient similaires à celles des individus sains, et celles-ci n’étaient pas associées avec

l’activité de la maladie. De plus, les quantités de PEV et de REV des patients LED étaient

élevées bien que non associées à l’activité de la maladie. Bien que les études s’accordent sur

l’augmentation des PEV chez les patients LED, leur association avec le SLEDAI est encore

débattue734,735. Nos résultats corroborent donc la publication qui n’associe pas les PEV avec

l’activité de la maladie735. Les quantités de REV PS+ nous ont permis de stratifier la cohorte

de patient LED en deux groupes, celui avec des quantités similaires aux individus sains et

l’autre qui présente des quantités élevées de EV plasmatique. Les patients LED avec des

quantités élevées de REV PS+ sont plus à risque d’avoir un historique de thrombose.

Les REV participent à la coagulation notamment par l’apport de thrombine et du facteur de

von Willebrand441,613,615,616. Ils sont aussi capables d’initier la coagulation dans certaines

conditions expérimentales441. Le recrutement des médiateurs de la cascade de coagulation

qui conduit à la production de thrombine par les REV est favorisé par la présence de la PS+

à leur surface440,441. Les quantités élevées de REV PS+ pourraient donc faciliter la coagulation

chez les patients LED et conférer une susceptibilité au développement de thromboses.

D’autre part, les EV sont associées à plusieurs étapes du développement de l’athérosclérose

qui la première cause des évènements thrombotiques677-679, notamment par l’apport de lipide

aux macrophages qui se transforment en cellules spumeuses710-713. En effet, les EV qui

présentent la PS sont plus sujets à leur internalisation par les macrophages733. Des quantités

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importantes de REV PS+ pourraient donc également stimuler le développement de

l’athérosclérose chez les patients LED par leur phagocytose par les macrophages.

Les patients LED qui ont des quantités élevées de REV PS+ présentent des concentrations

d’autotaxine plus élevées que les patients avec des quantités faibles de REV PS+. Les

quantités d’autotaxine pourraient ainsi avoir un impact sur les concentrations de LPA

plasmatiques et avoir des effets sur les cellules sanguines. L’activation des GR par le LPA

est une source potentielle de REV PS+. De plus, le LPA stimule la progression de plusieurs

étapes de l’athérosclérose qui est la cause majeure des évènements thrombotiques677-679. En

effet, le LPA stimule l’infiltration et l’activation des macrophages dans la paroi vasculaire et

leur différenciation en cellules de spumeuses par accumulation de lipides309,395,701. Il en

résulte un environnement pro-inflammatoire qui peut attirer d’autres cellules incluant des

neutrophiles. De plus lors de la rupture des plaques, le LPA est un des signaux d’activation

des plaquettes et promeut la coagulation et la formation d’un caillot thrombotique34,112,113. Ce

serait en accord avec notre observation que les patients avec des quantités élevées de REV

PS+ présentent également des concentrations de plaquettes circulantes plus faibles que le

groupe avec des quantités normales de REV PS+. Donc même si l’autotaxine ne semble pas

associée au développement du LED, elle pourrait tout de même être impliquée dans le

développement de l’athérosclérose et dans la formation de thromboses associées avec le

LED.

Les quantités élevées de REV PS+ pourraient également servir de source locale de

phospholipides pour la production de lyso-PS par des phospholipases capables d’utiliser les

phospholipides présents sur la membrane d’EV45,51. Cela est supporté par une étude récente

qui a fait état de quantité plus élevé de la PLA1 spécifique de la PS dans le sérum de patients

LED et que les quantités de la PLA1 spécifique de la PS étaient associées avec le SLEDAI736.

Les lyso-PS pourraient ensuite servir de précurseur pour la production de LPA par

l’autotaxine mais pourraient également présenter une activité biologique qui leur est propre,

notamment par l’intermédiaire de ses trois récepteurs couplés aux protéines G, P2Y10,

GPR34, et GPR174136,737. Ces récepteurs sont exprimés par les cellules hématopoïétiques. Le

lyso-PS à des effets répresseurs sur l’inflammation en inhibant la prolifération des

lymphocytes T738,739. Cependant il peut également promouvoir l’inflammation par

117

l’inhibition de la différenciation des lymphocytes T en lymphocytes régulateurs738,

l’activation du TLR2737,740, la libération d’histamine par les mastocytes, ainsi qu’en facilitant

la phagocytose et la libération de cytokines pro-inflammatoires par les macrophages737,741,742.

Les REV PS+ pourraient potentiellement avoir un rôle pro-inflammatoire et promouvoir

l’athérosclérose en étant une source de phospholipides pour la production de LPA et de lyso-

PS. En effet, l’inflammation pourrait augmenter la perméabilité vasculaire et facilité

l’extravasation des EV dans les tissus vasculaires.

Bien que notre étude n’ait pas montré d’augmentation des quantités plasmatiques

d’autotaxine, une augmentation de l’autotaxine sérique a déjà été rapportée pour des patients

LED avec des atteintes aux reins (néphrite lupique)743. L’autotaxine est sécrétée dans le

milieu extracellulaire et peut être trouvée sous forme libre, mais également associée à

différents transporteurs comme les plaquettes activées et les lipoprotéines. Les patients LED

présentent également une proportion de plaquettes activées et de plaquettes activées positives

pour l’autotaxine plus importante que les contrôles sains. Nos mesures ont été faites sur du

plasma sans plaquette et donc ne prend pas en considération l’autotaxine associée aux

plaquettes. Nos mesures pourraient donc sous-estimer les quantités d’autotaxine présentes

dans la circulation.

4 Perspectives

Les travaux de cette thèse ont souligné la complexité de la signalisation du LPA qui permet

des effets différents sur une même cellule en fonction de l’espèce moléculaire utilisée. De

plus, les différentes espèces moléculaires de LPA ont des concentrations différentes dans le

plasma et ne fluctuent pas de manière homogène, par exemple lors de l’activation des

plaquettes14,25,127. Cependant, l’étude des effets du LPA est encore souvent réalisé avec une

seule espèce moléculaire, dont l'idendité n'est pas toujours précisée 112,144,213,575. Il serait donc

important d’étudier l’effet de chaque espèce moléculaire majeure de LPA vasculaire sur les

cellules vasculaires comme les plaquettes ou les cellules endothéliales ainsi que dans les

fonctions vasculaires que ce soit l’inflammation ou la coagulation. Ces études seraient

bénéfiques pour une meilleure compréhension du rôle du LPA en situation pathologique.

118

Nous avons établi que certaines espèces moléculaires de LPA induisaient la libération de

deux populations de REV, les PS- et PS+. Il serait important d’une part de vérifié si elles sont

également pro-coagulantes et pro-inflammatoire à l’instar des REV issues de l’entreposage

de GR576,613,614,617,618. D’autre part, le LPA et les EV sont associées au développement de

l’athérosclérose et il est connu que les REV sont retrouvées dans les plaques 705. Il serait donc

pertinent d’évaluer les effets des REV induit par le LPA sur les cellules et fonctions

impliquées dans la progression des plaques d’athérosclérose comme les cellules

endothéliales, les cellules musculaires lisses ou les macrophages.

Nous avons observé que le groupe de patients LED avec des quantités élevées de REV PS+

avait une incidence de thrombose plus élevée. La construction de notre étude ne permet pas

de définir si les quantités importantes REV PS+ puissent être un facteur de risque ou

simplement une conséquence de la thrombose. Il serait donc nécessaire de compléter ce

travail avec une étude longitudinale pour définir si les quantités importantes de REV PS+ sont

un facteur de risque du développement de thromboses. Ce type d’étude sur plusieurs années

ou décennies nécessiterait une large cohorte de patients pour assurer qu’à son terme, le

nombre de patients suivis soit suffisant pour chaque groupe et qu’il y ait un nombre suffisant

de cas de thrombose pour tirer une conclusion.

Sachant que les REV sont impliquées dans la coagulation et peuvent induire un état d’hyper

coagulabilité dans des modèles murins de transfusion441,613,614, il serait à propos d’examiner

si de grandes quantités de REV PS+ dans le plasma de patients LED facilitent le

déclenchement de la coagulation par rapport à des plasmas de patients LED avec peu de REV

PS+ ou issus de donneurs sains. D’autre part, il serait intéressant d’établir si cette association,

entre la thrombose et les REV PS+, est retrouvée dans d’autres MRAS, notamment dans la

PAR. Également, les PEV ont déjà été associées à l’épaississement de la paroi vasculaire qui

est une conséquence de l’athérosclérose674. Puisque l’athérosclérose est une cause majeure

de thrombose, il serait pertinent d’évaluer une potentielle association entre les REV PS+ avec

l’épaississement de la paroi vasculaire chez les patients LED.

Enfin, les patients LED avec des quantités importantes de REV PS+ présentaient aussi des

concentrations d’autotaxine plasmatique plus forte que les patients LED avec de faibles

quantités de REV PS+. Il serait pertinent de mesurer les quantités de LPA présentes pour

119

chaque groupe. Cela permettrait d’une part d’évaluer si l’augmentation des concentrations

d’autotaxine se reflètent bien par une augmentation de LPA. Et d’autre part, cela permettrait

de déterminer les espèces moléculaires de LPA et d’étudier une potentielle association de ces

dernières avec les quantités de REV.

Conclusion

Les travaux présentés dans cette thèse ont permis la description des effets des espèces

vasculaires majeures de LPA sur l’activation des GR. Ces travaux ont mis en évidence des

effets activateurs et inhibiteurs du LPA sur l’exposition de PS par les GR et la production de

REV. Enfin, ces travaux ont permis, à notre connaissance, la première association d’une

population de REV, les PS+, à une situation pathologique qu’est la thrombose. Les

contributions de cette thèse sont soulignées dans le schéma récapitulatif (Figure 10).

120

Figure 110: Récapitulatif des contributions des travaux de cette thèse. Les flèches bleus (activation et

inhibition) et les encadrés bleus illustrent les apports de mes travaux de thèse.

La figure a été créée à l’aide de BioRender.com.

121

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Annexe I : Targeting the autotaxin - Lysophosphatidic

acid receptor axis in cardiovascular diseases

Yang Zhao 1 , Stephan Hasse 1 , Chenqi Zhao 2 , Sylvain G Bourgoin 3

Affiliations :

1 Centre de Recherche du Centre Hospitalier Universitaire de Québec - Université Laval,

Canada; Département de microbiologie, infectiologie et immunologie, Faculté de Médecine,

Université Laval, Québec, QC G1V4G2, Canada.


Canada.


Canada; Département de microbiologie, infectiologie et immunologie, Faculté de Médecine,

Université Laval, Québec, QC G1V4G2, Canada. Electronic address:

[email protected].

Keywords: Lysophosphatidic acid, Autotaxin, Atherosclerosis, Inflammation, Vascular

remodelling

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1 Abstract

Lysophosphatidic acid (LPA) is a well-characterized bioactive lipid mediator, which is

involved in development, physiology, and pathological processes of the cardiovascular

system. LPA can be produced both inside cells and in biological fluids. The majority of

extracellular LPA is produced locally by the secreted lysophospholipase D, autotaxin (ATX),

through its binding to various β integrins or heparin sulfate on cell surface and hydrolyzing

various lysophospholipids. LPA initiates cellular signalling pathways upon binding to and

activation of its G protein-coupled receptors (LPA1-6). LPA has potent effects on various

blood cells and vascular cells involved in the development of cardiovascular diseases such

as atherosclerosis and aortic valve sclerosis. LPA signalling drives cell migration and

proliferation, cytokine production, thrombosis, fibrosis, as well as angiogenesis. For instance,

LPA promotes activation and aggregation of platelets through LPA5, increases expression of

adhesion molecules in endothelial cells, and enhances expression of tissue factor in vascular

smooth muscle cells. Furthermore, LPA induces differentiation of monocytes into

macrophages and stimulates oxidized low-density lipoproteins (oxLDLs) uptake by

macrophages to form foam cells during formation of atherosclerotic lesions through LPA1-

3. This review summarizes recent findings of the roles played by ATX, LPA and LPA

receptors (LPARs) in atherosclerosis and calcific aortic valve disease. Targeting the ATX-

LPAR axis may have potential applications for treatment of patients suffering from various

cardiovascular diseases.

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2 Graphical abstract

LPA initiates cellular signalling pathways through LPA1-6 expressed by blood cells and

vascular cells, mediating the development of atherosclerosis. The ATX-LPA axis can be

targeted for potential treatment of CVDs.

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3 Lysophosphatidic acid and its receptors

Lysophosphatidic acid (LPA) is a bioactive lipid mediator required for the maintenance of

homeostasis in multiple physiological functions and pathological processes. LPA possesses

a glycerol backbone with an aliphatic fatty acid chain attached at sn-1 or sn-2 position

(Fig. 1). The sn-1 and the sn-2 positions are predominantly occupied by saturated and

unsaturated fatty acids, respectively [1]. The subclasses of LPA include the acyl-, alkyl-, and

alkenyl-LPA species (Fig. 1). Among them, acyl-LPA species are the most abundant

glycerophospholipid species [2]. The length and number of the fatty acid chain unsaturation

determine the diversity of molecular species of acyl-LPA. Common acyl-LPA species

include 16:0, 18:0, 18:1, 18:2, 18:3, 20:4, 20:5, and 22:6. Additionally, sn-2 acyl-LPA

species are unstable and easily undergo acyl chain migration to produce sn-1 acyl-LPA [1].

LPA biological activities rely at least in part on the fatty acid position on the glycerol

backbone, length, and degree of saturation [2].

Fig. 1. LPA structures. Basic structure of LPA includes a glycerol backbone, an aliphatic

acid chain, and a phosphate moiety. LPA comprises the acyl-, alkyl-, and alkenyl-LPA

species. Acyl-LPA can also be divided into 1-acyl and 2-acyl LPA according to the sn

position of the acyl chain.

174

LPA has important pro-atherosclerotic, pro-inflammatory, and pro-thrombotic properties

during development of various diseases. Functions of LPA are driven through the activation

of specific G protein-coupled transmembrane LPA receptors (LPARs). So far six LPARs

have been identified and named LPA1-6 (Fig. 2). According to their primary structure,

LPA1-3 belong to the endothelial differentiation gene family and LPA4-6 are related to the

purinergic P2Y receptor family. LPARs transmit downstream signals through at least four

heterotrimeric Gα proteins (Gα12/13, Gαq/11, Gαi/o, and Gαs) to mediate various

physiological and pathological conditions (Fig. 2). Binding preferences of LPA species to

LPAR subtypes have been reported. For example, alkenyl-LPA activates both LPA1 and

LPA2, alkyl-LPA activates LPA1, whereas acyl-LPAs are ligands for LPA1-3 [2]. Among

LPARs, LPA3 [3] and LPA6 [4] have relative binding preferences towards unsaturated acyl

species of LPA, such as 18:1, 18:2, 18:3, 16:1, and 20:4 2-acyl LPA, while LPA4 [5] is

strongly activated by 18:1 1-acyl LPA [6]. Although LPARs have specific and overlapping

functions, LPA-mediated responses are dictated by distinct LPAR expression patterns in

tissues and cells. LPA1-3 are ubiquitously expressed with high expression levels in nervous

system [7], immune organs [8], and reproductive organs [9]. LPA4-6 are also widely

expressed, albeit at lower levels in various organs and cells.

Fig. 2. LPA synthesis and LPA receptors. Phospholipase D (PLD) hydrolyzes various

phospholipids (PLs) to form intracellular phosphatidic acid (PA). PA can be converted into

LPA by a phosphatidic acid specific phospholipase A1 (PA-PLA1) or a cellular PLA2

(cPLA2). Synthesis of extracellular LPA depends on the hydrolysis of phospholipids such as

phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE),

producing lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), and

175

lysophosphatidylethanolamine (LPE) by phospholipases A1 (PLA1) or secreted

phospholipase A2 (sPLA2), respectively. Lysophospholipids are subsequently hydrolysed by

ATX to produce LPA. LPA is rapidly metabolized into monoacylglycerol (MAG) by the

ecto-activities of lipid phosphate phosphatases (LPPs). LPA induces various physiological

and pathophysiological responses through binding to and activation of six LPA receptors

(LPA1 to LPA6), which transmit downstream signals through at least four heterotrimeric Gα

proteins (Gα12/13, Gαq/11, Gαi/o, and Gαs).

4 LPA production pathways

Intracellular produced LPA works not only as a precursor or an intermediate in the synthesis

of cell phospholipids, but it can also serve as an intracellular signalling molecule. LPA can

be produced inside cells through sequential hydrolysis of phosphatidylcholine by a

phospholipase D (PLD) and a phospholipase A (PLA) [10] (Fig. 2). In this pathway, PLD

produced phosphatidic acid in the inner layer of cell membrane is subsequently deacylated

into LPA by a phosphatidic acid specific PLA1 [10].

LPA produced in the circulation acts in an autacoid way. Autotaxin (ATX) is responsible for

the production of extracellular LPA using various lysophospholipids as substrates (Fig. 2),

with plasma lysophosphatidylcholine (LPC) being the most abundant one. ATX, a member

of the nucleotide pyrophophatase/phosphodiesterase protein family, was originally isolated

from melanoma cells and characterized as a cell motility factor [11]. ATX is an enzyme with

unique lysophospholipase D activity that cleaves the choline group of LPC to produce LPA.

Structure of ATX consists of two N-terminal somatomedin B-like domains, a central catalytic

phosphodiester domain, and a C-terminal nuclease-like domain; these domains form a

hydrophobic channel containing the lysophospholipid-binding pocket [12]. The N-terminal

somatomedin B-like domain of ATX can bind to β integrins to access cell surface

lysophospholipids and to locally produce LPA [12].

There are three isoforms of ATX, namely ATX-α, -β, and -γ. ATX has a broad tissue

expression such as in brain, kidney, and lymphoid organs; and ATX can be produced locally

by a plethora of cell types [13]. ATX expression can be increased by growth factors, such as

fibroblast and epidermal growth factor, bone morphogenetic protein 2 (BMP-2) and the Wnt-

1 signalling pathway, but be inhibited by TGF-β and cytokines, such as interleukin-1 (IL-1),

176

IL-4, and IFN-γ [14]. In the vascular compartment, ATX-β is the most abundant ATX

isoform responsible for synthesizing plasma LPA [15]. Although the relative cellular origins

of plasma ATX are still uncertain, adipose tissues are a major source of ATX [16].

Megakaryocytes, the cells responsible for the production of platelets, do not express ATX

mRNA. But the enzyme was nevertheless found stored in α-granules of resting platelets and

was secreted upon platelet activation [17].

Plasma LPA was originally reported to be produced by activated platelets, especially under

pathological conditions such as inflammation and atherosclerosis [18]. During the

coagulation process, activated platelets release a large amount of lysophospholipids, which

are subsequently converted to 18:2 and 20:4 LPA by ATX [1]. Recently, a novel

phospholipase purified from thrombin activated human platelets, the acyl-protein

thioesterase 1 also known as lysophospholipase A1, was reported to possess PLA1 activity

[1]. Lysophospholipase A1 can generate sn-2-esterified LPC, which can be converted into

LPA by ATX. Almost half of serum LPA is produced in a platelet-dependent pathway

according to previous studies [10]. Furthermore, LPA can be generated in a cell-free system,

by cell-derived microparticles (MPs) [19]. Extracellular LPA is metabolized by the ecto-

activities of cell-associated lipid phosphate phosphatases (LPPs), which are responsible for

the rapid turnover of plasma LPA [20] and for maintaining the physiological concentration

of extracellular LPA in the low µM range.

5 The LPA-induced responses in cells of the cardiovascular

system

5.1. LPA and platelets

Research findings on LPARs expression in blood cells/vascular bed cells and LPA-induced

functional responses in context of cardiovascular diseases (CVDs) are summarized in Table

1. ATX has been reported to bind β1 and β3 integrins [12]. One of the consequences of

platelet activation is the release of ATX stored in α-granules [17]. ATX binding to activated

platelet β3 integrins promotes LPA production and LPA-dependent responses [12]. LPA

induces platelet shape change [21] and activation [22]. In blood, LPA-activated platelets form

platelet aggregates and platelet-monocyte aggregates [21], [23]. LPA also promotes the

177

stabilization of the platelet aggregates [24]. Furthermore, LPA enhances platelet fibronectin

binding and assembly, thereby suggesting a role in fibronectin matrix deposition following

vascular wall wounding [24]. Inhibition of lipid core plaque-mediated platelet aggregation

and activation by LPAR antagonists point to a role for LPA in thrombus formation upon

atherosclerotic plaque rupture [25]. Although platelets express all six LPA receptors (LPA1-

6), LPA effects are mainly mediated by LPA5 [26].

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5.2. LPA and endothelial cells

LPA contributes to the regulation of three endothelial cell-dependent processes: leukocyte

recruitment, angiogenesis, and vascular functions (Table 1). Silencing of LPA1 and LPA3 or

blocking those receptors with the selective antagonist Ki16425 attenuated LPA-induced

endothelial cell functional responses including chemokine C-X-C motif ligand 1 (CXCL1)

production [27], migration and proliferation [28]. LPA promotes leukocyte interaction with

the vascular wall through activation of LPA1/3 expressed on endothelial cells. Adhesion

proteins play a crucial role in leukocyte adhesion and migration across the vascular wall.

LPA increases the expression of the vascular cell adhesion molecules ICAM-1, VCAM-1,

and E-selectin [29], [30], [31]. LPA-mediated production of CXCL1 [27], IL-8 and monocyte

chemoattractant protein 1 (MCP-1) in endothelial cells also stimulates leukocyte adhesion to

the vascular wall [32]. IL-8 and MCP-1 are important mediators of leukocyte adhesion to the

endothelium under flow conditions [33]. In addition to these chemokines, LPA also induces

expression and secretion of the pro-inflammatory cytokine IL-1 in endothelial cells [31].

Studies using a mouse model of atherosclerosis suggested that LPA-mediated leukocyte

recruitment contributes to the initiation and the progression of atherosclerosis [27].

In vitro assays showed that LPA stimulated endothelial cell proliferation, migration, as well

as tube formation [28], [34]. LPA-induced cell migration, proliferation and tube formation

all contribute to angiogenesis, leading to new blood vessel formation from the existing

vasculature. LPA-induced angiogenesis depends on upregulation of the vascular endothelial

growth factor expression [35] and concomitant suppression of the angiogenesis repressor

CD36 expression in endothelial cells [36]. Taken together, these results suggest that the pro-

angiogenic properties of LPA contribute to development of imperfect intimal microvessels

commonly found in atherosclerotic plaques [37]. Those intimal microvessels are important

risk factors of plaque vulnerability since they are a source of intraplaque atherogenic lipids

and a cause for intraplaque haemorrhages [38]. Thus, LPA participates in atherosclerotic

plaque development and instability in part through its pro-angiogenic action and its ability to

recruit inflammatory cells.

Endothelial cell dysfunction is a characteristic of CVDs that is crucial to the initiation and

development of atherosclerosis. LPA influences some of endothelial cell functions that affect

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the structure and the properties of the vascular endothelium. LPA stimulation decreases

endothelial cell confluence [39] and increases their motility [40]. Measurement of hydraulic

permeability in rat vessels showed increased permeability of the vascular endothelium in

response to LPA [39]. LPA also induces the expression of matrix metalloproteinase 2 (MMP-

2) in endothelial cells [40]. MMP-2 contributes to remodelling of the extracellular matrix.

Therefore, LPA could alter vessel functions and increase vessel leakage through upregulation

of MMP-2. LPA impact on vessel function is not limited to modulation of vascular

endothelium permeability, it also impacts the vascular tone by inducing vasodilatation or

vasoconstriction [41], [42]. Injection of LPA in the lumen of isolated murine aortae resulted

in nitric oxide synthase dependent vasodilation. Genetic depletion of the nitric oxide synthase

or the mechanical removal of endothelial cells resulted in vasoconstriction [42]. LPA was

shown to induced smooth muscle cell contraction through liberation of thromboxane A2

which results in vasoconstriction [41]. In both studies, the use of an antagonist of LPA1/3,

Ki16425, and genetic deletion of LPA1 abolished LPA-induced vasoconstriction and

vasodilation [41], [42].

5.3. LPA and smooth muscle cells

Smooth muscle cells are a major cell type in the hyperplasic vascular lesions seen in

atherosclerosis [43]. LPA administered to mice and rat was reported to increase neointima

formation [44]. LPA-induced neointima formation is partly mediated through the recruitment

of progenitor cells and their differentiation into vascular smooth muscle cells. Similar as in

endothelial cells, LPA-mediated activation of smooth muscle cells induces the expression

and the release of several cytokines and chemokines such as MCP-1 [45], CXCL12 [44] and

IL-6 [46], which promote leukocyte recruitment and inflammation. The LPA1/3 antagonist

Ki16425 inhibits LPA-induced cytokine production and neointima formation as well (Table

1 and [44]). A transcriptomics study only detected the expression of LPA1 mRNA in human

aortic smooth muscle cells [47], thereby suggesting that the effects of LPA in these cells were

mediated by LPA1. LPA also increases the production of tissue factor [48], which is found

at high level in the atherosclerotic plaque and is an important initiator of atherothrombosis

[49].

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LPA stimulated smooth muscle cell proliferation and migration in vitro [50], [51] and in vivo

[51] in part through the induction of early growth response gene-1 expression and the

secretion of IL-6 [52]. In addition, smooth muscle cell growth was inhibited by an inhibitor

of NADPH oxidase, indicating a role for reactive oxygen species in LPA-dependent smooth

muscle cell proliferation [45].

5.4. LPA and monocytes

LPA also modulates monocyte recruitment and activation [53], [54] through the production

of reactive oxygen species, and the release of arachidonic acid and IL-1β [55]. LPA is an

important initiator of monocyte differentiation into macrophage [56] and formation of foam

cells [57], [58]. The LPA-dependent differentiation was associated with the inhibition of

high-density lipoprotein receptor SRB1 expression and generation of reactive oxygen species

[57], [58]. In atherosclerotic plaques, LPA enhances uptake of oxidized phospholipid by

macrophages [59]. Lipid uptake [59] and foam cell formation [57] are likely mediated

through LPA1 and/or LPA3. LPA may contribute to macrophage accumulation into

atherosclerotic plaques by inhibiting reverse transmigration across the endothelial layer [60].

Furthermore, LPA stimulates MMP-9 expression in THP-1 derived macrophages through

LPA2 [61]. MMP-9 also accelerates remodelling of the extracellular matrix of the artery wall,

resulting in progression and instability of the atherosclerotic plaque [61].

6 The ATX-LPA axis in cardiovascular diseases

ATX as well as the LPA-induced functional responses have been extensively studied in

various pathological conditions. Comprehensive reviews on the roles of lysophospholipids

in the development of atherosclerosis and other CVDs have been published previously [62],

[63]. Here we reviewed the most recent findings on the roles played by the ATX-LPA axis

to the development of cardiovascular pathologies such as atherosclerosis and calcific aortic

valve disease.

6.1. The ATX-LPA axis in atherosclerosis

LPA plays an important pro-thrombotic role and contributes to the development of

atherosclerotic plaques. Atherosclerosis is a slowly progressing arterial disease that is

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characterized by inflammatory and regenerative processes, resulting in vascular remodelling

and formation of atherosclerotic plaques. Although plasma LPA level is in low µM range,

unsaturated long chain acyl-LPA species can be predominantly accumulated in the

atherosclerotic lipid-rich core [22]. In atherosclerosis, LPA can be generated locally by

oxidization of low-density lipoprotein (oxLDL) [22]. OxLDL are transporters of oxidized

lipids and ATX in human plasma [64]. These oxidized lipids and ATX participate to the

production of LPA in atherosclerotic lesions, which subsequently promote several

pathological processes of atherosclerosis [22]. In addition to oxLDL-derived LPA, ATX

originated from various cell types also produces LPA within the atherosclerotic lesions.

Plasma ATX originating from adipose tissues together with ATX secreted by vascular cells,

such as endothelial cells [65], smooth muscle cells [47] and macrophages [66], can bind to

activated β1 and β3 cell integrins [12]. ATX binding to integrins increases its catalytic

activity and contributes to localized LPA production [12]. Furthermore, ATX also binds to

exosomes [67]. When incubated with phospholipases in vitro, cell-derived MPs are capable

of producing LPA [19]. During development of atherosclerosis, vascular inflammation

results in increased endothelial permeability, allowing circulating MPs in the blood to diffuse

within the vascular wall [68], [69]. In addition to oxLDL, higher levels of MPs originated

from vascular cells are accumulated at the atherosclerotic plaque and could contribute to

intra-plaque LPA production [70]. Whether MPs contribute to the ATX-dependent LPA

production in CVDs is yet to be confirmed.

The primary location of newly generated LPA at the cell surface can be in proximity to its

receptors [71]. On the other hand, inflammation associated with atherosclerotic lesions and

vascular injuries can increase the expression of ATX and LPARs. The enhanced LPA

production subsequently promotes neointima formation, which worsens the atherosclerotic

lesions [72]. A mouse model showed that exogenously administered LPA can increase

atherosclerotic plaque burden through LPA1/3 [27]. Extracellular LPA levels can be

decreased by LPP3, which localizes to the plasma membrane and serves as a negative

regulator of LPA signalling through its dephosphorylation catalyzed function. Enhanced

LPP3 expression in animal models was shown to decrease circulating LPA level [73]. In

atherosclerosis, alterations of LPP3 expression in monocytes and vascular wall cells are

closely related to circulating LPA levels [74]. LPP3 was therefore suggested to be involved

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in preventing the development of atherosclerosis, stabilizing atherosclerotic plaque, and

reducing the risks of complication associated with atherosclerosis [74]. Strategies to

stimulate LPP activity or protein expression could be envision for future treatments of CVDs.

Within the digestive tract, lysophospholipids coming from the diet are hydrolysed by ATX

to produce LPA in the intestinal lumen [75]. Intestinal LPC and LPA are absorbed and

transported into the plasma. These lysophospholipids, especially species with unsaturated

fatty acids such as arachidonic acid, can contribute to increased risks of CVDs by promoting

systemic inflammation and cell dysfunctions [76]. Atherosclerosis plaques in diabetic

patients are more vulnerable than those in non-diabetic patients with similar size of plaques.

One possibility is that carotid atheroma plaques of diabetic patients have a higher proportion

of polyunsaturated phospholipids such as 2-arachidonoyl-lysophosphatidylcholine [77]. This

lysophospholipid can be subsequently hydrolysed by ATX to produce C20:4 LPA, thereby

contributing to inflammation and to decreased atherosclerotic plaque stability. In addition,

ATX-derived LPA is also involved in mediating blood and vascular cell activation after

plaque rupture, further contributing to the progression and the complication of

atherosclerosis. LPA also serves as endogenous toll like receptor 4 ligand to activate NF-κB.

NF-κB signalling contributes to the development of atherosclerosis and the formation of

unstable plaques through enhanced inflammatory cytokine production and MMP-9

expression [78].

Acute coronary syndrome (ACS) is a life-threatening complication of atherosclerosis. In

ACS, most of the infarction is due to the formation of an occluding thrombus on the surface

of the atherosclerotic plaque. LPA has been suggested to increase the susceptibility of

atherosclerosis and its complications. For instance, a cross-sectional study of consecutive

patients showed a significant relationship between plasma LPA concentrations, especially

long chain unsaturated LPA species, and ACS [79]. Indeed, increased LPA levels were

detected in ACS patients, which were associated with ATX and platelet activation [79]. ATX

mass and enzymatic activity in the plasma from patients with coronary artery disease (CAD)

were associated with a higher risk of concurrent calcific aortic valve stenosis [80]. LPA levels

were higher in the plasma samples collected from coronary arteries than that from peripheral

arteries [81]. Elevated plasma LPA levels can be derived from different sources, to name a

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few, ATX-mediated production of 18:2 LPA, the platelet-related production of 20:4 LPA,

and other pathways, which might include not only LPC but also

lysophosphatidylethanolamine and lysophosphatidylglycerol as sources of 22:6 LPA [82].

Indeed, CAD susceptibility was also linked to the PPAP2B gene, which encodes the

expression of LPP3 and negatively regulates plasma LPA level [74]. A genome-wide

association study identified 13 novel loci harboring one or more single nucleotide

polymorphisms associated with CAD, and reported that among these 13 novel loci, PPAP2B

displayed risk allele frequencies at 0.91 that are highly associated with the risk of CAD [74].

Taken together, those studies suggest that ATX-LPA axis contributes to the pathogenesis of

ACS.

6.2. The ATX-LPA axis in calcific aortic valve diseases

Calcific aortic valve stenosis (CAVS) is the most common chronic and multifactorial

valvular disorder among the calcific aortic valve diseases (CAVDs). Pathological changes

associated with CAVD include progressive fibrosis, large mineral deposits in the lipid-rich

area of aortic valve, leading to gradual obstruction of the aortic valve orifice. Fibrosis and

valve mineralization are two intertwine factors that play crucial roles in the pathological

hemodynamic changes of CAVDs [83]. Mendelian randomization studies revealed a

significant association between the development of CAVD and the lipoprotein(a) gene

variant re10455872 in these patients [84], [85], [86]. CAVD patients with high lipoprotein

levels were at higher risk to develop aortic valve stenosis. Lipoproteins were reported to be

transporters of oxidized phospholipids and ATX, which accumulate not only in

atherosclerotic plaques but also in the mineralized aortic valves [64]. Plasma lipoprotein-

associated PLA2 is expressed in platelets [87] and macrophages [88]. Mineralized aortic

valve tissues express high levels of lipoprotein-associated PLA2 [88]. This phospholipase

and ATX both contribute to LPA production from plasma lipoproteins, promoting a pro-

inflammatory condition that drives mineralization of the aortic valve. LPA derived from

oxLDL was shown to promote aortic valve mineralization through the LPA1-RhoA/ROCK-

NF-κB signalling pathway [87]. In human CAVS tissues, LPA1 mRNA was increased by

1.5-fold compared to those in control non-mineralized aortic valves [87]. Activation of LPA1

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upregulates the expression of BMP2, a powerful morphogen signal that drives the osteogenic

program [88].

In addition to lipoproteins transported ATX, human explanted mineralized aortic valves

express high levels of ATX compared to non-mineralized valves. A 60% increase in ATX

enzymatic activity and a 1.5-fold increase in the levels of LPA in human mineralized aortic

valves were reported, respectively [64]. In a mouse model of CAVS, LPA promoted

inflammation and an osteogenic response through enhanced secretion of IL-6 and expression

of BMP2 [64]. Mass spectrophotometry analyses confirmed the enhanced LPA levels,

especially the unsaturated LPA species, in human aortic valve leaflets of CAVS patients [89].

A recent study revealed that the aggregation of platelets to endothelium-denudated aortic

valves contributes to the mineralization process of aortic valve through production of LPA

[90]. Platelets derived adenosine diphosphate can induce the release of ATX by valve

interstitial cells through P2RY1 receptors [90]. In turn, ATX binding to glycoprotein IIb/IIIa

(also known as integrin αIIbβ3) of platelets can promote the production of LPA during this

osteogenic process [90]. Both ex vivo and in vitro studies suggested that down regulation of

the LPP3 gene can promote the mineralization of aortic valves [91]. Medical interventions

targeting ATX and LPA1 have been suggested for treatment of CAVD.

7 Targeted ATX-LPA therapy

So far, no treatment targeting the ATX-LPA axis in CVDs has been fully developed.

However, pharmacological approaches targeting this pathway in other pathological

conditions, such as pulmonary and liver fibrosis, have been suggested (summarized below

and in Table 2). These examples of therapeutic approaches could potentially be developed

for the treatment of CVDs.

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7.1. ATX inhibitors

Several ATX inhibitors have been evaluated in animal models and phase 2 clinical trials

(Table 2). For example, EX_31, an orally administered ATX inhibitor, reduced plasma LPA

by 95% in a rat model of liver fibrosis [92]. However, EX_31 had no impact on markers of

inflammation and fibrosis in rat models of advanced fibrosis [92]. PF-8380 is one of the few

ATX inhibitors tested for the treatment of CVDs. This compound was assessed in a mouse

model of cardiomyopathy induced by a high fat diet [93]. Blood ATX activity, plasma LPA

levels, and cardiovascular symptoms were decreased in mice orally administered PF-8380

compared to non-treated mice [93].

A phase 2 clinical trial of idiopathic pulmonary fibrosis has been completed using the ATX

inhibitor GLPG1690 [94]. In this study, idiopathic pulmonary fibrosis patients were

administered GLPG1690 or a placebo. Participants who received GLPG1690 showed lower

18:2 LPA blood levels compared to the group who received placebo. Although not

significant, treatment of idiopathic pulmonary fibrosis patients with GLPG1690 for 12 weeks

slightly improved forced vital capacity when compared to the group who received placebo.

These studies only suggest that ATX inhibitors can decrease blood LPA levels. Further

clinical studies are required to determine whether those ATX inhibitors can halt or slow

disease progression.

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7.2. LPA sequestration

Systemic injection of anti-LPA antibodies was shown to reduce inflammation and

neurodegeneration in a zebrafish and in a mouse model of spinal cord lesion [95]. Mice

administered anti-LPA antibodies showed improved motor functions after spinal cord lesion

compared to non-treated mice [95]. LPA antibodies also diminished brain tissue damage and

inflammation in a mouse model of traumatic brain injury [96]. These studies suggest that

LPA antibody could be used to minimize injury-mediated neurone death and its associated

neurological dysfunctions.

7.3. LPA degradation

A genome-wide study highlighted a relation between CADs and the PPAP2B gene coding

for LPP3 [74]. Tetracyclines were shown in vitro to increase LPP1 and LPP3 cell surface

expression in malignant and non-malignant cell lines [73]. Interestingly, doxycycline

administration to rats accelerated the clearance of LPA from the circulation [73]. Enhancing

LPA degradation by vascular cells is a potential therapeutic option to reduce atherosclerotic

plaque development. Further studies are required to seriously evaluate the merits of this

strategy.

7.4. LPA receptors

The LPA1/3 antagonist Ki16425 has been used to study LPA signalling in CVDs [44] and

LPA-mediated responses in vascular wall cells [55]. So far Ki16425 has been used only in

preclinical studies. Other selective LPA1 antagonists such as ONO-7300243 [97] and ONO-

0300302 [98] have been tested for the treatment of benign prostatic hyperplasia and been

reported to reduce LPA-mediated increase in intraurethral pressure in rats and dogs.

Other LPAR antagonists have been evaluated in clinical trials. The LPA1 antagonist BMS-

986020 was used in a phase 2 clinical trial for the treatment of idiopathic pulmonary fibrosis

[99]. Patients receiving BMS-986020 showed a slower rate in respiratory capability decline

compared to the group administered placebo. However, the study was terminated before

completion due to serious side effects. On the other hand, a phase 2 clinical trials with another

LPA1/3 antagonist SAR100842 has been completed for the treatment of systemic sclerosis,

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a disease characterized by skin fibrosis [100]. Transcription of LPA-induced genes

(plasminogen activator inhibitor-1, Wnt-2, and sFRP-4) was attenuated in skin samples of

patients administered SAR100842. No significant clinical improvement was achieved

compared to the control group [100]. However, it should be highlighted that the phase 2

clinical trials mentioned above have several limitations including the duration of the

treatments and the size of the cohorts (Table 2). Further studies are required to assess the

beneficial effects of those compounds for the treatment of fibrotic diseases.

8 Conclusions

Taking together, emerging evidence shows that the ATX-LPA axis is involved in CVD

development through: (1) production of pro-inflammatory cytokines and mediators, (2)

neointima formation, (3) immune cells recruitment, and (4) oxidized phospholipids uptake.

All those actions are involved in and associated to atherosclerosis development and increased

risk of atherothrombosis. Therefore, ATX and LPARs, especially LPA1, are potential targets

to mitigate the development of CVDs. Since the ATX-LPA axis is druggable, future studies

should evaluate whether ATX inhibitors and LPAR antagonists represent promising

strategies for preventing CVDs such as atherosclerosis and CAVD.

Acknowledgements

This project was supported by a research grant from the Canadian Institutes for Health

Research (MOP-142210). YZ is supported by the scholarship from China Scholarship

Council (CSC).

Disclosure statement

The authors have declared no conflicts of interest.

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196

Annexe II :Phosphatidylserine-specific phospholipase

A1: A friend or the devil in disguise

Yang Zhao 1 , Stephan Hasse 1 , Sylvain G Bourgoin 2

Affiliations :

1 Centre de recherche du CHU de Québec-Université Laval, Centre ARThrite de l'Université

Laval, Département de microbiologie-infectiologie et d'immunologie, Université Laval,

Québec, G1V 4G2, Canada.

2 Centre de recherche du CHU de Québec-Université Laval, Centre ARThrite de l'Université

Laval, Département de microbiologie-infectiologie et d'immunologie, Université Laval,

Québec, G1V 4G2, Canada. Electronic address: [email protected].

Keywords: Phosphatidylserine, Lysophosphatidylserine, PLA1A, Lysophosphatidylserine

receptors, Autoimmunity, Cancer

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1 Abstract

Various human tissues and cells express phospholipase A1 member A (PLA1A), including

the liver, lung, prostate gland, and immune cells. The enzyme belongs to the pancreatic lipase

family. PLA1A specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or 1-acyl-

lysophosphatidylserine (1-acyl-lysoPS). PS externalized by activated cells or apoptotic cells

or extracellular vesicles is a potential source of substrate for the production of unsaturated

lysoPS species by PLA1A. Maturation and functions of many immune cells, such as T cells,

dendritic cells, macrophages, and mast cells, can be regulated by PLA1A and lysoPS. Several

lysoPS receptors, including GPR34, GPR174 and P2Y10, have been identified. High serum

levels and high PLA1A expression are associated with autoimmune disorders such as Graves'

disease and systemic lupus erythematosus. Increased expression of PLA1A is associated with

metastatic melanomas. PLA1A may contribute to cardiometabolic disorders through

mediating cholesterol transportation and producing lysoPS. Furthermore, PLA1A is

necessary for hepatitis C virus assembly and can play a role in the antivirus innate immune

response. This review summarizes recent findings on PLA1A expression, lysoPS and lysoPS

receptors in autoimmune disorders, cancers, cardiometabolic disorders, antivirus immune

responses, as well as regulations of immune cells.

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2 General introduction

Sato et al. named the enzyme phosphatidylserine-specific phospholipase A1 (PS-PLA1) in

1997 [1]. PS-PLA1 specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or

lysophosphatidylserine (lysoPS) [1]. PS-PLA1 has the name phospholipase A1 member A

(PLA1A) in databases. We will use the official HGCN gene nomenclature PLA1A

throughout the review.

Activated rat platelets release two types of lipases having phospholipase A2 (PLA2) and

lysoPS specific lysophospholipase A activities [2]. These lipases come from dense granules

or α-granules rather than the lysosomal compartments [2]. The lysophospholipase activity

hydrolyzes both 1-acyl- and 2-acyl-lysoPS, but no other lysophospholipids [2]. At the same

time, Higashi et al. also partially purified a lysophospholipase from thrombin-activated rat

platelets [3]. The lysophospholipase activity was towards 1-acyl-sn-glycerol-3-phospho-l-

serine but none other lysophospholipids [3]. Yokoyama et al. later reported that platelets

released one protein with phospholipase A1 and lysophospholipase activities [4]. This

enzyme worked with PLA2 to hydrolyze platelet phospholipids during blood clotting [4].

The cDNA sequences encoding rat and human PLA1A were published in 1997 [1,5]. Purified

PLA1A has an apparent molecular weight of 55-kDa on SDS-polyacrylamide gel

electrophoresis [1]. They assessed the enzyme activity towards PS and lysoPS and

highlighted the structural similarity between PLA1A and other mammalian lipases [1]. Nagai

et al. mapped the human PLA1A gene to chromosome 3q13.13–13.2 [6] and PLA1A was

identified as a gene previously named nmd [5]. The murine ortholog of Pla1a localizes to

mouse chromosome 16 [7].

Following PLA1A purification, several studies have assessed its biological functions. For

instance, Aoki et al. reported that the addition of recombinant PLA1A to activated rat

platelets accelerated lysophosphatidic acid (LPA) production, thereby suggesting that

PLA1A could contribute to serum LPA level during blood clotting [8]. PLA1A proteins from

rat, human, and mouse are highly conserved [7]. PLA1A molecular structure, expression, and

hydrolytic activities have been reviewed previously [[9], [10], [11], [12]] and will not be

discussed in depth in this review. There is a growing number of publications on the putative

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roles of PLA1A in disease states or its cellular functions. The involvements of

lysophospholipids and the potential roles of PLA1A in several diseases, such as acute

coronary syndrome (ACS), atherosclerosis, and gastric cancer, have been reviewed recently

[13]. This review will focus on more recent studies concerning the characterization of

PLA1A, expression in cells, and putative functions in various diseases, including cancer and

autoimmunity.

2.1. PLA1A belongs to the pancreatic lipase family

The pancreatic lipase family consists of six members: PLA1A, membrane-associated

phosphatidic acid-selective phospholipase A1a (mPA-PLA1a), mPA-PLA1b, hepatic lipase,

endothelial lipase, and pancreatic lipase-related protein 2. They are essentially extracellular

PLA1s [14,15]. PLA1A, mPA-PLA1a, and mPA-PLA1b consist of a subfamily with distinct

molecular features (a short lid domain and a deleted β9 domain) that distinguish them from

other lipases [15]. Extracellular PLA1s have no sequence homologies to intracellular PLA1s

and exhibit distinct functions [10]. We will not discuss the structures and roles of intracellular

PLA1s in this review.

The domain structure of pancreatic lipase family members determines their substrate

specificity and the ability to hydrolyze triglyceride and phospholipids. Crucial roles of lid,

β5, and β9 loops of pancreatic lipases in choosing substrate have been reviewed [9,12,14].

Fig. 1 shows the substrate specificity of the pancreatic lipases. Once the lipase contacts its

substrate, the lid undergoes a conformational change and adopts an open conformation with

β9 [9]. This conformational change allows the hydrophobic interaction with substrate acyl

chains and the ligand docking in the catalytic site [14]. Lipases are inactive in aqueous

solutions because the catalytic triad is obstructed by the lid [16].

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Fig. 1. Substrate preference of pancreatic lipase family members. PLA1A, mPA-PLA1a and

b consist of a subfamily exhibiting phospholipase activity towards PLs. PL and LPL can

hydrolyze TG. EL, PLRP2 and HL can hydrolyze both TG and PLs. PLs, phospholipids; TG,

triglyceride; PLA1A, phospholipase A1 member A; mPA-PLA1, membrane-associated

phosphatidic acid-selective phospholipase A1; EL, endothelial lipase; PLRP2, pancreatic

lipase-related protein 2; HL, hepatic lipase; PL, pancreatic lipase; LPL, lipoprotein lipase.

All pancreatic lipase family members have quite a distinct affinity to heparan sulfate

proteoglycans (HSPGs). PLA1A affinity to heparin was also reported [3,15]. Membrane-

bound PLA1A can hydrolyze externalized PS and be internalized into living mammalian cells

through binding to HSPGs. There is no specific inhibitor of PLA1A. Like phospholipases

with a catalytic serine, PLA1A is sensitive to inhibition by diisopropyl fluorophosphate [1].

There are five transcript variants in homo sapiens encoding for different isoforms of PLA1A,

NM_015900.4 (variant 1), NM_001206960.2 (variant 2), NM_001206961.2 (variant 3),

NM_001293225.2 (variant 4), and NR_120610.2 (variant 5, non-coding) (https://www-ncbi-

nlm-nih-gov.acces.bibl.ulaval.ca/gene/51365). Variant 1 represents the longest transcript

encoding the full-length protein. Isoform 2 has the same N- and C- terminal but is shorter

than the full-length PLA1A, whereas isoform 3 and 4 both have a shorter N-terminus.

Among the two PLA1A mRNAs characterized in vivo, the larger one encodes the full-length

PLA1A, and the second mRNA encodes a truncated form named PLA1AΔC [12]. PLA1AΔC

lacks two-thirds of the C-terminal domain, including the β5 loop and basic residues [12],

which eliminates enzyme ability to hydrolyze PS in liposomes but retains its

lysophospholipase activity towards 1-acyl-lysoPS in solution [6,12]. As for other lipases, the

β5 loop of PLA1A is likely required for interfacial binding to membrane leaflet [12,14]. The

N-terminal domain, which is conserved in both PLA1A and PLA1AΔC, encompasses the

catalytic triad and the heparin-binding site, thereby suggesting that both isoforms have

similar affinity to HSPGs [6]. PLA1A and PLA1AΔC are expressed in various human organs,

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tissues, and cells, with PLA1AΔC representing about 10–20% of total PLA1A [6].

PLA1AΔC and PLA1A can synergistically induce lipid signaling through hydrolyzing PS

exposed on damaged or activated cell surface and control the level of lysoPS [12].

2.2. Expression patterns of PLA1A

In rats, PLA1A is expressed in platelets, hearts, and lungs [10]. Rat platelets can release

PLA1A upon activation. Unlike rat, mouse and human platelets poorly express PLA1A [10].

Several human tissues express PLA1A

(https://gtexportal.org/home/gene/PLA1A#spliceVizBlock), including muscle, kidney, small

intestine, spleen, placenta, and testis, with the highest expression in the liver and prostate

gland [5,6]. Human fibroblasts, keratinocytes, melanomas, HepG2 and HeLa cells express

the PLA1A mRNA [6]. The sources of PLA1A in human serum have remained elusive [17].

One liver cancer study revealed that the serum PLA1A level was closely related to the PLA1A

mRNA level in non-hepatocellular carcinoma (HCC) tissues, indicating that the liver might

be a source of circulating PLA1A [18].

In 2010, Nakamura et al. generated anti-human PLA1A monoclonal antibodies [17]. Using

a novel enzyme immunoassay, they reported that the serum level of PLA1A in healthy

subjects was 33.8 ± 16.6 μg/L [17]. The concentration was significantly higher in men (13.8–

80.6 μg/L) than in women (12.1–68.8 μg/L), with no correlation between the age of the

subjects [17]. Furthermore, there was no association between serum PLA1A level and other

laboratory tests, such as total IgE concentration, platelet and leukocyte counts, tumor

markers, etc. [17]. Expression levels of PLA1A in pathophysiological conditions will be

reviewed in following paragraphs (summarized in Table 1).

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2.3. Structural basis of PLA1A substrate specificity

The very short lid [1] and the deleted β9 loop [11] in PLA1A allow the enzyme to retain a

PLA1 but not a triglyceride lipase activity [15]. The short lid domain is hydrophilic, with an

orientation towards the solvent that accommodates the phospholipid polar heads [19]. The

short β9 loop is also responsible for the recognition of phospholipids [12]. The

lysophospholipase activity of PLA1A requires the β5 loop, whereas other motifs contribute

to stringent substrate specificity [14]. The serine amino and carboxyl groups of PS make

interaction with amino acid residues of the catalytic pocket of PLA1A [14]. Basic residues

located in the β5 loop allow the formation of an oxyanion hole close to the catalytic triad that

contributes to catalytic activity [9,12]. The proposed mechanism of catalysis in the case of

PLA1 suggests that the β9 loop stabilizes the sn-1 acyl chain of phospholipids. The sn-2 acyl

chain is oriented along with the lid domain and remains in interaction with the lipid layer.

The polar head group is found between the sn-1 and sn-2 acyl chains and fits into the

hydrophilic active-site groove [9,19].

2.4. PS, lysoPS, and lysoPS receptors

PS exposure is very slow in dying cells and possibly dependent on a scramblase activated by

caspase cleavage and phosphorylation [20]. Exposure of PS on the outer membrane leaflet is

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the gold marker of cells undergoing apoptosis and is an eat-me signal for the clearance of

dying cells by macrophages (Mφ) [21], a process called efferocytosis. The engulfment of

apoptotic cells does not induce inflammation but promotes the secretion of anti-inflammatory

cytokines (IL-10 and TGFβ) and decreases the production of TNFα, IL-1β and IL-12 [[22],

[23], [24]]. PS functions as an immunosuppressive mediator for silent clearance of apoptotic

debris by Mφ.

The exposure of PS on the cell surface can occur in the absence of apoptosis. In that case,

cell surface exposure of PS is rapid and reversible, thereby preventing the engulfment of

viable PS exposing cells by Mφ and dendritic cells (DCs) [25,26]. The externalization of PS

has been reported in viable monocytes [27], activated mast cells [27,28], CD8+ T cells [29],

regulatory B cells [30], cancer cells and cancer cell-derived extracellular vesicles (EVs)

[31,32]. PS externalization on activated platelets is required for microparticles release and

plays a critical role in the recruitment and the activation of clotting factors with gamma-

carboxyglutamic domains [33,34]. Platelet-derived microparticles harboring IgG are

positively correlated with systemic lupus erythematosus (SLE) disease activity and vascular

damage [35]. EVs can be derived by various cell types and serve as cargos for the delivery

of proteins, lipids, and RNA to the target cells. In platelet-free plasma of SLE patients, the

amounts of EVs derived by platelets and red blood cells were significantly increased,

respectively (unpublished data). Among these EVs, the PS+ EVs were increased in SLE

patients compared to healthy controls (unpublished data). Besides, PLA1A level is increased

in active SLE patients [36]. Thus, PLA1A might impact immune regulatory processes and

inflammation through the production of 2-acyl-lysoPS converted from PS exposed at the cell

surface of activated cells, apoptotic cells, or various types of EVs (Fig. 2 and [[37], [38],

[39]]).

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Fig. 2. Overview of the potential contributions of PLA1A, lysoPS, and ATX-derived LPA to

various diseases. In cells undergoing apoptosis or during cell activation, externalized PS on

surface of activated/apoptotic cells or EVs is converted by secreted PLA1A to lysoPS, which

can be subsequently metabolized into LPA by ATX. Circulating PLA1A can originate from

liver or be secreted by other cells. PLA1A, lysoPS and LPA participate in many pathological

processes, including autoimmune disorders, cancers, cardiometabolic disorders, and antiviral

innate immune responses, etc. EV, extracellular vesicles; PS, phosphatidylserine; PLA1A,

phospholipase A1 member A; lysoPS, lysophosphatidylserine; ATX, autotaxin; LPA,

lysophosphatidic acid; HCV, hepatitis C virus.

The sn-1 and the sn-2 positions of phospholipids are predominantly occupied by saturated

and unsaturated fatty acids, respectively [40]. Compared with secreted phospholipase A2

group IIa (sPLA2-IIa), PLA1A produces lysoPS more efficiently and is more potent in

inducing histamine release from rat peritoneal mast cells [11] and stimulating alkaline

phosphatase-tagged TGFα release [41]. 2-acyl-lysoPS is a lipid mediator for mast cells, T

cells, and neural cells [11]. The 2-acyl-lysoPS can undergo the spontaneous intra-molecular

acyl-migration to form the 1-acyl-lysoPS, which subsequently becomes the substrate of

PLA1A [42]. PLA1A can also hydrolyze 1-acyl-lysoPS produced by PLA2, which is crucial

for controlling the amount of 1-acyl-lysoPS and the subsequent activation of its receptors

[14,41]. Furthermore, PLA1A-derived lysoPS can be metabolized to LPA by circulating

autotaxin (ATX) in cancer ascites [43], or by cells expressing ATX, leading to autocrine

signaling in human fibroblast-like synoviocytes (Zhao et al., manuscript in preparation).

ATX is the primary extracellular LPA producing enzyme, and we previously reviewed the

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roles and involvements of ATX-LPA receptor axis [44]. LPA receptors have ligand

selectivity and can be activated differentially by the LPA species [45]. LysoPS receptors also

show preference for lysoPS species. Several G protein-coupled receptors for lysoPS were

identified, including GPR34 (LPS1), P2Y10 (LPS2), A630033H20 (LPS2L), and GPR174

(LPS3) [46,47]. A630033H20 is expressed in mouse but is a pseudo-gene in human [46].

GPR34 prefers lysoPS with an unsaturated fatty acid at the sn-2 position like those produced

by PLA1A [41]. GPR174 is highly activated by 16:1 lysoPS and P2Y10 can be activated

similarly by 16:0 and 18:1 lysoPS [48]. We will discuss the functions of lysoPS and its

receptors in the following paragraphs.

3 Expression of PLA1A and lysoPS receptors in cells

3.1. Immune cells

Treg cells and memory CD4+ T cells, at a much lower level, express PLA1A

(https://www.proteinatlas.org/ENSG00000144837-PLA1A). A few reports suggested that

PLA1A and lysoPS receptors could be involved in immune cell rep rogramming, maturation,

and modulation of immune cell functional responses [38,39,49].

Unsaturated PS could inhibit mitogen-induced T cell activation, and serum PLA1A might

enhance the inhibitory effects through the generation of unsaturated lysoPS [38,39]. Of note,

among the tumor-infiltrating lymphocytes, there was a high enrichment of PLA1A mRNA in

tumor-promoting CD8+ T cells (31.1-fold) compared to native peripheral blood CD8+ T cells

[49]. Whether high levels of PLA1A expression are associated with reduced CD8+ T cell-

dependent cytotoxicity and poor prognostic survival are yet to establish. LysoPS species

(C18:0 > C18:1 > C16:0) and PLA1A transcripts were abundant in lymphoid tissues (spleen,

thymus) [39]. LysoPS suppressed T cell proliferation, Treg generation and homeostasis

through activation of GPR174, and the subsequent elevation of intracellular cAMP levels

[39,50]. GPR174 weakened the capacity of Treg to suppress the functions of Th1 subsets and

controlled tissue-specific immune responses [51]. TGFβ signals suppressed the expression

of GPR174 by Treg [51]. The blockade of GPR174 might be a potential treatment option for

autoimmune disorders [39].

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Human and mouse DCs were reported to express PLA1A [39,52]. Treg cells can interfere

with the induction of mature DCs [52]. In immature Treg-DCs, which drive CD4+ T cells

polarization towards a regulatory phenotype, PLA1A was among the most downregulated

genes compared to mature DCs [52].

The TLR4 agonist lipopolysaccharide (LPS) enhanced PLA1A mRNA expression in human

THP-1 derived Mφ [53]. Immunosuppressive agents, such as corticosteroids, prednisolone,

6α-methylprednisolone, dexamethasone, and beclomethasone reduced LPS-mediated PLA1A

expression in THP-1 derived Mφ [53]. In LPS-induced peritoneal Mφ from microsomal

prostaglandin E synthase-1 knockout mice, the expression of Pla1a was modulated [54].

Kawamoto et al. found that nerve growth factor (NGF) could stimulate the release of

histamine from rat peritoneal mast cells incubated with activated rat platelets [55]. NGF

antibodies or inhibition of NGF receptor tyrosine kinase activity can completely block

histamine release by rat peritoneal mast cells [55]. Furthermore, histamine release by rat

peritoneal mast cells incubated with PS+ erythrocytes and NGF required the presence of

PLA1A, thereby suggesting a role for PLA1A-derived lysoPS in mast cell activation [55].

GPR34 is a highly expressed functional lysoPS receptor in rat mast cells [56]. PLA1A-

mediated production of 2-acyl-lysoPS may also contribute to histamine release by rat

peritoneal mast cells in response to cross-linking of the high-affinity IgE receptor, known as

FcεRI [57]. Histamine release was blocked by heparin, suggesting that PLA1A activity

required binding to cellular HSPGs [57].

3.2. Other cells

LysoPS was showed to promote the NGF-induced neural differentiation of PC12 cells [58].

NGF and lysoPS stimulated the growth of PC12 cells and enhanced neurite length [58]. The

cells developed only 1–2 neurites instead of the multipolar appearance induced by NGF alone

[58]. LysoPS was showed to stimulate intracellular calcium increase and chemotactic

migration in U87 human glioma cells [59] and L2071 mouse fibroblasts [60]. These effects

were dependent on the activation of PI3K, p38 MAPK, and JNK pathways [59,60]. A

pertussis toxin-insensitive but phospholipase C-dependent cascade was involved in the

intracellular calcium increase [60]. Pertussis toxin-sensitive chemotactic migration was

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mediated through the PI3K and ERK pathways [60]. The lysoPS receptors expressed by

L2071 fibroblasts were not characterized.

4 Expression of PLA1A in disease states

Whether PLA1A has beneficial or harmful functions in healthy and disease states remains to

be determined. Under physiological conditions, the PS in the inner cell membrane layer is

not accessible to secreted PLA1A [11,12]. However, in cells undergoing apoptosis or during

cell activation, externalized PS can be converted by secreted PLA1A to lysoPS, which

potentially induces lysoPS receptor-dependent functional responses in various cell types

[11,12]. The PLA1A chromosome locus is not associated with human diseases [11]. However,

high PLA1A expression levels in melanoma cells or subtypes of prostate cancers,

autoimmune disorders such as SLE and Graves' disease suggest a role for PLA1A in the

regulation of tumor growth and autoimmunity [5,36,61,62]. The following paragraphs will

review the expression of PLA1A and the lysoPS receptors in autoimmune disorders, cancers,

cardiometabolic disorders, virus immunomodulation, and other diseases (summarized in

Table 1, Table 2, Table 3 and Fig. 2).

4.1. Autoimmune disorders

Serum PLA1A level was significantly higher in SLE patients compared to healthy controls

and patients with other systemic autoimmune rheumatic diseases, such as active rheumatoid

arthritis (RA), Sjögren syndrome, and systemic sclerosis [63]. Serum PLA1A level

significantly correlated with SLE disease activity index [36]. Patients with lupus nephritis

and diabetic nephropathy had similarly elevated plasma levels of PLA1A [63]. In the cohort

of patients with lupus nephritis, serum level of PLA1A was not correlated with activity index

or chronicity index, proteinuria, kidney survival, SLE disease activity index, and other

clinical laboratory data such as anti-double-stranded DNA antibody and complement proteins

(C3 and C4) [63]. Disease treatment reduced serum PLA1A levels [36,63]. However, serum

PLA1A level inversely correlated with the daily dose of prednisolone [63]. We have

compared plasma PLA1A levels between early diagnosed RA patients and a cohort of SLE

patients with age- and gender-matched controls. PLA1A levels were higher in early

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diagnosed RA patients and SLE patients, with no sex-based differences. PLA1A levels in

synovial fluids from patients with RA and psoriatic arthritis were higher than those from

osteoarthritis and gout patients (Zhao et al., manuscript in preparation). Therefore, we do not

exclude that the levels of serum/plasma PLA1A in systemic autoimmune rheumatic diseases

would correlate with high systemic inflammation levels or tissue damages [36].

Graves' disease, also known as Basedow's disease, is an autoimmune disorder characterized

by overactive thyroid and the presence of antibody against the thyroid stimulating hormone

receptor [64]. Serum PLA1A level was higher in patients with Graves' disease and strongly

correlated with thyroid hormone levels [62]. Treatment with anti-thyroid reagents can lower

serum PLA1A levels, suggesting a possible link between PLA1A and the inflammation in

chronic autoimmune thyroiditis [62]. Changes in serum PLA1A levels also occurred in

patients with subacute thyroiditis or silent thyroiditis [62].

Intestinal inflammation modulates the expression of PLA1A and lysoPS receptors [65].

PLA1A expression was increased in inflamed ulcerated mucosa from Crohn's disease, even

in their noninflamed macroscopically normal-looking mucosa [66]. The PLA1A mRNA,

protein, and lipase activity were all enhanced in primary intestinal microvascular endothelial

cells and human colonic Caco-2 epithelial cells treated with inflammatory stimulations, such

as TNF-α, IL-1β, and LPS [65]. Besides, expression of all lysoPS receptors (GPR34,

GPR174, P2RY10) increased in Caco-2 cells after inflammatory stimulation [65].

The serum of systemic sclerosis patients showed an elevated level of PLA1A [36]. Serum

PLA1A is possibly associated with low-grade systemic inflammation. PLA1A may

contribute to progressive skin fibrosis in systemic sclerosis patients, as the expression of

PLA1A gene was increased 2.63-fold in response to the TLR3 agonist poly(I:C) [67].

Expression of PLA1A is elevated in various diseased tissues and is possibly induced in

response to inflammatory stimuli [57]. By hydrolyzing PS, PLA1A might contribute to

inflammation or interfere with processes involved in inflammation-resolution mechanisms

through coving the recognition of apoptotic cells/debris by other immune cells, such as Mφ

and DCs, leading to the development of autoimmunity [36,62]. The roles played by PLA1A

in the pathophysiology of many diseases remain to be established. Whether PLA1A is a

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promising biomarker or a treatment target for autoimmune diseases needs further

investigation.

4.2. Cancers

There is growing information pointing to a role for PLA1A in tumor progression, such as

melanoma, prostate, liver, gastric, colorectal, and glioma cancers

(https://portals.broadinstitute.org/ccle/page?gene=PLA1A). PLA1A mRNA or protein level

was correlated with tumor progression and the poor disease outcomes [43,68]. PLA1A can

promote tumor progression through the generation of lysoPS and GPR34-induced activation

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of the PI3K/Akt pathway [68] or through ATX-mediated conversion of lysoPS to LPA [43],

a lipid mediator associated with cancer progression and metastasis [69].

PLA1A was highly expressed in poorly-metastatic melanoma cell lines [5,70], suggesting a

link to cancer cell fate. Of note, the levels of serum PLA1A and that of ATX were

significantly higher in melanoma subjects and correlated with the clinical stages in females

[71]. In a recent study, increased PLA1A expression was positively correlated to disease

severity and routine diagnostic markers of metastatic melanoma [72].

Transmembrane serine protease 2/erythroblast transformation-specific transcription factor

(TMPRSS2/ERG) gene fusion events play a role in the initiation and progression of prostate

cancer [73]. The chromatin and gene expression are distinct between TMPRSS2/ERG positive

and non-TMPRSS2/ERG prostate cancers in the regions proximal to the PLA1A gene [74],

which is one of the target genes regulated by ERG [61,73]. Poly ADP-ribose polymerase 1

and the catalytic subunit of DNA protein kinase were required for ERG mediated

transcriptional activities, and the expression of PLA1A in VCaP cells decreased following

siRNA knockdown of ERG or incubation with small molecule inhibitors of poly ADP-ribose

polymerase 1 and catalytic subunit of DNA protein kinase [73,75,76]. However,

overexpression of various ΔERG constructs had no significant effect on PLA1A mRNA

expression [75]. In VCaP cells, the vitamin D receptor agonists induced expression of ERG

target genes but had mixed outcomes on PLA1A expression [77]. In PCa cells, PLA1A gene

expression was not induced in response to stimulation with androgen [78].

Elevated levels of PLA1A mRNA were detected in HCC tissues [18] and were associated

with higher serum PLA1A levels when compared to healthy controls, but no relationship

with clinical parameters was observed [18]. However, elevated serum PLA1A level

correlated to background PLA1A mRNA expression in normal tissues adjacent to the tumors,

but not in the HCC tissues [18]. The relationship between high serum PLA1A and hepatic

enzyme levels suggested that liver tissue injury could contribute to serum PLA1A in HCC

patients [18]. High GPR34 expression in HCC tissues correlated with poorly differentiated

HCC tumors [18]. Pla1a is part of a signature gene set modulated by nongenotoxic hepatic

tumorigens in rats, including peroxisome proliferator-activated receptor agonists and steroid

hormones [79]. Pla1a expression was increased in response to hepatotoxic agents such as

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pirinixic acid, nafenopin, or phenobarbital, and was decreased by chloroform, pravastatin, or

methapyrilene [80].

In ascites of gastric cancer patients, the levels of lysoPS and PLA1A were positively

correlated [43]. Although ascites from gastric tumor patients and control cirrhosis patients

expressed similar amounts of PLA1A, higher levels of C18:0 and C18:1 lysoPS were

observed in the gastric tumor group [43].

PLA1A and lysoPS have different effects on colorectal cancer (CRC) cell growth and tumor

metastasis [68,81]. PLA1A expression was associated with tumor invasion, hematogenous

metastasis, and poor disease-free survival in CRC patients [81]. LysoPS failed to stimulate

CRC cell proliferation, but it stimulated tumor cell migration through GPR34 and PI3K/Akt

pathway [68]. These results possibly correlate with different expression patterns of lysoPS

receptors among cancer cell lines. For instance, CRC cell lines expressing only GPR34 were

less sensitive to high dose lysoPS-mediated inhibition of proliferation than cell lines

expressing all lysoPS receptors [68].

Differential expression of phospholipases and lysophospholipases correlated with alterated

glycerophosphocholine lipid metabolism in gliomas [82]. Expression of PLA1A was

increased in low-grade glioma but reduced in high-grade glioma compared to normal brain

tissues [82]. The distinct expression of PLA1A and other phospholipase genes (PLA2G4A

and LYPLA1) in low- and high-grade could help to grade astrocytomas [82].

Altogether the studies suggest a role for PLA1A in tumor invasion and metastasis through

GPR34-induced signaling [68,81]. The roles of PLA1A should take the tumor

microenvironment into account [43,69,71]. Surface exposure of PS, the release of PS-

positive EVs by tumor cells, and PLA1A can negatively impact tumor immunity and tumor-

killing through lysoPS-mediated suppression of immune cell functions and lysoPS

conversion into LPA by ATX [[37], [38], [39],69,83].

4.3. Cardiometabolic disorders

PLA1A might participate in ACS pathogenesis [13]. High PLA1A levels in serum samples

from culprit coronary arteries of patients with ACS significantly correlated with C18:0 and

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18:1 lysoPS levels [84]. The monitoring of plasma serotonin levels in 141 consecutive

patients undergoing coronary angiography emphasized that serotonin, a biomarker of platelet

activation, was significantly associated with plasma lysoPS level in the ACS group [85].

Aspirin intake was without effect on plasma lysoPS levels, but plasma LPA and other

lysophospholipid levels were lower in patients who had taken aspirin regularly [85].

Correlation between serum PLA1A and plasma lysoPS levels was observed only in the ACS

group [85]. There was no significant difference in serum PLA1A level among the patients

with normal coronary arteries, stable angina pectoris, and ACS groups, thereby suggesting

that PLA1A might only contribute to plasma lysoPS during ACS [85].

In rats, PLA1A is released by activated platelets since it is only detected at high levels in the

serum but not in the plasma [10]. During blood coagulation, PS+ platelet-derived EVs provide

a catalytic surface for tenase and prothrombinase complexes [4]. Besides, these platelet-

derived EVs expose the PS substrate to the extracellular phospholipases, such as PLA1A and

sPLA2, which are responsible for lysoPS production [4]. LysoPS might activate platelets and

serotonin release through the P2Y10 receptor [13]. Incubation of washed activated rat

platelets reduced PS content but enhanced lysoPS accumulation, which was inhibited

partially by a PLA2 inhibitor [4]. Besides, blood clotting time was decreased, and thrombin

formation was increased, thereby suggesting a role for PLA1A in blood clotting [4]. Platelet

activation also initiated an upsurge in polyunsaturated (18:2 and 20:4) LPA production [40].

Incubation of activated rat platelets with recombinant PLA1A, sPLA2-IIa, and ATX induced

a drastic decrease in PS and an increase in LPA, indicating that PLA1A works with sPLA2

for producing lysophospholipids including various lysoPS species [4,8].

Evidence suggests an independent association between plasma triglyceride concentrations

and increased atherosclerosis risk [86]. In addition to its immunomodulatory functions,

lysoPS may contribute to atherosclerosis by influencing Mφ and platelet functions [13]. A

mouse insertional lipid defect (lpd) mutation led to hypertriglyceridemia and a fatty liver

phenotype [7]. Although PLA1A has homology to triglyceride lipases, the mouse Pla1a gene

is distinct from the lpd locus [7]. Lpdl, another lipase gene in the lpd locus, and its human

homolog LPDL have high sequence homology with PLA1A [86]. Nine DNA polymorphisms

in PLA1A were identified in a Caucasian population [87]. Although the sample size studied

213

was small (10 patients) to draw a valid conclusion, DNA sequence comparison of Caucasian

descents with hypertriglyceridemia and clinically normal subjects revealed no association

between PLA1A polymorphisms and hypertriglyceridemia [87].

Removal of excess cholesterol from cells and the capacity of high-density lipoproteins to

transport cholesterol for elimination by the liver is thought to be atheroprotective [88].

PLA1A likely participates in the high-density lipoprotein metabolism in vivo [89].

Overexpression of Pla1a in human apoA-I transgenic C57BL/6 mice led to increases in serum

phospholipids/apoA-I ratio and cholesterol efflux [89]. The high high-density lipoprotein

cholesterol levels and phospholipids/apoA-I ratio were associated with enhanced cholesterol

efflux via the scavenger receptor class BI and reduced efflux via the ATP-binding cassette

transporter 1, respectively [89].

Mesenchymal stromal cells contribute to the homeostasis of many organs. Proteomic

analyses of proteins secreted by mesenchymal stromal cells isolated from bone marrows,

visceral and subcutaneous adipose tissues only identified PLA1A in mesenchymal stromal

cells from tissues of high-fat but not normal diet-fed mice [90]. Thus, obesity can modify the

secretome content of MSCs [90]. Future studies using Pla1a knockout mice will establish

whether PLA1A plays a role in atherosclerosis, cardiometabolic syndromes, and obesity.

4.4. Antiviral innate immune responses

Several phospholipases contribute to hepatitis C virus (HCV) replication [[91], [92], [93],

[94], [95]]. Serum levels of PLA1A were higher in HCC patients with HCV-related liver

injury than in those with hepatitis B virus or non-hepatitis B virus, non-HCV-related liver

diseases [18]. PLA1A expression was upregulated by HCV infection [95], and PLA1A levels

were higher in the liver from HCV-infected patients [96]. In cells, PLA1A showed a reticular

pattern reminiscent of the endoplasmic reticulum that was not disturbed by HCV infection

[95]. PLA1A facilitated viral assembly through direct binding to HCV E2, NS2, and NS5A

proteins, leading to stabilization of the NS2-E2 and the NS2/NS5A complexes during

infection [95,96]. An amino acid motif essential for viral RNA replication in the C-terminal

domain I of NS5A drove the interaction with PLA1A [96]. The reduced expression of PLA1A

by siRNA silencing resulted in reduced HCV replication, decline in intracellular HCV RNA

214

level and viral proteins expression [95]. Though the impact of catalytically inactive PLA1A

was not tested on HCV replication, the effect of PLA1A silencing was reversed by addition

of lysoPS [95]. Furthermore, PLA1A might participate in the antiviral immune response

through TANK-binding kinase 1-mediated signaling [97]. PLA1A modulated the recruitment

of TANK-binding kinase 1 and its interactions with interferon regulatory factor 3 and

mitochondrial antiviral signaling proteins [97]. The knockdown of PLA1A reduced

recruitment of TANK-binding kinase 1 and interferon regulatory factor 3 to mitochondria,

leading to mitochondria morphology changes and inhibition of signaling for type I interferon

production [97].

HIV-1 Tat protein released by infected cells can affect bystander uninfected T cells functions,

contributing to HIV pathogenesis [98,99]. PLA1A mRNA was enhanced in Jurkat T cells [99]

and human brain microvascular endothelial cells exposed to Tat protein [100]. In the

cerebrospinal fluid of rhesus macaques with simian immunodeficiency virus-infected central

nervous system, fatty acids and phospholipids (C18:2, C16:0, and C18:0) were increased

[101]. These increases in lipid metabolites were concomitant with enhanced expression of

PLA1A (9.3-fold) and PLA2G4C (6.4-fold) in the hippocampus [101].

4.5. Other diseases

In patients with non-alcoholic fatty liver diseases, high serum PLA1A levels were reported

[18]. Non-alcoholic fatty liver diseases are common liver diseases. Multiple factors can

contribute to oxidative-stress-induced liver fibrosis, such as viruses, alcohol, high-fat diet,

obesity, insulin resistance, and gut-derived LPS [102,103]. LPS administered to rats

differently modulated numerous genes in the liver, including a 24.3-fold increase in Pla1a

expression compared to pair-fed animals, but in the alcohol-fed rats, LPS-mediated

expression of Pla1a was suppressed [104].

Vitreous eye fluids from proliferative diabetic retinopathy patients expressed higher PLA1A

compared to nondiabetic controls [105]. PLA1A might contribute to the development of

proliferative diabetic retinopathy through the synthesis of bioactive lysophospholipids [105].

Increased expression of PLA1A seems to be associated with fibrosis in multiple forms of

chronic disease, including liver, eyes, and kidneys [18,105,106].

215

Treatment of skin diseases includes the use of all-trans retinoic acid, but the all-trans retinoic

acid adverse effects on the epidermal barrier function limit its use [107]. In mouse skin

treated with all-trans retinoic acid, corneocytes and keratinocytes contained abnormal lipid

droplets in the cytoplasm, which is related to a hyperproliferative state [108]. Pla1a is one of

the upregulated epithelial barrier-associated genes in the all-trans retinoic acid-treated mouse

skin [108]. Besides Pla1a, several other phospholipases were either upregulated (Pla2g4e)

or downregulated (Pla2g2e and Lypla) [108]. PLA1A is highly expressed in skin samples of

discoid lupus patients compared to psoriasis patient samples, suggesting that specific

pathological traits regulate its expression [109]. Exposure of mouse skin to Staphylococcus

aureus strongly induced Pla1a transcripts [110]. Among human skin cells, PLA1A is only

expressed in melanocytes but not in keratinocytes or fibroblasts [111]. PLA1A expression in

diseased or infected skins might be related to recruitment of T-cell subsets expressing PLA1A

in skin lesions [49,109], stimulation of skin fibroblasts by TLR3 microbial ligands [67], or

TLR4 ligand-induced PLA1A expression in tissue-resident Mφ [53]. Further studies are

required to understand the roles played by PLA1A in skin immunity, inflammation, and

repair.

Pla1a was the most upregulated gene in peripheral blood cells isolated from long-term

surviving rats following allogeneic heart transplant compared to syngeneic graft

transplantation [112]. Increased expression of PLA1A was also observed in human biopsies

undergoing antibody-mediated rejection after heart transplantation [113]. In biopsies from

patients post kidney transplantation, elevated PLA1A expression in renal proximal tubule

epithelial cells and INFγ-stimulated endothelial cells during antibody-mediated rejection

have been reported [114,115]. INFγ-activated renal epithelial cells were likely the principal

source of PLA1A transcripts in acute allograft rejection [115]. Expression of PLA1A

correlated with histological lesions [115]. Urinary exosomes were released into the

extracellular environment by podocyte epithelial cells [116]. Proteomics analysis identified

PLA1A in enriched podocyte vesicles isolated from human urine [116]. The assessment of

PLA1A expression can help diagnose antibody-mediated rejection after organ transplantation,

possibly predicting future failure [115,117].

216

5 Other enzymes regulating serine phospholipid metabolism in

neural system

ABHD (α/β-hydrolase domain) containing-protein family plays a crucial role in lipid

metabolism. Mutations of the ABHD protein family members led to inherited inborn lipid

metabolic disorders [118]. Among the ABHD family members, ABHD16A and ABHD12

play crucial roles in the metabolism of serine phospholipids in mammalian brains as the

foremost PS lipase [119] and lysoPS lipase [120], respectively. Deficiencies of ABHD16A

(PS production) and ABHD12 (PS degradation) are associated with metabolic syndrome and

inflammatory neurodegenerative disease, respectively [[118], [119], [120]]. Mutation in

ABHD12 causes the neurodegenerative disorder PHARC, which means polyneuropathy,

hearing loss, ataxia, retinitis pigmentosa, and cataract [121]. The brain of ABHD12−/− mice

displayed at a younger age (2–6 months) a massive increase of long-chain lysoPS, which

acted as a TLR2 agonist in the microglia [120]. Activation of microglial TLR2 was

responsible for the subsequent age-dependent proinflammatory responses and neural death

in the ABHD12−/− mouse model, which was associated with auditory and motor defects

[120]. Both ABHD16A and ABHD12 are localized in the endoplasmic reticulum in the

central nervous system, especially in the cerebellum that is the most atrophic brain region in

PHARC patients [122]. ABHD16A [123] and ABHD12 [121,124] also exhibited

monoacylglycerol lipase activity towards 2-arachidonoylglycerol, responsible for neural

pain. Thus, ABHD16A and ABHD12 were responsible for the intracellular serine

phospholipids metabolism, and the ABHD16A-ABHD12-lysoPS pathway was assessed as

an emerging lysophospholipid signaling network for neuro-immunological disorders,

indicating their potential therapeutic relevance [119]. It is unknown that the conversion of

lysoPS to LPA by ATX in the brain contributes to neurodegenerative diseases.

DO264, a selective inhibitor of ABHD12, elevated the lysoPS level in mice brain and induced

manifestations similar to PHARC [125]. In contrast, a reversible inhibitor of ABHD16A (12-

thiazole abietanes) inhibiting the lipase activity towards PS reduced the lysoPS level and

brain inflammation [126]. GPR34 is expressed in microglial cells [127,128]. Deletion of

GPR34 impaired glial cell morphology, functional responses such as phagocytosis [127], and

217

the production of inflammatory cytokines involved in neuropathic pain [128]. Altogether, the

available information suggests that the altered metabolism of PS lipids and lysoPS-mediated

signaling in the brain can play a role in the pathogenesis of neurodegenerative disorders.

6 Conclusions

There is growing evidence suggesting roles for PLA1A in many pathological conditions,

including autoimmune disorders, cancers, cardiometabolic disorders, antiviral innate

immune responses, and other diseases. Elevated PLA1A expression and high PLA1A protein

levels are associated with various pathologies. Increased PLA1A expression and release in

tissues and biological fluids result in hydrolysis of surface exposed PS. Production of high

level of lysoPS might subsequently contribute to disease development through lysoPS

receptor activation. However, the roles of PLA1A in disease mechanism remain to be

established. Future studies will determine whether PLA1A is a valuable clinical biomarker

for disease diagnosis or a drug target.

Acknowledgments

This work was supported by the Canadian Institutes for Health Research (MOP-142210). YZ

was the recipient of a scholarship from the China Scholarship Council (CSC).

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Annexe III: Platelet-derived extracellular vesicles contain

an active proteasome involved in protein processing

for antigen presentation via class I major

histocompatibility molecules

Genevieve Marcoux1,2, Audrée Laroche1,2, Stephan Hasse1,2, Marie Bellio1,2,

Maroua Mbarik1,2, Marie Tamagne3,4,5, Isabelle Allaeys1,2, Anne Zufferey1,2, Tania

Lévesque1,2, Johan Rebetz6, Annie Karakeussian-Rimbaud7,8, Julie Turgeon7,8,

Sylvain G. Bourgoin1,2, Hind Hamzeh-Cognasse9, Fabrice Cognasse9,10, Rick

Kapur11, John W. Semple6,12, Marie-Josée Hébert7,8, France Pirenne3,4,5, Herman S.

Overkleeft12, Bogdan I. Florea12, Mélanie Dieude7,8,14, Benoit Vingert3,4,5, Eric

Boilard1,2,8.

Affiliations :

1 Centre de Recherche du Centre Hospitalier Universitaire de Québec-Université Laval,

Québec, QC, Canada.

2 Centre de Recherche Arthrite, Faculté de Médecine de l'Université Laval, Québec, QC,

Canada.

3 Univ Paris Est Créteil, INSERM, IMRB, F-94010 Créteil, France

4 Etablissement Français du Sang, Ivry sur Seine, F-94200, France

5 Laboratory of Excellence GR-Ex, Paris, France

6 Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden

7 Research Centre, Centre hospitalier de l'Université de Montréal (CRCHUM), Montréal,

Québec, Canada.

8 Canadian Donation and Transplantation Research Program, Edmonton, Alberta, Canada

9 Université de Lyon, Université Jean Monnet, INSERM U1059, Saint-Etienne, France

10 Établissement Français du Sang Auvergne-Rhône-Alpes, Saint-Etienne, France.

11 Sanquin Research, Department of Experimental Immunohematology, Amsterdam and

Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the

Netherlands.

226

12 Departments of Pharmacology and Medicine, University of Toronto, Toronto, Canada

13 Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre,

Leiden, The Netherlands.

14 Département Microbiologie, Infectiologie et Immunologie, Faculté de Médecine,

Université de Montréal, Montréal, Québec, Canada.

Keywords: platelet, extracellular vesicles, proteasome, MHC-I, antigen

presentation, immunity

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1 Abstract

In addition to their hemostatic role, platelets play a significant role in immunity. Once

activated, platelets release extracellular vesicles (EVs) formed by budding of their

cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to

perform diverse functions. It is unknown, however, whether the proteasome is transferred

from platelets to PEVs or whether its function is retained. We hypothesized that functional

protein processing and antigen presentation machinery is transferred to PEVs by activated

platelets. Using molecular and functional assays, we show that the active 20S proteasome is

enriched in PEVs along with MHC-I and lymphocyte costimulatory molecules (CD40L and

OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not

increase in platelet concentrates that caused adverse transfusion reactions. They were,

however, augmented after immune complex injections in mice. The complete biodistribution

of murine PEVs following injection into mice revealed that they could principally reach

lymphoid organs such as spleen and lymph nodes, in addition to the bone marrow, and to a

lesser extent liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA)

and loaded its antigenic peptide onto MHC-I molecules which promoted OVA-specific

CD8+ T lymphocyte proliferation. These results suggest that PEVs contribute to adaptive

immunity through cross-presentation of antigens and have privileged access to immune cells

through the lymphatic system, a tissue location that is inaccessible to platelets.

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2 Introduction

Platelets are the second most abundant lineage in the blood and are best known for

their role in hemostasis.744 Platelets are small fragments produced by the large

multinucleated megakaryocyte in the bone marrow. They bear receptors that permit

recruitment of immune cells and carry an extensive set of immune and inflammatory

molecules (e.g. cytokines/chemokines, lipid mediators, hormones) stored in their

granules, cytoplasm, or synthesized by mRNA translation following platelet

activation. Thus, while platelets may mount an innate immune response against

injury, which is critical to combat pathogen invasion, organ and tissue damage may

also favor platelet activation and inflammation in chronic inflammatory diseases.745-

751

Albeit anucleate, the platelet cytoplasm includes numerous molecules comprising

the proteasome, which are transferred from megakaryocytes to their progeny. The

proteasome is a high molecular weight cylindrical protein complex through which

unwanted or damaged proteins are degraded.752,753 The central complex part, called

the 20S proteasome, is made up of twenty-eight distinct subunits,522 comprising the

three catalytic subunits necessary for the degradation of proteins into peptides of

three to fifteen amino acids in length.522,754 Proteasome activity in megakaryocytes

is required for platelet production755,756 and in platelets, the proteasome regulates

platelet lifespan,757 activation758-760 and the release of PEVs.761,762 The platelet

proteasome can hydrolyze proteins into smaller peptides,522,763,764 thereby enabling

peptide loading onto the platelet major histocompatibility complex (MHC) class I

molecules (MHC-I).765-767 Components of the peptide loading complex are also

expressed in platelets and are found in close proximity with MHC-I during platelet

activation.520,521 As platelets can efficiently form an immunological synapse with T-

lymphocytes to activate lymphocyte proliferation,520,768,769 they are known to fulfill

roles in cross-presentation of antigens in adaptive immunity. In a similar manner,

megakaryocytes cross-present antigens to CD8 T-lymphocytes, thereby suggesting

that they may also play a dual role in innate and adaptive immunity.767,770-772

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Extracellular vesicles, produced in abundance by platelets, are small (up to 1µm in

diameter) membrane-bound vesicles released from the plasma membrane or

endosomal compartments of activated cells. Platelet EVs are heterogeneous in

terms of surface molecules and content (e.g. nucleic acids, lipids, transcription

factors, enzymes, mitochondria) and as such, may play diverse functions beyond

hemostasis.538,773,774 For instance, PEVs convey mitochondrial components that are

associated with inflammation and adverse transfusion reactions (ATRs).442,540,775

Despite the fact that platelets are restricted to the blood circulation, PEVs can cross

tissue barriers and enter synovial fluid,447,776 lymph535,777 and bone marrow778 where

they can deliver platelet-derived molecules and modulate target cells.773 For

instance, PEVs promote the formation of germinal centers and the production of IgG

by B-cells.543,544 They also interact with and modulate regulatory T cell differentiation

and activity.541,542 Thus, PEVs may be able to transport platelet-derived molecules

relevant to adaptive immunity into lymphoid organs. However, it is unknown whether

the proteasome and the molecules necessary for antigen presentation are also

transferred during the budding of PEVs. In this study, we evaluated whether

functional protein processing and antigen presentation machinery is transferred to

PEVs by activated platelets.


More details are presented in supplemental methods.

Labelling of murine platelets, DCs and PEVs

Platelets were isolated from C57BL/6J mice by retro-orbital or cardiac puncture in 200µL

ACD, 350µL Tyrode’s buffer pH 6.5. Whole blood was centrifuged at 600xg for 3min and

then at 400xg for 2min to remove red blood cells. Supernatant was spun at 1,300xg for 5 min

and the platelet-containing pellet was gently resuspended in 600µL Tyrode’s buffer pH 7.4.

Platelets were either left nonactivated or activated with thrombin (0.1U/mL) after addition of

5 mM of calcium for 90 min at RT (time based on kinetics of CD41+ Proteasome+ PEV

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release shown in Supplementary figure 3C). Platelet EVs were obtained by two rounds of

centrifugation of stimulated platelets at 1,300xg for 5min at RT. Either activated platelets,

EVs or DCs were pulsed with 100µg/mL OVA protein (Sigma-Aldrich), 200µg/mL of OVA

peptide (SIINFEKL [Invivogen]) or left unpulsed for 4h at RT. These conditions were either

left unlabelled for lymphoproliferation and intracellular staining experiments or labelled for

Hs-FCM experiments.

Five µL of PEVs or platelet suspensions were labelled with 250nM LWA300 proteasome

probe in a total volume of 100μL for 90min at 30°C. Samples were then incubated with the

following antibodies for 30min at RT prior to dilution in Annexin V binding buffer and

analysis by Hs-FCM: BUV395 anti-CD41, BV650 anti-CD62p, BUV395 anti-CD41,

BV650 anti-CD62p, BV711 Annexin V, BV421 anti-OX40L, BUV737 anti-CD154 (all BD

Biosciences), PeCy7 anti-CD40, PeCy7 anti-MHC-I (AF6-88.5) and PE anti MHC-I bound

to OVA peptide (25D1.16) (all from Biolegend).

4 Results

PEVs contain functional proteasome

Following platelet activation by thrombin, remnant platelets were eliminated by

centrifugation and larger EVs were isolated by a second high-speed centrifugation

(18Kxg fraction). The supernatant obtained was further centrifuged at 100,000g and

smaller EVs (likely exosomes) were obtained from this pellet. We found that 98.10.5

% of proteins were retrieved in the larger EV 18Kxg fraction. Immunoblotting

confirmed that human PEVs from this fraction were enriched in proteasome 20S α

subunit, in addition to mitochondria (indicated by TOM20 expression) and CD41, but

lacked TSG101 (putative marker of exosomes) (Figure 1A-E). Using platelets as a

positive control, we assessed proteasome function in these PEVs. Proteasome-

associated trypsin-, caspase- and chymotrypsin-like activities were detectable in

platelets and significatively increased in the PEV fraction, but were undetectable

after treatment with epoxomicin, a proteasome inhibitor (Figure 1F). Visualization of

immunogold-labelled proteasome 20S α subunit by transmission electron

microscopy confirmed the presence of proteasomes in PEVs (Figure 1G). These

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data suggest the catalytically active proteasome was transferred to PEVs upon their

release from platelets.

LWA300 is a conjugate between epoxomicin and BODIPY FL fluorophore that

generates an activity-based, plasma membrane-permeable inhibitor that can identify

the proteasome in cells.779,780 Using LWA300, we detected and quantified active

proteasome-containing PEVs directly in the platelet secretome.779,780 High-sensitivity

flow cytometry (Hs-FCM) confirmed PEV heterogeneity following platelet activation

by thrombin (Figure 1H). Approximately 16.66.5% of the larger (i.e. 500–900nm)

PEVs781 contained proteasome whereas smaller vesicles (i.e. less than 500nm) had

no detectable proteasome (Figure 1H and Supplementary figure 1). The detection

specificity of proteasome-containing PEVs by hs-FCM was confirmed using a

combination of controls. We confirmed efficient competition of the LWA300 probe by

unlabelled epoxomicin, and we determined the particulate nature and membrane

moiety of proteasome-containing PEVs, as they were respectively pelleted by

ultracentrifugation and sensitive to detergent treatment (Figure 1I-J). Confocal

microscopic visualization of platelets as positive controls, and PEVs from thrombin-

activated platelets labelled with LWA300 revealed that both platelets and a

subpopulation of PEVs contained active proteasome (Figure 1K).

Hs-FCM was further used to characterize proteasome-containing PEVs in terms of

surface markers and mitochondrial content. Approximately half of the proteasome-

containing PEVs exposed phosphatidylserine while the vast majority expressed

surface P-selectin (Supplementary figure 2A). Furthermore, 68.37.8% of the

proteasome-containing PEVs also contained mitochondria (Supplementary

figure 2A). Investigation of the mechanisms underlying release of active

proteasome-positive PEVs revealed that the total number of PEVs (with and without

proteasomes) were significantly reduced in the presence of actin inhibitors

(cytochalasins B, D, E and latrunculin A) but not by the tubulin polymerization

inhibitor nocodazole (Supplementary figure 2B). Proteasome release in PEVs was

not unique to thrombin stimulation as ADP, cross-linked collagen related peptide

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(CRP-XL) and heat-aggregated IgG (HA-IgG) also triggered release of proteasome-

containing PEVs (Supplementary figure 2C).

Identification of proteasome-containing PEVs under physiological and

pathological conditions

The presence of proteasome-containing PEVs was assessed under conditions

conducive to platelet activation and PEV release. A mean of 1.82×106

(range:1.13×105 to 8.11×106, n=6) proteasome-containing PEVs/mL were detected

by hs-FCM in the blood of healthy individuals, which corresponded to 2.61.8% of

the total PEVs in blood. PEVs were quantified in platelet concentrates (PCs) known

to have caused ATRs and compared with control PCs that did not induce ATRs.

Given the reported increase in mitochondria-containing PEVs in ATRs,442,540 we also

determined their levels. High levels of proteasome-containing PEVs were found in

all tested PCs (Figure 2A) but the concentrations of proteasome-containing PEVs

(with or without mitochondria) were not significantly elevated in PCs that induced

ATR (Figure 2A). In contrast, compared with controls, the concentrations of

mitochondria-containing PEVs were increased in ATR-associated PCs, consistent

with prior findings.442,540

Transfusion-related acute lung injury (TRALI) is a potentially lethal adverse reaction

that can result from transfusion of PCs.782 Thus, we quantified proteasome-

containing PEVs in murine bronchoalveolar lavages in an inducible TRALI

model.783,784 Proteasome-containing PEVs were detected in bronchoalveolar

lavages from both TRALI and control mice (Figure 2B), however, no significant

difference was observed between the two groups (Figure 2B). This suggests that

proteasome-containing PEVs are not increased during lung inflammation in this

model and therefore may not participate to acute inflammation that characterizes the

pathogenesis.

Our in vitro investigations pointed to the high potency of immune complexes (HA-

IgG) in generating proteasome-containing PEVs (Supplementary figure 2C).

Although mice lack FcγRIIA, this is the only Fcγ receptor expressed by human

233

platelets that is capable of responding to immune complexes.785 Recent findings

indicate that circulating immune complexes stimulate the release of mitochondria-

containing PEVs in mice expressing the FcγRIIA transgene.492,786 Compared with

diluent injected control mice, there were significantly elevated levels of proteasome-

containing PEVs in plasma of mice with immune-complexes challenge (Figure 2C).

These findings confirmed that proteasome-containing PEVs are present under

various physiological and pathological conditions.

Protein processing by proteasome-containing PEVs

In order to study proteasome function in PEVs, we investigated its ability to process

proteins into smaller peptides by assessing their successful loading into the antigen-

binding groove of MHC-I molecules. We confirmed the expression of MHC-I on

resting and thrombin-activated murine platelets and verified whether MHC-I is

maintained on PEVs present in the platelet secretome. We found that washed resting

platelets did not express MHC-I on their surface (Figure 3A-B), however, thrombin

activation led to a significant increase in surface MHC-I expression (Figure 3A-B),

consistent with the reported presence of this molecule in α-granules and its release

upon activation.520,521,787,788 A small proportion (0.930.13%) of the spontaneously

released PEVs expressed MHC-I, but this proportion significantly increased upon

platelet activation with thrombin (means of 4.640.98%).

To determine whether PEV MHC-I can indeed load small peptides, we pulsed PEVs

present in the platelet secretome with the ovalbumin (OVA) peptide SIINFEKL and

monitored its association with MHC-I molecules using the 25D1.16 monoclonal

antibody, which specifically recognizes MHC-I/SIINFEKL complexes.789 Similarly

with platelets, PEVs loaded the SIINFEKL peptide onto their MHC-I molecules

(Figure 3C-D). Native OVA was also efficiently processed by platelets and the

SIINFEKL peptide was loaded in MHC-I (Figure 3C-E), consistent with prior work.520

We found that an average of 2.53±0.74% of CD41+ PEVs pulsed with the peptide

and 1.83±0.24% of CD41+ PEVs pulsed with OVA (n=18) were positive for 25D.1.16.

Of interest, incubation of native OVA with PEVs resulted in proteolysis of the former

and retrieval of the SIINFEKL peptide from MHC-I molecules expressed by the

234

PEVs. Taken together, the data show that PEVs can process native proteins into

smaller peptides thereby enabling antigen presentation through MHC-I.

Proteasome-containing PEVs can reach lymphoid organs and circulate through

the lymphatic system

Intravenously injected PEVs have a limited circulation time in human blood, ranging

from 10 min to hours depending on studies.790,791 It is unclear, however, whether

they can reach lymphoid organs. Fluorescently labelled PEVs generated from

activated mouse platelets were intravenously injected into mice and their presence

in blood and different organs was monitored. We could identify free PEVs (unbound

to cells) for up to 2 minutes in blood (Figure 4A and Supplementary figure 4A-C).

PEVs in blood were also mainly found bound to platelets and to leukocytes, mainly

Ly6G+ neutrophils, and to a lesser extent lymphocytes, but were mostly undetectable

by 60min (Figure 4A). Screening of individual PEVs in whole tissue sections in

different organs identified spleen and lymph nodes (popliteal and inguinal) as

primary targets, followed by liver, bone marrow, lungs, and kidneys, while none were

found in brain (Figure 4B-C and Supplementary figure 4D-E). Moreover,

aggregates of PEVs (i.e. larger than 1µm2 and up to 541µm2) were mainly observed

in spleen (mean size 2.84±0.16µm2), popliteal (4.16±0.40µm2) and inguinal

(4.08±0.30µm2) lymph nodes, followed by bone marrow (2.75±0.15µm2), lung

(33.51±7.15µm2) and liver (3.40±0.21µm2). This may reflect their accumulation into

smaller vessels or the internalization of numerous PEVs within single cellular

recipients in these organs (Figure 4B-C and Supplementary figure 4D-E).

Platelet EVs can circulate through the lymphatic system and the levels of PEVs in

lymph are increased in mouse models of atherosclerosis and autoimmune

inflammatory arthritis.535,773,777 Using the lymph from mice, we evaluated whether

PEVs were associated with proteasome and MHC-I molecules. We found that a

fraction of the PEVs in lymph expressed MHC-I (11.22.2%) and contained an active

proteasome (12.03.9%). Remarkably, a detectable proportion (1.60.7%) of the

lymph PEVs contained both proteasome and MHC-I molecules (Figure 4D-E) and

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this was significant given the substantial number of PEVs in lymph (mean of

2.5×107/mL in mice777).

Proteasome-containing PEVs express lymphocyte co-stimulatory molecules

Efficient stimulation of adaptive immunity requires both recognition of the antigen-

MHC-I complexes by the T-cell receptor (TCR) and the activity of co-stimulatory

molecules. We evaluated whether platelets or proteasome-containing PEVs loaded

with SIINFEKL expressed co-stimulatory molecules in addition to other known PEV

markers displayed by CD41+ Proteasome+ EVs. Compared with PEVs that had

undetectable SIINFEKL loading, both platelets and PEVs present in the platelet

secretome loaded with SIINFEKL (25D1.16-positive) expressed higher levels of

proteasome (Figure 5). Moreover, in contrast to thrombin-activated platelets, where

phosphatidylserine expression is increased when loaded with SIINFEKL, both PEVs

bearing SIINFEKL and those negative for SIINFEKL expressed similar levels of

phosphatidylserine (Figure 5). Furthermore, both platelets and SIINFEKL-bearing

PEVs expressed higher levels of P-selectin, and the co-stimulatory

molecules CD40L, CD40 and OX40L (Figure 5). Thus, among the different subtypes

of PEVs, those with a higher density of antigen–MHC-I complexes show more

abundant expression of lymphocytes co-stimulatory molecules and bear a higher

content of active proteasome.

Proteasome-containing PEVs can support antigen-specific T cell activation

T cells isolated from OT-1 mice100 were co-incubated for 18h with PEVs present in

the platelet secretome that were either pulsed or not with the SIINFEKL peptide or

native OVA. Dendritic cells and platelets were treated similarly as positive controls

and for comparison (Figure 6A). The T cells (CD3+CD8+) were then washed and the

expression of CD40, OX40, IL-2 and IFN-γ was evaluated to assess T-cell activation.

Compared with DCs and platelets, PEVs could induce a significant release of IFN-γ

when pulsed with the OVA peptide, whereas native OVA led to an increase in IFN-γ

236

but did not reach statistical significance (Figure 6B). Moreover, DCs and, to a lesser

extent, platelets and PEVs were only capable of inducing significant CD40

expression by T lymphocytes previously pulsed with the OVA peptide (Figure 6C).

In contrast, OX40 and IL-2 expression were not induced by DCs, platelets or PEVs

under these experimental conditions (Figure 6D-6E).

Whether PEVs could stimulate T cell proliferation, a hallmark response by the

lymphocyte antigen-MHC-I complex was evaluated. T cells from OT-1 mice were

labelled with CFSE to monitor cellular division and co-incubated for 5 days with either

DCs, activated platelets or PEVs, which were either pulsed or not with either

SIINFEKL or native OVA (Figure 7A). Lymphoproliferation would be represented by

a decrease in the mean fluorescence intensity histogram, i.e. a dilution of CFSE

fluorescence. (Figure 7B).

As expected, we found that the proportion of lymphoproliferative cells was

significantly higher when OT-1 T lymphocytes were incubated with peptide- or native

OVA-pulsed DCs or activated platelets (Figure 7B-C). Of particular note is that PEVs

also supported T cell proliferation when pulsed with either the SIINFEKL or native

OVA (Figure 7B-C). In addition, when PEVs present in the platelet secretome were

removed from pulsed conditions by ultracentrifugation, no proliferation was

observed, confirming that the pulsed proteins alone, or the platelet secretome devoid

of PEVs, cannot support proliferation (Figure 7D). Furthermore, inhibition of PEV

proteasome by epoxomicin before pulsing with native OVA inhibited the ability of

PEV to induce T cell proliferation (Figure 7E left panel). The effect was directed

toward PEV proteasome, as addition of epoxomicin prior to peptide pulsing at the

same concentration used on DCs did not inhibit proliferation (Figure 7E right

panel). Thus, PEVs are capable of proteosome-dependent processing of native

proteins, thereby enabling peptide loading onto MHC-I. Platelet EVs express co-

stimulatory molecules, and their interaction with T lymphocytes promotes

lymphocyte cytokine production and proliferation.

237

5 Discussion

Megakaryocytes and platelets are emerging as active players in innate and adaptive

immunity.750,770,771 The platelet’s role in immunity is mainly confined to the blood

circulation, while megakaryocytes are localized in bone marrow and lungs. The latter

location potentially provides the megakaryocyte with more direct access to airborne

pathogens and allergens.772,792,793 In contrast, PEVs can additionally disseminate

into organs and tissues and this may be possibly due to their small dimensions and

the presence of unique surface molecules. In this study, we found that the

proteasome and the necessary machinery to process and present antigens to CD8+

T cells are packaged into PEVs by platelets. Thus, PEVs may extend the immune

functions played by platelets and megakaryocytes outside the confines of the blood.

Platelet EVs are heterogeneous in terms of surface molecules and their platelet-

derived content. The presence of mitochondria within PEVs is well

documented,442,527,540 but it was unknown whether other organelles were also

transferred from the platelet. The proteasome is much more abundant than

mitochondria, at around 800,000 copies per cell794 in contrast to approximately 3–7

mitochondria per platelet.442 Further investigation will be necessary to determine if

the presence of multiple organelles within a single vesicle is the result of a specific

sorting mechanism, or because those vesicles are larger and may have more

storage capacity. Nonetheless, we observed that the release of proteasome-

containing PEVs requires cytoskeleton remodeling via intact actin microfilament

dynamics and that a broad array of platelet agonists induce the release of

proteasome-containing PEVs.442

The presence of an extracellular proteasome has already been documented in

normal human blood, and elevated levels have been found in patients suffering from

autoimmune diseases, sepsis or trauma.795 Moreover, the 20S proteasome core is

present and active within EVs derived from apoptotic endothelial cells and regulates

tertiary lymphoid structure formation, autoantibody production and graft rejection

following transplantation.754 While some evidence supports that a circulating

238

extracellular proteasome may be transported by EVs, we show that EVs of platelet

origin, among the most abundant EVs in blood, do contain the proteasome. We

further suggest, based on our characterization of these EVs, that platelet

microvesicles, not exosomes, contain the proteasome. Consistent with this, mass

spectrometry analysis of the human PEV proteome identified numerous proteasomal

subunits.796-798 These include subunits of the 20S catalytic core and

immunoproteasome subunit (PSMB8), subunits of the 11S and 19S regulator and

the 26S proteasome.796-798 Moreover, with calnexin, calreticulin, ERP57 and ERP29,

other members of the ubiquitin-proteasome pathway were identified in PEVs such

as members of the E1 and E2 ubiquitin-conjugating enzyme family.796,798

Considering the presence of these proteins and the fact that intact ovalbumin needs

to be ubiquitinated for degradation by the proteasome,799 it points to the occurrence

of functional ubiquitination in PEVs. To our knowledge, there is no evidence of

protein TAP-1 and TAP-2 (related to TAP transporter) presence in PEVs. Further

investigations are required to see if the TAP transporter is present in PEVs and/or if

the processing pathway of the antigen differs in extracellular vesicles since there is

no reported endoplasmic reticulum in PEV. Thus, while proteomic data points to

ubiquitin-proteasome system proteins in PEVs, the present work unequivocally

demonstrates its presence and documents that the extracellular proteasome in

PEVs is functional and can contribute to antigen processing.

We used complementary approaches and developed a Hs-FCM-based assay to

detect active proteasome at the single EV level, thereby permitting quantification and

assessment of other molecules expressed by the EVs. In particular, the proteasome-

containing PEVs also expressed MHC-I and co-stimulatory molecules, which

enabled lymphocyte activation/proliferation and cytokine generation. These findings

demonstrate a novel and potentially important role for PEVs in adaptive immunity.

While our work suggests that PEVs may be involved in adaptive immunity through

antigen presentation, it does not necessarily exclude that other cells may release

proteasome-containing EVs capable of playing this role. Indeed, EVs derived from

DCs, B and T lymphocytes, macrophages and NK cells can perform cross-

presentation, suggesting that they also contain the necessary antigen

239

processing machinery.800-805 Further studies will be necessary to determine the

impact and the importance of PEVs as antigen presenting elements.

We identified proteasome-containing PEVs in the blood of healthy donors. As most

PEVs in blood under healthy conditions are suggested to originate from

megakaryocytes,482,806 the latter may also constitutively release proteasome-

containing EVs. Moreover, we found that numerous stimuli of human platelets, as

well as in vivo stimulation of mouse platelets could induce release of proteasomes

in PEVs, suggesting that proteasome release is at least conserved in both humans

and mice and takes place via platelet activation. Furthermore, platelets can actively

induce immunity against the Plasmodium berghei parasite520 and megakaryocytes

can be infected by Dengue virus807 and can also phagocytose E. coli.772 Given their

small size, intact microorganisms may not necessarily be present inside PEVs, but

PEVs might process cytosolic microbial proteins derived from intact

platelets/megakaryocytes that lack the ability to enter the lymphatic system. Thus,

PEVs may be implicated in immune surveillance and might contribute to presentation

of microbial antigens within lymph tissues. Future studies are however needed to

determine whether exposure to PEVs suffices to establish immunity in vivo, such as

to less immunodominant antigens than OVA, or whether co-stimulation by

inflammation or infection are needed to establish sustained immune response.

Self-antigens may also be presented by PEVs. Mitochondria were identified in a

proportion of the proteasome-containing PEVs, and although prior studies showed

that mitochondria-containing PEVs are rare in lymph (0.41±0.25% (n=4) of the PEVs

in mouse lymph contain mitochondria)777 in comparisons to proteasome-containing

PEVs 13.61±4.27% (n=6), these proportions might be augmented in certain

diseases. It would be interesting to determine whether PEVs contribute to the

formation of mitochondrial autoantibodies that are described in autoimmune

diseases, such as systemic lupus erythematosus.808 Furthermore, the presence of

proteasome-containing PEVs in platelet concentrates was not associated with

increased risks of ATR or TRALI in a mouse model. It remains to be verified whether

the presentation of platelet antigens (e.g. CD41 or CD61) by PEVs from PCs might

240

contribute to generation of anti-platelet immunity in transfused recipients, although

this has been shown with megakaryocytes.56 It is also not excluded that PEV might

also participate in other immune responses such as autoantibody production or in

tissue remodeling, or if they could be used a platform for cell-based

vaccines.434,436,437 The cross-presentation of PEVs presented here may allow for

new therapeutic possibilities such as in anti-tumor or anti-viral immunity or to induce

cytotoxic immunity by vaccination.809 For example, PEVs have already been

proposed as antigen carriers for vaccination,810,811 and our results suggest that these

types of PEVs are also endowed with cross-priming properties that offer new

prophylactic or therapeutic vaccination.

Human platelets injected into WT mice circulate less than 2h, in contrast to mouse

platelets transfused into mice that can circulate for several days.812,813 We thus used

mouse PEVs in our transfusion experiments, and yet most were undetectable from

the blood circulation after 15min, pointing to their rapid uptake in surrounding tissues.

In blood, the main absolute cellular target was the platelets, mostly because platelets

outnumber leukocytes, which might suggest that PEVs might recycle molecules back

to platelets. PEVs were also found in bone marrow, consistent with recent findings

that pointed to their role in the stimulation of megakaryocyte biogenesis.778 The main

organs that were targeted were the lymphoid organs. Our findings in mouse lymph

revealed that proteasome-containing PEVs can circulate in the lymphatic system,

potentially explaining their accumulation in lymphoid organs following intravenous

injection. This access to the lymphatic system by proteasome-containing PEVs may

reveal a new immune route for PEVs to reach lymphoid organs or infected tissues.

Our study highlights the diversity of PEVs and supports the concept that different

subtypes of PEVs may play different roles depending on their cargo and tissue

distribution.

Acknowledgments

We are grateful to the blood donors and patients who participated in this study. We

acknowledge the generous technical help provided by Nicolas Tessandier and

Carolanne Gélinas. We are thankful to Julie-Christine Lévesque from the Cytometry

241

and Microscopy platform (CHU de Quebec) and Richard Janvier from the

Microscopy platform (Université Laval).

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7 Figures

Figure 1. Platelets and PEVs contain proteasome

247

(A) Proteasome 20S α subunit, CD41, TOM20, TSG101 and actin in human platelet

extracellular vesicles (PEVs) (18Kxg fraction) and platelet (PLTs) preparations (20 μg

protein per lane) were assessed by immunoblotting. Results are representative of five distinct

preparations. (B-E) Protein quantifications were assessed by densitometry using image lab

software (Biorad), results were normalized to actine and expressed as arbitrary units (AU).

Mean ± SEM, n=5, paired t-test *P < 0.5. (F) Proteasome function was assessed by measuring

trypsin-like, caspase-like or chymotrypsin-like activity of PEVs and platelets treated or not

with epoxomicin using the Proteasome-Glo™ chymotrypsin-like, trypsin-like and caspase-

like cell-based assays. Twenty and 10 μg of proteins were used for platelets and PEVs,

respectively. Mean ± SEM, n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney.

(G) TEM visualization of immunogold labelling of proteasome 20S α subunit in PEVs

released from thrombin (0.5U/mL)-activated platelets. Data are representative of three

independent experiments. (H) High-sensitivity flow cytometry (hs-FCM) analysis of resting

platelets and thrombin (0.5U/mL)-activated platelets. Two distinct populations of PEVs, i.e.

larger PEVs (approximately 17% of these PEVs contain active proteasome) and smaller

PEVs, not containing active proteasome. (n = 20 data are presented as mean ± SEM, ** P <

0.01, *** P < 0.001 and **** P < 0.0001, Kruskal–Wallis). (I-J) Controls were performed

to assess the specificity of PEV detection using hs-FCM. Sensitivity of CD41+Proteasome+

PEVs to competition by epoxomicin, ultracentrifugation (Ultracentri) or 0.05% Triton X-100

and unlabelled samples are presented as % of untreated (Control). Data are presented as mean

± SEM of 5 independent experiments, paired t-test ****P < 0.0001 compared with the

control. (K) Confocal microscopy visualization of proteasome content associated with

platelets (left panel) and PEVs (right panel). Visualization of CD41, wheat germ agglutinin

(WGA) to determine plasma membrane surface, proteasome (LWA300) and merge is

displayed in the region of interest (ROI). Populations originating from dashed lines squares

and represented in ROI are triple positives (white arrowheads) or CD41- and WGA-positive

but proteasome-negative (white arrows).

Figure 2. Identification of proteasome-containing PEVs under physiological and

pathological conditions

248

(A) Proteasome-containing PEVs detected by hs-FCM are found in PFP from platelet

concentrates that have caused adverse transfusion reaction (ATR) in recipients and in control

concentrates that did not induce ATR. The total number of proteasome-containing PEVs

(containing or not mitochondria (mito)), proteasome+mito-PEVs or proteasome+mito+PEVs

does not significantly differ between control and ATR, while proteasome- mito+ PEVs are

increased in ATR (no adverse reaction group [n = 33] vs. adverse reaction group [n = 34]

matched in terms of storage duration; data are presented as mean ± SEM, NS non-significant,

**** P < 0.0001, Student's t-test). (B) Proteasome-containing PEVs detected by hs-FCM are

found in bronchoalveolar lavages from mice after induction of transfusion related acute lung

injury (TRALI) with 34-1-2s and AF6-88.5.5.3 antibody and in control mice (n = 5, data are

presented as mean ± SEM, NS non-significant, Student's t-test). (C) Proteasome-containing

PEVs are detected at significantly higher levels in mice 1-hour post i.v. injection of HA-IgG

vs. control (diluent) mice. (n = 3, **P< 0.01, data are presented as mean ± SEM, Student’s t-

test)

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Figure 3. Platelets and PEVs load and process OVA onto MHC-I

(A-B) Thrombin (0.1 U/mL)-activated murine platelets and their PEVs express MHC-I

(detected by hs-FCM). (n = 19, data are presented as mean ± SEM, **** P < 0.0001, Mann–

Whitney). Activated platelets and their PEVs are able to load the SIINFEKL peptide (C-D)

or to process and load ovalbumin (OVA) (C-E) onto MHC-I. (n = 19, **** P < 0.0001, data

are presented as mean ± SEM, Kruskal-Wallis test comparisons between pulsed (+) to

unpulsed (-))

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Figure 4. PEVs in blood circulation can reach lymphoid organs and circulate in lymph

(A-C) Fluorescently labelled PEVs generated from activated mouse platelets were

intravenously injected into mice and their presence in blood (A) and different organs (B-C)

was monitored after 2, 15 and 60 minutes. Free PEVs (unbound to cells) were identified by

flow cytometry for up to 2 minutes in blood as well as PEVs bound to platelets and to

leukocytes (mainly Ly6G+ neutrophils, few lymphocytes), but were mostly undetectable by

60 minutes. Dashed lines represent mean of vehicle (n=9-13), n = 11 (2 min), n = 5 (15 min)

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and n = 6 (60 min), data are presented as mean ± SEM, * P < 0.05, ** P < 0.01, *** P <

0.001, Kruskal-Wallis). (B) Representative images of CMFDA-labelled (Green) individual

PEVs (White arrowhead) and PEV aggregates (* white asterisk) in whole tissue sections

(Spleen, popliteal LN (PLN), inguinal LN (ILN), bone marrow, lungs and liver) at 15 and 60

min by confocal microscopy, nuclei (Hoeschst 3342) are in blue. Results are representative

of observations made in 5-6 mice per group. (C) PEVs and aggregates were quantified using

5 different sections for lymph nodes (PLN and ILN) (representing a total surface of at least

1.5mm2), 8 zones of 500,000 µm2 each, randomly assigned on 2 different sections for femurs

(total surface of 4 mm2) and 10 zones of 500,000 µm2 each, randomly assigned on 2 different

sections for lungs, spleen, kidneys and brain and 1 section for liver (total surface of 5 mm2)

using Zen 3.3 software. (n=6 (PBS 60min), 5 (15 min) and 6 (60 min, data are presented as

mean ± SEM, * P < 0.05, ** P < 0.01, Kruskal-Wallis). (D-E) PEVs in lymph were detected

by hs-FCM. (D) Gating strategy to analyze expression of MHC-I and proteasome (LWA300)

on CD41+ EVs in lymph and representative dot plot of labelled and unlabelled (CD41 only)

lymph. (E) Expression of MHC-I and proteasome (LWA300) on CD41+ EVs in lymph was

determined. +/+ double positive and −/− double negative for MHC-I and proteasome. (n = 6,

data are presented as mean ± SEM).

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Figure 5. Platelets and PEVs with loaded OVA peptide express activation and co-

stimulatory molecules

(A-B) Activated platelets and (C-D) PEVs loaded with OVA peptide (25D1.16+) express

higher levels of proteasome (LWA300), and activation (Annexin V, P-selectin) and co-

stimulatory molecules (CD40, CD40L, OX40L). (A,C) Mean fluorescence intensity (MFI)

of the different markers assessed by hs-FCM (n = 7, data are mean ± SEM, NS non-

significant, * P < 0.05, Student t test). (B,D) Representative MFI histogram of the 25D1.16

negative and positive populations for each marker shown on CD41+ Proteasome+ events.

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Figure 6. PEVs can induce antigen-specific T cell activation and cytokine production

through antigen presentation

(A) Schematic representation of the experimental plan. Cells and PEVs used for the

stimulation of lymphocytes assessed by intracellular cytokines staining (ICS). DC: dendritic

cells, NS: unpulsed, OVA: ovalbumin, O/N: overnight. (B-E) Expression of receptors or

cytokines by CD3+ CD8+ T cells co-incubated with either DCs, activated platelets (PLTs) or

PEVs, left unpulsed or pulsed with SIINFEKL (PP) or ovalbumin (OVA). (B) Interferon

gamma (IFN-γ) production, (C) CD40 expression, (D) OX40 expression and (E) IL-2

production (n = 6, 7 or 9; data are presented as mean ± SEM. * P < 0.05, ** P < 0.01

Wilcoxon vs. unpulsed). Dashed lines are unstimulated conditions.

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Figure 7. PEVs loaded with native OVA process and present OVA peptide to induce

antigen-specific T cell lymphoproliferation

(A) Schematic representation of the experimental plan. DC: dendritic cells, NS: unpulsed,

OVA: ovalbumin, CFSE: Carboxyfluorescein succinimidyl ester. (B) Histogram showing

CFSE fluorescence shift of CD3+ CD8+ T cells populations when co-incubated with either

dendritic cells (DCs), activated platelets (PLTs) or PEVs left unpulsed or pulsed with

SIINFEKL peptide (PP) or ovalbumin (OVA) for 7 days. (C) Percentage of CD3+ CD8+

lymphoproliferative cells after co-incubation with either DCs, PLTs or PEVs unpulsed or

pulsed with PP or OVA for 7 days. (n = 14; data are presented as mean ± SEM. NS non-

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significant, ** P < 0.01, *** P < 0.001, ****P < 0.0001, Friedman test followed by Dunn’s

post-test for multiple comparisons to unpulsed). (D) Percentage of CD3+ CD8+

lymphoproliferative cells after 7 days co-incubation with either PP pulsed DCs or supernatant

(surn) depleted of PEVs by ultracentrifugation, left unpulsed or pulsed with PP or OVA.

(n=5; data are presented as mean ± SEM, ** P < 0.01, Mann-Whitney vs. unpulsed). (E)

Proportion of CD3+ CD8+ lymphoproliferative cells after 7 days co-incubation with OVA-

pulsed PEVs treated or not with epoxomicin (epoxo) for 2 hours and PP-pulsed DCs (DC +

PP) treated or not with epoxomicin (epoxo). (n = 9 for PEVs and n = 3 for DC; data are mean

± SEM, NS non-significant, ** P < 0.01, Wilcoxon). Dashed lines are unstimulated

conditions.

Étude de l'implication de l'acide lysophosphatidique par

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