The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions

doi:10.1182/blood-2010-12-324756Prepublished online May 6, 2011;

Ornella Azzolino, Fiorella Altruda, V. Marco Ranieri and Emilio HirschCarlotta Costa, Giulia Germena, Erica L. Martin-Conte, Ivan Molinaris, Eleonora Bosco, Stefano Marengo, functionsThe RacGAP ArhGAP15 is a master negative regulator of neutrophil

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1

The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions

Short title for running heads: ArhGAP15 is a neutrophil master regulator

Carlotta Costa1#*, Giulia Germena1*, Erica L. Martin-Conte2, Ivan Molinaris1, Eleonora Bosco1,

Stefano Marengo1, Ornella Azzolino1, Fiorella Altruda1, V. Marco Ranieri2, Emilio Hirsch1#.

1. Department of Genetic, Biology and Biochemistry and Molecular Biotechnology Center.

University of Torino. Via Nizza 52. Torino 10126. Italy

2. Department of Anesthesiology and Critical Care, University of Torino, Ospedale S. Giovanni

Battista-Molinette. Corso Dogliotti 14. Torino 10126. Italy

* These authors equally contributed to this work. # Co-corresponding authors

Corresponding authors:

Carlotta Costa, PhD Department of Genetic, Biology and Biochemistry and Molecular Biotechnology Center (MBC) University of Torino, Via Nizza 52, 10126 Torino, Italy Tel. 0039 011 670 6425 Tel. 0039 011 670 6432 Email: [email protected] Emilio Hirsch, PhD Department of Genetic, Biology and Biochemistry and Molecular Biotechnology Center (MBC) University of Torino, Via Nizza 52, 10126 Torino, Italy Tel. 0039 011 670 6425 Tel. 0039 011 670 6432 Email: [email protected]

Blood First Edition Paper, prepublished online May 6, 2011; DOI 10.1182/blood-2010-12-324756

Copyright © 2011 American Society of Hematology




2

Abstract

In phagocytes, GTPases of the Rac family control crucial antimicrobial functions. The

RacGAP ArhGAP15 negatively modulates Rac activity in leukocytes but its in vivo role in innate

immunity remains largely unknown. Here we show that neutrophils and macrophages derived

from mice lacking ArhGAP15 presented higher Rac activity but distinct phenotypes. In

macrophages, the loss of ArhGAP15 induced increased cellular elongation and membrane

protrusions but did not modify chemotactic responses. Conversely, the lack of ArhGAP15 in

neutrophils affected critical Rac-dependent antimicrobial functions, specifically causing

enhanced chemotactic responses, straighter directional migration, amplified ROS production,

increased phagocytosis, and improved bacterial killing. In vivo, in a model of severe abdominal

sepsis, these effects contributed to increase neutrophil recruitment to the site of infection, thereby

limiting bacterial growth, controlling infection spread, reducing systemic inflammation and

ultimately improving survival in ArhGAP15-null mice. Altogether, these results demonstrate the

relevance of ArhGAP15 in the selective regulation of multiple neutrophil functions, suggesting

that ArhGAP15 targeting might be beneficial in specific pathological settings like severe sepsis.




3

Introduction

Rac proteins are members of the Rho family of GTPases and are key mediators of

phagocyte functions, through their involvement in the control of migration to the site of

infection, phagocytosis and reactive oxygen species (ROS) production1. In neutrophils, Rac is

needed for proper gradient sensing2-4 and F-actin polymerization2 during migration. On the other

hand, in macrophages, Rac can alter cell morphology and mode of migration but is not essential

for chemotaxis5. In phagocytosis, Rac plays an important role in neutrophils6 and macrophages

by localizing to the phagosomal membrane and controlling the closure of the phagocytic cup7.

Rac also mediates phagocyte-mediated bacterial killing, through its ability to stimulate ROS

production3,6,8, as Rac can directly bind to p67phox, thereby inducing the activation of the

NADPH oxidase complex9. Consistent with these roles, human patients carrying a dominant

negative Rac mutation show defective neutrophil chemotaxis, reduced superoxide production and

increased susceptibility to infections10,11.

Like all GTPases, Rac proteins can oscillate between two states: the GDP-bound inactive

and GTP-bound active state. The switch between these two conditions is regulated by guanine

nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs): while GEFs

promote the exchange of GDP for GTP thus causing Rac activation, GAPs turn off Rac activity

by accelerating the hydrolysis of GTP. It is estimated that 0.5% of all predicted human genes

encode putative GAPs, thereby suggesting that these proteins have widespread and important

roles in GTPase regulation12; however, how RacGAPs regulate phagocyte functions remains

largely unknown.

In phagocytes the best characterized RacGAPs are Bcr and Abr, which have been found to

have overlapping roles. In particular, these proteins negatively control most of the Rac-

dependent functions in macrophages13, while, conversely, they only regulate specific activities in

neutrophils14,15. Nonetheless, whereas the presence of other RacGAPs in neutrophils has been

reported16,17, their activity has not been thoroughly investigated.

ArhGAP15 is a RacGAP activated by membrane recruitment triggered by docking, through

its PH domain, to the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3)18,19. Our

previous studies identified ArhGAP15 as protein expressed in macrophages19, but did not define




4

a specific role of this protein in phagocyte functions. To explore the role of this protein in vivo,

we generated ArhGAP15-deficient mice and found them to be viable and fertile. Analysis of

phagocyte functions showed that macrophages lacking ArhGAP15 exhibited an atypical

morphology but normal chemotaxis. Conversely, knockout neutrophils displayed multiple

alterations, including increased directional migration, phagocytosis, ROS production and

bacterial killing. Consistently, the improved neutrophil migration to the site of infection and the

subsequent control of bacterial colonization and dissemination protected ArhGAP15-null mice

from severe polymicrobial abdominal sepsis.




5

Materials and Methods

Details regarding the generation of the ArhGAP15 knockout mice, RT-PCR, Western blot analysis,

immunohistochemistry and FACS analysis are available in Supplementary data.

Cell purification

BMDMs were derived and used as previously described19. To isolate neutrophils, murine bone

marrows were re-suspended in saline +0.1% fetal calf serum (FCS) and then centrifuged (1100g, 30

min, 4°C) through discontinuous Percoll gradients (72%, 64% and 52%) in Hank’s buffered salt

solution (HBSS). Mature neutrophils were derived from the 64-72% interface and cell purity was

determined by flow cytometric analysis (FACS).

Rac GST-PAK Pull-down assay

A glutathione-S-transferase (GST)-PAK-CD (PAK-CRIB domain) fusion protein, containing the Rac

binding region from human PAK1 was used to determine Rac activity. Briefly, BMDMs and

neutrophils were stimulated with 20nM or 100nM C5a, respectively. Cell lysates were centrifuged at

4°C for 10 min at 13,000 rpm and the supernatant was incubated with GST-PAK glutathione-coupled

Sepharose 4B beads (Amersham Biosciences, Buckinghamshire, UK) for 30 min at 4°C. Proteins

bound to the beads were washed three times in lysis buffer, and the amount of bound proteins was

quantified by western blot analysis.

Immunofluorescence of Actin

BMDMs were plated on glass coverslips for 24 hrs then starved for 12 hrs prior to stimulation with

20nM C5a. Cells were incubated with phalloidin TRITC (Sigma, St. Louis, USA), followed by Hoechst

(Sigma, St. Louis, USA) to stain nuclei. Images were acquired with an Axio Observer Zeiss

microscope (Zeiss, Switzerland) equipped with a MRm Axiocam at a 63x magnification. MetaMorph

software (Meta Imaging Series Version 6.1, Universal Imaging Corporation, Downingtown, PA, USA)

was used to examine differences in macrophage morphology, including the elongation ratio,

automatically calculated by the software program as a ratio of the longest/shortest cellular cross-section




6

of each cell, and cellular perimeter, automatically calculated by the software program as the distance

around the outer edge of each cell. Over 60 macrophages per genotype were analyzed.

Knock-down of ArhGAP15 in the RAW 264.7 macrophage-like cell line was obtained using GAP15-1

and GAP15-2 shRNAmir sequences19. High titer lentiviral vector stock was produced as previously

described19. Stimulation, staining and morphological analysis was then performed as described for

BMDMs.

Migration assays

Chemotactic migration of BMDMs and neutrophils towards 100nM CXCL2 (Sigma, St. Louis, USA),

10μM fMLF (Sigma, St. Louis, USA), 50nM C5a (Sigma, St. Louis, USA) or culture medium

(negative control) was tested using Boyden chambers as previously described19. For chemokinesis

experiments, macrophages were seeded in the presence of 50nM C5a in both chambers. After

incubation at 37°C for 1 hr for BMDMs or 2 hrs for neutrophils, migration was quantified by counting

cells that migrated to the lower surface of the filter, on which five random fields were examined.

In vivo neutrophil migration toward the chemoattractants IL-8 and E.coli were tested as previously

described20. Directional migration was determined by the under-agarose method21. 1x106 neutrophils

were loaded to the right and 10pmol of C5a or 5pmol of CXCL2 was loaded to the left in two opposing

points in the agarose along the x-axis and incubated for 1 hr at 37oC. Gels were then transferred to a

Zeiss Axiovert 200M (Zeiss, Switzerland) inverted microscope equipped with a CCD cooled camera

(Princeton Instrument, USA), a heated enclosure (Incubator PM S1, Zeiss, Switzerland) and imaged

with a phase contrast 32x objective. Cells were filmed for 1 hr with one frame every 4 sec. Analysis

was performed using a purpose-made software (available at http://cbu.mbcunito.it/ctrack/), which

generated x-y coordinates for each cell at each time point, in order to calculate the chemotactic index

(C.I.) defined by the displacement along the x-axis (Δx) (migration distance towards the

chemoattractant) divided by the total migrated distance (Σdist). Analysis was determined on low cell

density fields to avoid pathway interference between cells; experiments were excluded from the

analysis, if manual inspection showed incorrectly reconstructed cellular paths. The elongation ratio of

migrating cells was analyzed during the first 5 min, examining one frame every 12 sec.

To assess the cellular ability to turn, similar under agarose experiments were performed except for a

third smaller well that was cut below the x-axis at an equal distance between the two original wells22.

Along the x-axis, 1x106 neutrophils were loaded to the right and 5pmol of CXCL2 was loaded to the

left. Gels were incubated for 1.5-2 h, allowing neutrophils to migrate into a target area, located between




7

the two wells in the x-axis. Subsequently, cell tracks were followed as described above. A baseline

recording was taken for 10 min, 5pmol of C5a was then added to the lower middle well, and the

cellular migration was recorded for an additional 50 min. The resulting time lapse image series were

then analyzed calculating the displacement along the y-axis (Δy) (migration towards C5a) divided by

the total migrated distance (Σdist).

ROS production, MPO release, phagocytosis and in vitro killing

Production of ROS was determined by priming isolated neutrophils (5x106/mL) with LPS (1μg/mL) for

1 hr and then pre-incubating them with luminol (20μg/mL) and horseradish peroxidase (35μg/mL) in

RPMI medium +10% FCS. Cells were subsequently stimulated with 2.5μM fMLF, 50nM C5a or IgG-

opsonized zymogen particles (ratio of 10:1 zymogen:neutrophil) (Invitrogen, Carlsbad, USA) and light

emission was recorded by a Berthold MicroLumat Plus luminometer (Berthold Technologies,

Hartfordshire, UK).

Myeloperoxidase (MPO) release was assessed by using tetramethylbenzidine based on a previously

established technique23 following stimulation with 2.5μmol cytochalasin B (Sigma, St. Louis, USA)

and 100nmol C5a, in the presence of 50U/mL of superoxide dismutase (Sigma, St. Louis, USA).

To assess phagocytosis, E.coli bacteria conjugated to Texas Red (Invitrogen, Carlsbad, USA) were

serum opsonized or IgG opsonized and mixed with neutrophils at a ratio of 10:1 (bacteria:neutrophil).

Phagocytosis was initiated by incubating the cells at 37°C for 1 hr, and was stopped by placing the

mixture on ice immediately after incubation. Cells were washed twice with cold PBS, fixed, and

stained for the neutrophil marker Gr-1 (BD Immunocytometry, San Jose, USA). Similarly phagocytosis

was assessed in macrophages using serum opsonized bacteria. Images were acquired with an Axio

Observer Zeiss microscope (Zeiss, Switzerland) equipped with an MRm Axiocam at a 63x or 40x

magnification for neutrophils and macrophages, respectively; mean fluorescence intensities (MFIs) for

Gr-1 positive populations were compared.

The ability of neutrophils to kill bacteria in vitro was assayed by incubating E.coli bacteria with

neutrophils (ratio of 100:1 bacteria:neutrophil) in HBSS +10% FCS +1mM Hepes for 1hr at 37°C.

Serial dilutions of the incubated mixture were plated on Luria-Bertani plates and incubated overnight at

37oC. Colony forming units (CFUs) were then counted.

Cecal ligation and perforation-induced sepsis




8

Mice were anesthetized with 100 mg/kg ketamine (Pfizer, Milan, Italy) and 5 mg/kg xylazine (Bayer

S.p.A., Milan, Italy) and randomized to either cecal ligation and perforation-induced sepsis (CLP) or

laparotomy (Sham) surgery. To induce severe sepsis, the cecum was isolated and punctured twice with

an 18 gauge needle, following which a small amount of fecal matter was extruded from each puncture

site. Sham animals received a laparotomy with no manipulation of the cecum. Animals were sacrificed

6 hrs following surgery, at which time the peritoneal cavity was washed with 5mL of sterile PBS+1mM

EDTA, the spleen was isolated and homogenized in sterile PBS+1mM EDTA, and blood serum was

collected and snap frozen for later analysis. Aliquots of the peritoneal lavage and spleen homogenate

were then serially diluted, plated on Muller-Hinton agar dishes and incubated at 37°C. CFUs were

counted after 24 hrs and results were expressed as the total CFU per peritoneal cavity or spleen.

Separate aliquots of the peritoneal lavage fluid were assessed for cellular influx by automated cell

counter Countess (Invitrogen, Life Technologies, Paisley, UK). Additionally, the percentage of

neutrophil influx was determined by FACS using the targeting antibody Gr-1. To assess systemic

inflammation, serum concentration of the cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF,

CXCL2, and IFNγ were measured using a commercially available BioPlex kit (Bio-rad, Hercules,

USA). In a separate cohort of animals, the lung, liver, kidney and ileum and cecum were collected after

18 hours of CLP, fixed in 4% paraformaldehyde overnight, embedded in paraffin, sectioned and stained

by hematoxylin and eosin, and assessed at 20x and 40x magnification by light microscopy on an

Olympus BH2-RFCA microscope equipped with a Leica DFC320 camera for images acquisition. The

overall pathophysiological effect was assessed by Kaplain-Meier analysis of survival over seven days,

during which mice were supplemented with fluids subcutaneously at 6 hrs and every subsequent 12 hrs

following surgery.

Statistical analysis

Values were presented as mean ± standard error of the mean (SEM). P-values were calculated using the

non-parametric two-tailed Mann–Whitney U-test, Student t test and one and two way ANOVA,

Wilcoxon test and Kaplain-Meier when appropriate (GraphPad Software, La Jolla, CA, USA). The

number of experiments is indicated by the n values and a p value of <0.05 was considered statistically

significant, where #= p<0.05, *= p<0.01, and **= p<0.001. Each experiment was independently

performed at least three times.




9

Results

Generation of ArhGAP15 knockout mice

To investigate the functional role of ArhGAP15 in vivo, a knockout mouse line was

generated (Figure S1A-B). The mutation disrupted the ArhGAP15 gene by insertion of a LacZ

and neomycin resistance cassette in the first coding exon (Figure S1A). Fertile homozygous

mice (ArhGAP15-/-) were obtained with the expected Mendelian ratios (Figure S1C) and did not

show any overt phenotype. While ArhGAP15 expression was confirmed in wild-type

macrophages and neutrophils, no ArhGAP15 mRNA and protein could be detected in either

ArhGAP15-/- spleen or phagocyte lineages, respectively (Figure 1A-B).

In ArhGAP15-/- mice, circulating neutrophils and monocyte/macrophages were decreased by

36±6% and 43±9%, respectively, while lymphocytes by 28±4% (Table I). Despite this effect, the

number of CD18 positive leukocytes as well as F4/80 positive macrophages in various organs,

including the liver, kidney and heart, was similar between wild-type and ArhGAP15-deficient

mice (Figure S2A-B and data not shown). In addition, analysis of surface markers such as CD88,

CD11b (MAC-1) and F4/80 in bone marrow-derived macrophages (BMDMs) and Gr-1 in bone

marrow-purified neutrophils were equally expressed in both mutant and control cells (Figure

S3A-D), suggesting that there was no effect of the loss of ArhGAP15 on phagocyte

differentiation.

Nonetheless, Rac1 activation after C5a stimulation in ArhGAP15-/- BMDMs and mutant

neutrophils was significantly higher than wild-types (Figure 1C-F), thus indicating that

ArhGAP15 is a crucial negative regulator of phagocyte Rac1 in response to GPCR agonists like

C5a.

Elongated morphology but normal migration in ArhGAP15-/- macrophages

In macrophages, Rac1 is thought to control membrane protrusions and cell shape5.

Consistently, in the absence of ArhGAP15, BMDMs developed a significantly higher number of

membrane protrusions than wild-type controls and showed a more elongated shape, either in the

absence or presence of C5a stimulation (Figure 2A). In agreement with these findings, mutant

BMDMs displayed an increased perimeter (Figure 2B) and a greater elongation ratio (Figure




10

2C). Conversely, these cells showed no difference with wild-type controls in chemotactic

responses induced by either MCP-1 or C5a (Figure 2D). To test if this lack of difference was due

to adaptation to chronic loss of ArhGAP15, similar experiments were performed in RAW 264.7

cells where ArhGAP15 was acutely down-regulated by a lentiviral-transduced siRNA

expression. In agreement with genetically modified BMDMs, RAW 264.7 expressing one of two

different shRNAmir sequences (shRNAmir GAP15-1 and shRNAmir GAP15-2) showed a

similar atypically elongated shape but normal migration in comparison to controls (Figure S4A-

B). These results thus indicate that, in macrophages, ArhGAP15 mediates cellular morphology

but not chemotactic responses.

Increased migration of ArhGAP15-/- neutrophils

In neutrophils, analysis of chemotactic responses in Boyden chamber assays revealed that

migration towards different chemo-attractants like C5a and fMLF, as well as chemokines like

CXCL2 were significantly higher in ArhGAP15-/- than wild-type controls (Figure 3A). Analysis

of CD88 and CXCR2 expression revealed equal expression in both mutant and control

neutrophils (Figure 3B), thus excluding their involvement in the observed differences of cell

migration.

To further test the role of ArhGAP15 as a negative regulator of neutrophil chemotaxis,

migration assays were performed in vivo following the administration of IL-8 in pre-formed

subcutaneous air pouches. After 4 hrs, neutrophil accumulation was significantly higher in

ArhGAP15-/- mice than in wild-type controls (Figure 3C). In addition, ArhGAP15-/- mice also

displayed increased neutrophil influx in vehicle-treated controls (Figure 3C), as well as in non-

treated air-pouches (data not shown), thus suggesting a stronger local response to air-pouch

induction.

Neutrophil recruitment was subsequently assessed 12hr following the peritoneal

administration of E.coli, demonstrating that the amount of recruited leukocytes was two-fold

higher in mutant than in wild-type peritoneal lavages (Figure 3D). Analysis of cellular sub-

populations showed that, while macrophages and lymphocytes responded equally in both

genotypes, neutrophils appeared three-fold more abundant in mutant than in wild-type mice, thus

accounting for differences in total cell numbers (Figure 3E).

Enhanced directional migration in ArhGAP15-/- neutrophils




11

To better address the specific aspects of neutrophil migration affected by the loss of

ArhGAP15, neutrophil chemotaxis was studied by time-lapse microscopy in under agarose

chemotactic assays. Results from these experiments showed that, 1h after exposure to C5a,

24±3% of ArhGAP15-null neutrophils reached the last horizon (blue dotted line) while only

13±2% of wild-type controls traveled the same space (Figure 4A), thus indicating that mutant

neutrophils migrated to a greater distance than wild-type controls. As migratory speed did not

reveal differences between the genotypes (ArhGAP15+/+: 11.45±3.44 μm/min, ArhGAP15-/-:

10.23±4.32 μm/min), tracks of migrating cells were further analyzed to test potential differences

in path convolution. This revealed that ArhGAP15-/- neutrophils migrated in straighter paths than

wild-type controls (Figure 4A-B). In agreement, quantification of the ratio between cell

displacement in the correct direction (Δx) and the total length of the migration path indicated that

the loss of ArhGAP15 reduces path tortuosity (Figure 4C). Similar results were obtained with

CXCL2 (Figure S5A-B). Of note, these experiments did not show any difference between

genotypes in the displacement in the y-axis, thus indicating that loss of ArhGAP15 does not

affect gradient sensing during migration.

To test if this increased directional migration was caused by differences in cell polarization,

the elongation ratio of these migrating cells was measured. Interestingly, ArhGAP15-null

neutrophils showed a limited but significant increase (3.3±0.1%) in cell polarization compared to

wild-type controls (ArhGAP15+/+: 1.519±0.014, ArhGAP15-/-: 1.571±0.019; p<0.05).

Next, cells moving towards a specific chemotactic cue were challenged by the addition of a

second perpendicular chemotactic gradient causing the bending of migratory paths. ArhGAP15-

deficient neutrophils, stimulated first with CXCL2 and subsequently with C5a, changed direction

significantly less than wild-type cells (Figure 4D, ArhGAP15+/+ video1 and ArhGAP15-/-

video2). Consistently, analysis of individual migratory paths showed that the ratio between the

displacement of a cell toward the second chemo-attractant (Δy) and the total length of its

migration path was significantly lower in ArhGAP15-/- neutrophils (Figure 4E), thus indicating

that the loss of ArhGAP15 leads to more persistent directional migration.

Increased phagocytosis, oxidase activation and bacterial killing in ArhGAP15-/- neutrophils

The finding that ArhGAP15 altered migration suggested that other Rac-dependent neutrophil

functions, such as phagocytosis and ROS generation, could be affected by the loss of this protein.

Measurement of in vitro phagocytosis revealed that ArhGAP15−/− neutrophils engulfed two-fold




12

more serum-opsonized E.coli cells than wild-type controls (Figure 5A-B). Conversely, no

difference was detected when bacteria were coated with IgG alone (Figure S6A) or when

phagocytosis of serum-opsonized particles was assessed in macrophages (Figure S6B).

On the other hand, superoxide production analysis revealed that while the kinetics of ROS

production was similar in both genotypes, the generation of oxygen species in response to either

fMLF or C5a showed a peak 3-fold higher in ArhGAP15-null neutrophils than in wild-type

controls (Figure 5C-D). In contrast, ROS production in response to IgG-zymosan was equal in

ArhGAP15 deficient and wild-type neutrophils (Figure S6C), thus further supporting a more

substantial involvement of ArhGAP15 in GPCR than in FcγR mediated signaling. In agreement

with increased ROS production after GPCR stimulation, C5a triggered in ArhGAP15 null

neutrophils significantly higher MPO release than in wild-type controls (Figure 5E).

Since previous studies have shown that phagocytosis, ROS production and azurophil granule

release are more dependent on Rac2 than on Rac1, the C5a-mediated activation of Rac2 was

determined in ArhGAP15-null neutrophils. Rac2 activity reached at 30 s higher levels in mutant

cells than in controls (Figure 5F-G) and presented a kinetics similar to that observed in ROS

production (Figure 5D). Enhanced Rac2 activity could thus explain the increased response

detected in mutant phagocytes and imply that ArhGAP15-deficient neutrophils may have

stronger bactericidal properties. In agreement with this hypothesis, in vitro killing of live E.coli

cells was found to be 42±7% higher in ArhGAP15-deficient neutrophils than in wild-type

controls (Figure 5H).

Protection of ArhGAP15-/- mice from severe, polymicrobial, abdominal sepsis

The enhancement of neutrophil functions caused by ArhGAP15 deficiency suggested that

blockade of this RacGAP may be beneficial in pathological conditions that critically depend on

rapid neutrophil recruitment, such as severe abdominal polymicrobial sepsis. To test this

hypothesis, mutant and wild-type mice were exposed to a model of cecal ligation and perforation

(CLP)-inducing severe sepsis. In this pathological setting, ArhGAP15-null mice showed in

sections of the cecum an increased neutrophil load (Figure S7A) and in the peritoneum a 3-fold

higher neutrophil recruitment than wild-type controls (Figure 6A), as well as a dramatic

reduction in peritoneal bacterial load (Figure 6B). Furthermore, the spleens of ArhGAP15-/-

septic mice contained significantly less bacteria than wild-type controls (Figure 6C), thus

indicating decreased bacterial decompartmentalization in the absence of ArhGAP15. Following




13

CLP, no differences were observed between genotypes in neutrophil recruitment to the lung,

liver, kidney or ileum (data not shown).

Septic injury and mortality are strongly associated with exaggerated systemic inflammation

and involve the increased production of multiple cytokines and chemokines24. Interestingly,

serum concentrations of cytokines known to be involved in the septic response such as IL-1β, IL-

2, IL-4, IL-5, IL-6, CXCL2, IL-10, GM-CSF and INF-γ were significantly lower in ArhGAP15-

null than wild-type mice following CLP (Figure 6D, Figure S7B). Consistently, while all wild-

type controls died within 48 hrs after CLP, 40% of ArhGAP15-deficient mice survived more

than 7 days (Figure 6E). Overall, these results demonstrate that, in severe sepsis, the loss of

ArhGAP15 significantly reduces local infection as well as systemic inflammation, ultimately

improving survival.




14

Discussion

The human genome is predicted to encode 60-70 RhoGAPs25, far exceeding the 22 genes

encoding Rho family GTPases26. The excess of RhoGAPs over their target Rho GTPases

suggests that each RhoGAP may selectively regulate a specific Rho GTPase signaling pathway

activated during defined cellular events12. ArhGAP15 is a RhoGAP with in vitro specificity

towards Rac18 that is regulated by means of membrane recruitment through its PH domain18,19.

While the expression of ArhGAP15 has been reported in inflammatory cells19, its in vivo

function has so far remained unclear. In this study, the effects of the lack of ArhGAP15 were

analyzed in macrophages and neutrophils derived from ArhGAP15-deficient mice.

Despite the potential presence of numerous other RacGAPs in phagocytes, ArhGAP15 was

found to play a non-redundant role in controlling Rac deactivation in both macrophages and

neutrophils. In resting conditions, Rac1 activity was elevated in macrophages but not in

neutrophils, suggesting a different regulation of Rac1 in the two cells types. In response to

GPCR agonists like C5a, both mutant macrophages and neutrophils showed a higher peak of

Rac1 activity. These observations thus indicate that, in neutrophils but not in macrophages,

ArhGAP15 down-regulates Rac1-GTP only in response to specific stimuli.

Alteration of Rac activity is thought to differently affect functions of distinct phagocyte

populations27. In agreement, our findings showed that the loss of ArhGAP15 causes less severe

phenotypes in macrophages than in neutrophils. For example, in BMDMs, ArhGAP15 deficiency

only triggered abnormal elongated morphology. This is consistent with the previously

established role of Rac in controlling macrophage cell shape5 and similar to what was detected in

macrophages concomitantly lacking the two RacGAPs Abr and Bcr13. As the single loss of either

of these two molecules fails to induce an effect, our results point to a more dominant role of

ArhGAP15 in this process. Consistently, ArhGAP15 and these other two RacGAPs may

modulate parallel pathways eventually converging in controlling macrophage morphology.

Nonetheless, the effect of the loss of ArhGAP15 on cell shape did not significantly alter the

ability of macrophages to migrate towards a chemotactic gradient. Consistent with this

observation, neither the knockdown of ArhGAP1519 nor the loss of Rac proteins5 affect

macrophage chemotaxis. Although the absence of the RacGAPs Abr and Bcr in double-knockout

macrophages is reported to cause increased cell migration13, our results reinforce the view that

Rac proteins are not crucial for macrophage chemotaxis and raise the possibility that the effects




15

seen in Abr/Bcr-deficient cells are due to mechanisms potentially independent from the

modulation of Rac activity.

In further agreement with the notion of different Rac functions in specific phagocyte

populations, the absence of ArhGAP15 in neutrophils severely affected their responses to

inflammatory mediators and infectious agents. Our results showed that ArhGAP15 plays a

crucial negative role in the control of chemotaxis in vitro and in vivo. Previous observations

support the model that, in neutrophils, active Rac accumulates at the front of the cell through a

positive feedback involving the PI3K-product PIP3, thus locally driving leading edge formation,

cell polarization and subsequent directional migration28-30. By down-modulating Rac activity,

ArhGAP15 might function as a negative regulator of this feedback. Through PIP3-mediated

membrane recruitment18,19, ArhGAP15 could locally block Rac activity, destabilize the leading

edge and negatively influence directional migration. In support of this hypothesis, our results

showed that the increased chemotaxis of ArhGAP15-null neutrophils results from the combined

effects of a series of different events, including increased polarization and improved straightness

in the migratory path. However, it is possible to envisage that, while the absence of ArhGAP15

improves the efficacy of chemotactic migration; its loss might also come at a cost to other

neutrophil responses. Supporting this hypothesis, the lack of ArhGAP15 reduced the ability of

cells to modify their trajectory. Indeed, while ArhGAP15-null neutrophils showed more efficient

migration towards a single chemoattractant source, their ability to change direction in response to

multiple chemotactic agonists appeared significantly impaired. Therefore, the inhibitory function

of ArhGAP15 on Rac may be essential to provide neutrophils with the plasticity required to

finely regulate and/or alter their directional movements as well as prioritize various chemotactic

cues.

Of note, the absence of ArhGAP15 not only affected neutrophil migration but also other

critical antimicrobial activities of these cells, such as phagocytosis and respiratory burst, thus

suggesting that this RacGAP is a master regulator of neutrophil function. ArhGAP15-null

neutrophils displayed increased phagocytosis of serum-opsonized bacteria, indicating that a

single RacGAP can affect neutrophil-mediated phagocytosis. Indeed, the lack of Bcr is not

sufficient to alter neutrophil-mediated bacterial ingestion15. Nonetheless, when Ig-opsonization

was used to trigger phagocytosis, the absence of ArhGAP15 did not cause an abnormal response.

Although our results cannot exclude the possibility that, besides immunoglobulins, other tyrosine

kinase signaling agonists present in the serum might activate ArhGAP15, this result suggests that




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ArhGAP15 is more substantially activated downstream of GPCRs. Nonetheless, the finding that

ArhGAP15 can block phagocytosis in a more physiological response to serum-opsonized

bacteria is in keeping with the observation that bacterial toxins can mimic RhoGAP activity to

reduce their internalization and subsequent killing by neutrophils. For example, ExoS and ExoT,

two toxins from Pseudomonas aeuriginosa, possess RacGAP activity that blocks phagocytosis

by disrupting RhoGTPase-mediated actin rearrangement31.

In neutrophils, the main Rac isoform involved in phagocytosis is Rac26 and in agreement

with increased bacterial ingestion, ArhGAP15-deficient neutrophils showed enhanced GPCR-

induced Rac2 activity. Rac2 is also critically required for NADPH oxidase complex assembly

and consequent ROS production3,9. Consistent with ArhGAP15 being a negative regulator of

Rac2, the lack of this protein caused a significant increase in ROS production after stimulation of

different GPCRs but interestingly not of FcγR. This reinforces the view that, within the RacGAP

family, ArhGAP15 possesses unique signaling properties mainly involving Rac2 modulation

after GPCR signaling, a concept further supported by the observation that C5a stimulation led to

the enhancement of azurophile granule release, a process known to depend on Rac223. Overall, as

GPCR engagement is typically occurring in vivo in response to invading pathogens, our results

explain the increased ability of mutant neutrophils to kill bacteria in vitro.

Bactericidal activity of neutrophils following recruitment to the site of infection is critical

in conditions like severe polymicrobial sepsis, where systemic spread of a localized bacterial

contamination becomes life threatening. In this setting, insufficient neutrophil recruitment and

bacterial elimination has been shown to increase mortality32. Therefore, it could be envisaged

that enhancing polymorphonuclear leukocyte functions, as observed in the absence of

ArhGAP15, may be beneficial. In line with this hypothesis, in a model of polymicrobial

peritoneal sepsis, induced by severe cecal ligation and perforation, the absence of ArhGAP15

increased neutrophil migration to the site of infection, decreased bacterial growth and

decompartmentalization, lowered systemic inflammation, and ultimately improved survival at

one week. Although this is in apparent contrast to the increased mortality observed in Abr/Bcr

double knockout mice at 48 hours following mild sepsis14, several reasons could account for this

discrepancy. Firstly, the functions of ArhGAP15 and Abr/Bcr likely do not completely overlap

due to structural differences, such as the additional presence, in both Abr and Bcr, of a DH motif

and a PDZ-binding domain. Secondly, there are significant differences reported between mild

CLP, used in the Abr/Bcr study, and severe CLP, used in this study, with survival and the pro-




17

inflammatory response varying depending on the point of ligation, size of puncture, number of

punctures, fluid resuscitation, and antibiotic treatment33. Thirdly, while both studies show

heightened neutrophil activity, the loss of ArhGAP15 in sepsis improved recruitment to the site

of infection (cecum and peritoneal cavity), thereby decreasing bacterial decompartmentalization

and systemic inflammation, but had no effect on recruitment to distal organs (lung, liver, kidney,

ileum), suggesting that decreased systemic inflammation is the dominant cause of improved

survival. In contrast, the loss of Abr/Bcr increases unwanted migration to distal organs resulting

in unwarranted inflammation, organ damage and death.

In conclusion, this study shows that ArhGAP15 plays a non-redundant role as a regulator of

multiple neutrophil functions, providing a balance between anti-microbial activities and allowing

the versatility to respond to distinct chemotactic signals. Therefore, it is tempting to speculate

that in pathological conditions with a severe focalized infection, where neutrophil-mediated anti-

bacterial activity is imperative, ArhGAP15 may be beneficial as a novel therapeutic target.




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Acknowledgements

The authors have no conflicting financial interests. This work was supported by AIRC, Piedmont

Region, Fondation Leducq, the European Union Sixth Framework Program EUGeneHeart and

Fondazione Cariplo 2009-2455.

Authorship contribution

C.C., G.G., E.M. designed and performed research, analyzed data, and wrote the manuscript; I.M.,

E.B., S.M. and O.A. performed research and analyzed data; M.R. and F.A. analyzed data; E.H.

designed research and wrote the manuscript.

Disclosure of conflicts of interest

No potential conflicts of interest were disclosed.




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20. Hirsch E, Katanaev VL, Garlanda C, et al. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science. 2000;287(5455):1049-1053. 21. Heit B, Kubes P. Measuring chemotaxis and chemokinesis: the under-agarose cell migration assay. Sci STKE. 2003;2003(170):PL5. 22. Heit B, Robbins SM, Downey CM, et al. PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils. Nat Immunol. 2008;9(7):743-752. 23. Abdel-Latif D, Steward M, Macdonald DL, Francis GA, Dinauer MC, Lacy P. Rac2 is critical for neutrophil primary granule exocytosis. Blood. 2004;104(3):832-839. 24. Bozza FA, Salluh JI, Japiassu AM, et al. Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis. Crit Care. 2007;11(2):R49. 25. Bernards A. GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila. Biochim Biophys Acta. 2003;1603(2):47-82. 26. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247-269. 27. Costa C, Germena G, Hirsch E. Dissection of the interplay between class I PI3Ks and Rac signaling in phagocytic functions. ScientificWorldJournal;10:1826-1839. 28. Niggli V. A membrane-permeant ester of phosphatidylinositol 3,4, 5-trisphosphate (PIP(3)) is an activator of human neutrophil migration. FEBS Lett. 2000;473(2):217-221. 29. Wang F, Herzmark P, Weiner OD, Srinivasan S, Servant G, Bourne HR. Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils. Nat Cell Biol. 2002;4(7):513-518. 30. Weiner OD, Neilsen PO, Prestwich GD, Kirschner MW, Cantley LC, Bourne HR. A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity. Nat Cell Biol. 2002;4(7):509-513. 31. Deng Q, Barbieri JT. Modulation of host cell endocytosis by the type III cytotoxin, Pseudomonas ExoS. Traffic. 2008;9(11):1948-1957. 32. Alves-Filho JC, de Freitas A, Spiller F, Souto FO, Cunha FQ. The role of neutrophils in severe sepsis. Shock. 2008;30 Suppl 1:3-9. 33. Rittirsch D, Hoesel LM, Ward PA. The disconnect between animal models of sepsis and human sepsis. J Leukoc Biol. 2007;81(1):137-143.




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Table I

Blood parameters in ArhGAP15+/+ and ArhGAP15-/- mice.

Data are expressed as mean ± SEM with n=15 in each group.

Parameter ArhGAP15+/+ ArhGAP15-/-

Hematocrit (%) 42.88±0.81 40.21±1.19 WBC (103/μl) 5.63±0.49 3.81±0.34*

Neutrophils (103/μl) 1.07±0.11 0.68±0.09* Lymphocytes (103/μl) 4.0±0.37 2.89±0.31* Monocytes (103/μl) 0.17±0.03 0.097±0.01* Eosinophils (103/μl) 0.14±0.02 0.10±0.01 Basophils (103/μl) 0.014±0.003 0.009±0.002 Platelets (103/μl) 1145±49 998±28*




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Figure legends

Figure 1. ArhGAP15 regulates Rac activity in phagocytes A) Quantitative RT-PCR analysis of

ArhGAP15 expression in spleens derived from ArhGAP15+/+ and ArhGAP15−/− mice, n=3/genotype.

B) Representative detection of ArhGAP15 western blot from macrophage (MØ) and neutrophils

(NØ) of ArhGAP15+/+ (WT) and ArhGAP15-/- (KO) mice. Only the lower band, exclusively present

in WT lanes, corresponds to the correct size of ArhGAP15. Equal loading was monitored by

vinculin expression. n=3 C-F) Determination of Rac1 activation following C5a stimulation in

macrophages (C-D) and neutrophils (E-F). Representative detection of active (Rac1-GTP) and total

Rac1 are shown. Normalization was obtained by setting the percent stimulation of ArhGAP15+/+

cells after 1 min for macrophages and after 5 sec for neutrophils to 100%; at each point,

n=8/genotype.

Figure 2. Loss of ArhGAP15 affects macrophage morphology but not migration. A)

Representative images of ArhGAP15+/+ and ArhGAP15-/- BMDMs were stimulated with C5a and

stained for F-actin (red) and nuclei (blue). B-C) Perimeter analysis and elongation ratio of

ArhGAP15+/+ and ArhGAP15-/- BMDMs before and after C5a stimulation (n>60 cells/genotype).

D) Boyden chamber chemotaxis and chemokinesis analysis of ArhGAP15+/+ and ArhGAP15-/-

BMDMs, n=3/genotype.

Figure 3. Loss of ArhGAP15 causes increased in vitro and in vivo neutrophil migration. A)

Boyden chamber chemotaxis analysis of ArhGAP15+/+ and ArhGAP15-/- neutrophils, n=5/genotype.

B) Representative FACS analysis of neutrophil CD88 and CXCR2 expression, n=3/genotype. C)

Neutrophil recruitment to air pouches after saline (control) or IL-8 injection, n=6/genotype. D-E)

Peritoneal total cell recruitment and cellular subpopulation (MØ macrophages, NØ neutrophils and

LY lymphocytes) analysis following i.p. administration of 1x107 CFU of E.coli, n=14/genotype.

Figure 4. ArhGAP15 null neutrophils showed increased directional migration but decreased

turning ability A-E) Under-agarose migration experiments A) Center-zeroed tracks of

ArhGAP15+/+ and ArhGAP15-/- neutrophils migrating toward C5a (left). B) Single cell tracks of a




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typical ArhGAP15+/+ and ArhGAP15-/- neutrophil. C) Measurement of chemotactic index towards

C5a (Δx) D) Center-zeroed tracks of ArhGAP15+/+ and ArhGAP15-/- neutrophils migrating from a

primary CXCL2 gradient in the x-axis (left) towards a secondary C5a gradient in the y-axis

(bottom) E) Measurement of chemotactic index towards C5a (Δy). For each experiment n=4

movies/genotype.

Figure 5. ArhGAP15 regulates neutrophils-mediated phagocytosis, ROS generation and in vitro

bacterial killing. A) Representative images of E.coli (red) phagocytosed by neutrophils (Gr-1

staining in green), B) Quantification of Texas Red fluorescence (E.coli) in ArhGAP15+/+ and

ArhGAP15−/− neutrophils, n>100 cells/genotype. C-D) ROS generation by neutrophils induced by

(C) 2.5μM fMLF or (D) 100nM C5a, n=5/genotype. E) Quantification of MPO release by

ArhGAP15+/+ and ArhGAP15−/− neutrophils, n=4/genotype. F-G) Determination of Rac2 activation

following C5a stimulation in neutrophils. Representative detection of active (Rac2-GTP) and total

Rac2 are shown. Normalization was obtained by setting the percent stimulation of ArhGAP15+/+

neutrophils after 15 sec for to 100%, n=5/genotype. H) In vitro killing of E.coli by isolated

ArhGAP15+/+ and ArhGAP15−/− neutrophils, n =4/genotype.

Figure 6. ArhGAP15 null mice are protected against CLP-induced severe sepsis. A) Number of

neutrophils recruited to the peritoneal cavity following CLP, n=7/genotype. B) Bacterial load of

peritoneal lavage, n=7/genotype. C) Bacterial load of isolated spleens, n=7/genotype. D) Serum

cytokine concentrations 6 hrs following CLP, n=7/genotype. E) Kaplan-Meier survival analysis of

ArhGAP15+/+ and ArhGAP15−/− mice following CLP-induced sepsis, n=7/genotype.




The RacGAP ArhGAP15 is a master negative regulator of neutrophil functions

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