jiy589.pdf - Oxford Academic

Heparin-Binding Protein Release Is Strongly Induced

by Leptospira Species and Is a Candidate for an Early

Diagnostic Marker of Human Leptospirosis

Mônica L Vieira, Sandra Persson, Mônica Lopes-Ferreira, Eliete C Romero, Karin

Kirchgatter, Ana Lucia T O Nascimento, Heiko Herwald

Dow

nloaded from https://academ

ic.oup.com/jid/article/219/6/996/5123686 by guest on 28 July 2022

http://akrurl.com/.2zpmo

The Journal of Infectious Diseases

996 • JID 2019:219 (15 March) • Vieira et al

Heparin-Binding Protein Release Is Strongly Induced by Leptospira Species and Is a Candidate for an Early Diagnostic Marker of Human LeptospirosisMônica L. Vieira,1,2 Sandra Persson,1 Mônica Lopes-Ferreira,3 Eliete C. Romero,4 Karin Kirchgatter,5 Ana Lucia T. O. Nascimento,2 and Heiko Herwald1

1Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden; and 2Laboratório Especial de Desenvolvimento de Vacinas and 3Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, 4Centro de Bacteriologia, Instituto Adolfo Lutz, and 5Nucleo de Estudos em Malária, Superintendência de Controle de Endemias, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil

Background. Leptospirosis, caused by spirochetes of the genus Leptospira, is one of the most widespread zoonoses worldwide. Efficient diagnostic methods for early diagnosis of leptospirosis are still lacking, and acute disease presents with nonspecific symp-tomatology and is often misdiagnosed. The leptospires pathogenic processes and virulence mechanisms remain virtually unknown. In severe infections, hemostatic impairment is frequently observed, and pathophysiological complications often develop when the host response is modulated by the pathogen. The neutrophil heparin-binding protein (HBP) is an inflammatory mediator and potent inducer of vascular leakage.

Results. In this study, we found that leptospires and their secreted products induce the release of HBP from stimulated neutro-phils through a controlled degranulation mechanism. We acknowledged 2 leptospiral proteins as able to induce HBP degranulation. These findings have clinical implications, as high levels of HBP were detected in serum from patients with leptospirosis, especially at the early phase of the disease.

Conclusion. In conclusion, we describe a new mechanism by which the leptospirosis pathophysiological complications may arise, such as vascular leakage and edema formation. We also propose HBP as a new early screening biomarker for human leptospirosis.

Keywords. Leptospira; inflammation; infection; neutrophils; host-pathogen-interactions.

Leptospirosis, caused by the pathogenic spirochete Leptospira, is one of the most prevalent zoonotic diseases. After entering the host via skin or mucosa, leptospires rapidly disseminate through the bloodstream. Although the pathogen is immedi-ately cleared in the circulation, leptospires can disseminate into the surrounding tissues. Infections may vary from nonsymp-tomatic to severe forms characterized by jaundice, hypotension, acute lung injury, hemorrhages, and multiorgan failure [1–4]. The pathophysiological mechanisms responsible for leptospi-rosis symptomatology are poorly understood, although the host immune response and inflammation seem to be main determinants.

Heparin-binding protein (HBP), also known as azuroci-din or cationic antimicrobial protein (CAP37), is an inac-tive member of the serine protease family [5]. HBP is stored in the secretory and azurophilic granules of neutrophils and can be released upon neutrophil activation [6]. While initially

investigated for its antimicrobial activity, HBP has been recog-nized as an important multifunctional inflammatory mediator. It is secreted from neutrophils upon contact with the capillary endothelium, inducing cytoskeletal rearrangements of endo-thelial cells, which leads to disruption of cell barriers and an increased vascular permeability [7]. Also, it is able to recruit and activate monocytes and macrophages [8–10], enhance monocyte cytokine production [11], mobilize T cells [12], and induce morphological changes and aggregation in endothelial cells and fibroblasts [13].

HBP has been shown to be mobilized from polymorphonu-clear neutrophils (PMNs) in the presence of bacteria or their supernatants [14–17]. Clinical studies revealed the release of HBP in many bacterial diseases, and this is considered as an important marker for severe infections, especially sepsis [18–23].

Because of the pathophysiological signs of severe leptospi-rosis, we hypothesized that HBP release could be involved in disease progression. We show that leptospires and their secreted products induce massive HBP release from human PMNs, and we recognize Lsa63 and LipL45 as leptospiral proteins able to trigger HBP degranulation and increase endothelial perme-ability in vitro. Our data further demonstrate that Lsa63 has inflammatory effects in vivo. Moreover, we reveal that serum samples from individuals with leptospirosis contain elevated levels of circulating HBP. Our findings help to explain the

M A J O R A R T I C L E

© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: [email protected]: 10.1093/infdis/jiy589

Received 19 June 2018; editorial decision 24 September 2018; accepted 6 October 2018; published online October 8, 2018.

Correspondence: M. L. Vieira, Laboratório Especial de Desenvolvimento de Vacinas, Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, Sao Paulo, SP, Brazil ([email protected]; [email protected]).

The Journal of Infectious Diseases® 2019;219:996–1006

Dow



mailto:[email protected]?subject=

mailto:[email protected]?subject=

Leptospira Induces HBP Release • JID 2019:219 (15 March) • 997

physiopathology of the disease and suggest that HBP can be used as a new biomarker for early leptospirosis screening.

METHODS

Reagents

Recombinant human HBP and rabbit antiserum (410A) to recombinant HBP were prepared as described elsewhere [8, 24]. Genistein, wortmannin, SB203580, AG1478, GF109203, H-89, PD98059, and U73122 were obtained from R&D Systems. BAPTA-AM, EGTA, and E-64 were obtained from Sigma Aldrich. Antibody IB4 was obtained from Santa Cruz Biotechnology. H-1998 was obtained from Bachem.

Leptospirosis Serum Samples

Confirmed leptospirosis human samples were obtained from the Instituto Adolfo Lutz sera collection (São Paulo, Brazil). We used paired samples collected from the same patients at the early phase (defined by negative results of a microscopic agglutination test [MAT]) and convalescent phase (defined by positive results of a MAT) of leptospirosis. The MAT was per-formed according to the protocol published by Faine et al [1] (Supplementary Materials).

Ethics Statement

The patients’ serum samples were donated for research pur-poses, and their use in this study was waived from requiring official ethics approval by the Ethical Committee for Human Research of Universidade de Sao Paulo, as the study does not involve procedures regulated by the Brazilian National Commission for Research Ethics (resolution 466/2012). We had no access to patient data. Informed consent was obtained from all healthy donors. Animal experimentation was approved by the Butantan Institute’s Ethics Committee on Animal Use (São Paulo; protocol CEUAIB 7885070417).

Bacterial Strains and Culture Conditions

Virulent Leptospira interrogans serovar Copenhageni strain L1-130, culture-attenuated L. interrogans serovar Copenhageni, and saprophytic Leptospira biflexa serovar Patoc were kindly donated by Dr Mathieu Picardeau (Institute Pasteur, Paris, France). Leptospires were cultured as previously described [25]. Virulent leptospires were used up to the third passage.

Leptospiral Recombinant Proteins

Proteins were expressed and purified as previously reported [26, 27].

HBP Release in Whole Blood

Blood specimens were collected from healthy volunteers in tubes containing sodium citrate. Bacteria were washed, and 1 × 108 leptospires/mL (in HEPES buffer) were added to the same volume of human blood. Alternatively, culture superna-tants were used. Buffer alone or fresh culture medium were

used as controls for cells or supernatant as stimuli, respectively. Samples were incubated at 37°C for different times (0, 0.5, 1, 2, or 4 hours), following centrifugation (at 1000 ×g for 20 minutes) to remove blood cells. The HBP content of the supernatants was determined as previously described [6].

To assess the signal transduction pathways involved in the HBP release by neutrophils, inhibitors were mixed with blood 5 minutes before the addition of L1-130 cells or culture super-natants. Stimulation was performed for 1 hour at 37°C, cells were removed by centrifugation, and the HBP concentrations were determined. The HBP contents of samples incubated with Leptospira cells or supernatants alone were considered 100% HBP release for graph normalization. The inhibitor concentra-tions are shown in Supplementary Table 1.

When membrane protein fractions or recombinant proteins were used as stimuli for HBP release, equal volumes (containing 0.1, 1, and 10 µg/mL) were mixed with blood and incubated for 1 hour at 37°C. Blood stimulated with phosphate-buffered saline (PBS) served as a negative control, while 0.5% Triton X-100 was used as a control indicative of 100% HBP release, owing to cell lysis.

Leptospiral Membrane Protein Isolation and Purification

L. interrogans Copenhageni L1-130 membrane proteins were isolated by the detergent Triton X-114 (TX-114) method as previously detailed [26]. The TX-114 soluble phase was frac-tionated by anion exchange chromatography in a HiTrap Q HP column (GE Healthcare), eluted by increasing NaCl concentra-tions, as described elsewhere [26].

Superoxide Anion (O2−) Production Assay

Human neutrophils were purified from citrated blood by using Lymphoprep density gradient media (Axis-Shield). A total of 5 × 105 cells/100 µL were seeded into 96-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium. Neutrophils were labeled by adding 100 µL of DCF-DA (Sigma-Aldrich) to a final concentration of 100 μM, after which they were incubated at 37°C for 20 minutes while protected from light. Negative controls received only culture medium. Cells were centrifuged (at 1000 ×g for 5 minutes) and the supernatants disposed. The remaining neutrophils were stimulated with 100 µL of RPMI 1640 medium containing 25 nM PMA as a positive control for neutrophil activation, L. interrogans Copenhageni L1-130 (1 × 108 or 1 × 107 cells/mL), or no additions. Fluorescence (excitation, 485 nm; emission, 530 nm) was measured every 15 minutes for 1 hour. Supernatants were stored at −20°C until analysis by the lactate dehydrogenase (LDH) cytotoxicity assay and Western blot.

LDH Cytotoxicity

The test was performed by a kit, according to the manufacturer instructions (Pierce; Supplementary Materials).

Dow



http://academic.oup.com/jid/article-lookup/doi/10.1093/infdis/jiy589#supplementary-data


HBP Western Blot

Supernatants obtained from the purified neutrophils stimulated for the O2

− assay were separated by 10% sodium dodecyl sul-fate polyacrylamide gel electrophoresis, transferred onto mem-branes, and blocked for 2 hours at room temperature with PBS plus 0.05% Tween 20 containing 2% bovine serum albumin. Membranes were incubated overnight at 4°C with antibodies against human HBP (410A) and, after a washing step, with sec-ondary antibodies.

Endothelial Permeability Assay

The in vitro vascular permeability assay used in this study was purchased from Millipore. Neutrophils (1 × 106 cells in 0.7 mL) were stimulated for 1 hour at 37°C with 5 µg/mL leptospiral recombinant proteins in RPMI 1640 medium supplemented with 10% plasma. Supernatants were used in the permeability assay. EA.hy926 cells were cultured until confluence on rat-tail collagen 1–coated culture inserts (pore diameter, 1 µm) in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum (FBS). Inserts were washed in PBS and transferred to 24-well plates. Supernatants from previously stimulated neu-trophils or RPMI 1640 medium plus 10% FBS (Control and NCM = no cell monolayer, inserts without cells) were added into the inserts (100 µL) and 500 µL of culture medium to receiver wells, incubated for 24 hours at 37°C in 5% CO2. Inserts were aspirated and transferred into new 24-well plates; 500 µL of RPMI 1640 medium plus 10% FBS was added to the receiver wells, 150 µL of FITC-dextran in RPMI 640 medium plus 10% fetal bovine serum was added on top, and cells were incubated for 20 minutes at room temperature while protected from light. The volume of FITC-dextran that passed through the mono-layer was measured by a fluorescence assay (excitation, 485;

emission, 535 nm). Cell culture medium without FITC-dextran was included to obtain the background value.

Intravital Microscopy

The dynamics of alterations in the microcirculatory network of the cremaster muscle of BALB/c mice were determined as detailed in the Supplementary Materials and previously described [28]. Recombinant proteins (150 or 75 µg in 30 µL of PBS) or PBS alone were superfused onto the exposed cremaster muscle. The muscle was then observed for up to 30 minutes by means of an inverted microscope, to determine the number of rolling and adherent leukocytes in the venule and to observe arteriole dynamics.

Statistics

Comparisons between 2 groups were performed by the Student t test, whereas comparisons among multiple groups were per-formed by 1‐way analysis of variance. P values were 2‐sided, and a P < .05 was considered statistically significant. All data are expressed as means ± standard deviation.

RESULTS

Leptospira and Their Secreted Products Strongly Induce HBP Release in

Human Blood

The only blood cells that have been reported to produce HBP are PMNs [29]. Thus, we incubated leptospires and culture super-natants with human blood to investigate their ability to stim-ulate HPB release from PMNs. Both virulent and attenuated L. interrogans serovar Copenhageni and L. biflexa serovar Patoc were able to induce massive and time-dependent HPB release (Figure 1A). No significant differences were observed between the strains tested. Similar results were obtained when culture

800

600

400

200

0

A

HB

P le

vel (

ng/m

L)

Cells

L. inter

rogan

s L1-1

30

L. inter

rogan

s

L. bifle

xaBu�

er

0 h0.5 h1 h

4 h2 h

0 h0.5 h1 h

4 h2 h

1000

800

600

400

200

0

B

HB

P le

vel (

ng/m

L)

Supernatants

L. inter

rogan

s L1-1

30

L. inter

rogan

s

L. bifle

xa

EMJH

Figure 1. Heparin-binding protein (HBP) is released in response to Leptospira cells and culture supernatants. Virulent Leptospira interrogans serovar Copenhageni strain L1-130, culture-attenuated L. interrogans, or saprophytic Leptospira biflexa (108 cells/mL in HEPES buffer [A] or its secreted products [B]) were mixed (1:1 v/v) with human blood and incubated for different time points at 37°C, under gentle agitation. After removal of the cells by centrifugation, the HBP content in the resulting supernatants was determined by capture enzyme-linked immunosorbent assay. Standard curves using purified HBP were used for quantification. Buffer alone or fresh culture medium (EMJH) were used as controls. The bars represent means ± standard deviations of 3 independent experiments performed in duplicate.

Dow




supernatants were used (Figure 1B). The data suggest that the release of HBP from neutrophils is triggered by leptospiral agent(s) that seem to be surface associated and/or secreted.

Leptospires Stimulate HBP Release by Promoting PMN Degranulation

PMNs can release their contents by cell lysis or through con-trolled degranulation pathways [30]. To test the mechanism driving the observed HBP release, we evaluated the viability of bacteria-treated PMNs by means of their ability to generate O2

− and secrete LDH. PMNs treated with different concentrations of L. interrogans Copenhageni L1-130 did not affect O2

− production (Figure 2A), whereas HBP release was dependent on Leptospira dose (Figure 2B), suggesting that PMNs remain viable when treated with the bacteria. PMA and RPMI 1640 medium stimula-tion induced only minimal HBP release from PMNs (Figure 2B).

LDH is a cytosolic enzyme that is released into extracel-lular space when the plasma membrane is damaged [31]. Corroborating the maintenance of PMN viability upon Leptospira stimulation, we found that findings of the LDH cyto-toxicity assay did not differ between bacteria-treated cells and controls (ie, cells treated with PMA or RPMI 1640 medium alone; Figure 2C).

Release of HBP From Human PMNs Is Modulated by Signal Transduction

Mediators

Knowing that Leptospira-induced HBP release is not due to cell lysis, we next investigated the underlying molecular mechanisms leading to its mobilization. To this end, the effect of a number of signal transduction inhibitors was analyzed (Supplementary Table 1). We found that genistein (a tyrosine kinase inhibi-tor), wortmannin (a PI3K inhibitor), SB203580 (a p38 MAPK inhibitor), AG1478 (an EGF receptor tyrosine kinase inhibi-tor), and GF109203 (a protein kinase C inhibitor) markedly reduced HBP secretion in response to both L. interrogans

Copenhageni L1-130–infected cells and culture supernatants. H-89 (a cAMP-dependent protein kinase inhibitor), PD98059 (a MAPK pathway inhibitor), and U-73122 (a phospholipase C inhibitor) did not interfere with the secretion of HBP (Figure 3 and Supplementary Table 1). Together, the results suggest (i) that the release of HBP in human blood is induced by leptospires and their secreted products, (ii) that the HBP release is dependent on the interaction with a receptor like structure at the neutrophil surface, and (iii) that the mechanism involves downstream PI3K and p38 MAPK intracellular signaling. More than 1 mechanism is likely to be involved in HBP release, as various signal trans-duction inhibitors exerted effect.

Leptospira-Induced HBP Release Is Ca2+ Dependent

An increase in cytosolic Ca2+ promotes PMN degranulation by mobilizing granules and cytoskeleton rearrangement [32]. To study the effect of intracellular and extracellular calcium on Leptospira-induced HBP degranulation, BAPTA-AM (intracellular calcium) and EGTA (extracellular calcium) were added to the blood. These treatments inhibited the mobilization of HBP when both L. inter-rogans Copenhageni L1-130 and culture supernatants were used as stimuli. When EGTA was used in the absence of BAPTA-AM, it also blocked HBP release to similar extents (Figure 3C and 3D and Supplementary Table 1). These results suggest that leptospire-in-duced HBP release is dependent on Ca2+ influx.

Leptospira-Induced HBP Release Is Not Mediated by Fibrinogen and

β2-Integrins

The first and most studied bacterial protein that provokes HBP release, streptococcal M protein, forms complexes with fibrin-ogen that bind to and activate neutrophils via β2-integrins [14, 33]. However, our results suggest the HBP release stimulated by leptospires or culture supernatants does not involve fibrin-ogen complexes or β2-integrins cross-linking. The fibrinogen

600 000

400 000

200 000

0

–200 000

A

Flu

ores

cenc

e

LD

H c

ytot

oxic

ity a

ssay

findi

ng (A

490)

B C

0 15 30Time (min)

45 60

Unlabeled

Cells (107)Cells (108)

108 107

RPMI

RPMI

PMA

PMA

L. interrogans1.00.90.80.7

0.3

0.2

0.1

0.0

0.6

PMA

Cells (

108 )

Cells (

107 )

RPMI

LDH

Figure 2. Leptospires induce polymorphonuclear neutrophil (PMN) heparin-binding protein (HBP) degranulation without interfering with cell viability. Purified PMNs were labeled with DCF-DA and then either treated with 25 nM PMA or Leptospira interrogans serovar Copenhageni cells (1 × 107 or 1 × 108 cells/mL) or left untreated. Unlabeled cells were used as controls. A, Cell fluorescence relative to superoxide anion production was measured. B, The supernatants of the stimulated PMNs were assayed as HBP contents by Western blot. C, PMNs supernatants were also analyzed by a lactate dehydrogenase (LDH) cytotoxicity assay. Purified LDH was used as a positive control. The data represent the means ± standard deviations of 2 independent experiments performed in duplicate. The Western blot depicts a representative experiment. RPMI, Roswell Park Memorial Institute 1640 medium.

Dow








peptide H-1998 did not influence HBP mobilization, and anti–β2-integrin antibodies (IB4) resulted in a statistically significant but physiologically irrelevant inhibitory effect when culture supernatants were used as a stimulus (Supplementary Figure 1). A peptide cysteine proteinase inhibitor (E64) and antimouse antibodies were used as nonrelated controls.

The Leptospiral Proteins Lsa63 and LipL45 Trigger HBP Release

Aiming to identify the proteins able to trigger HBP release, we purified the L. interrogans L1-130 membrane proteins by the TX-114 solubilization method, following anion exchange chro-matography fractionation. Each fraction, eluted with increasing NaCl concentration, was tested for its ability to trigger HBP release from neutrophils in human blood (Supplementary Figure 2). The positive fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the suspected candidates were identified by mass spectrometry to be the proteins Lsa63 and LipL45. The proteins were recombinantly expressed and purified by affinity chromatography as previously reported [26]. The leptospiral protein LipL21 was included as a control. Each

purified recombinant protein was mixed with human blood, and the ability to stimulate PMNs to release HBP was evaluated. LPS and M1 streptococcal protein were used as negative and positive controls, respectively [14]. Although not as potent as M1 protein, Lsa63 and LipL45 both significantly induced HBP degranulation, with Lsa63 being more potent than LipL45 (Figure 4A).

In the next series of experiments, we used signaling pathway inhibitors to determine the mechanisms by which Lsa63 and LipL45 trigger HBP release from PMNs. Except for genistein, which did not exert any effect, we found that the same inhibitors that reduced HBP secretion in response to both Leptospira cells and culture supernatants (ie, wortmannin, SB203580, AG1478, and GF109203) had an inhibitory effect when Lsa63 and LipL45 where used as stimuli (Figure 4B and 4C). Additionally, treat-ment with intracellular and extracellular calcium chelators inhibited the mobilization of HBP induced by both recombi-nant proteins (Supplementary Figure 3). Together, these results suggest that Lsa63 and LipL45 induce HBP release by interfer-ing with controlled signaling pathways of the PMNs, as demon-strated before using whole bacteria stimulation.

150

100

50

0

A

HB

P re

leas

e (%

)

Cells

Genist

ein

SB2035

80

AG1478

GF1092

03H-89

PD9805

9

U7312

2

Contro

l

Wor

tman

nin

*

150

100

50

0

C

HB

P re

leas

e (%

)

*

*

** * *

Bu er

Contro

l

EGTA

BAPTA-AM

/EGTA

Bu er

150

100

50

0

HB

P re

leas

e (%

)* *

Contro

l

EGTA

BAPTA-AM

/EGTA

EMJH

150

100

50

0

B

HB

P re

leas

e (%

)

Supernatants

Cells D Supernatants

Genist

ein

SB2035

80

AG1478

GF1092

03H-89

PD9805

9

U7312

2

Contro

l

Wor

tman

nin

* *

*

**

EMJH

Figure 3. Influence of various inhibitors on Leptospira-induced release of heparin-binding protein (HBP) in human blood. Culture-attenuated Leptospira interrogans (A and C) or its secreted products (B and D) were mixed (1:1 v/v) with human blood in the presence of different inhibitors and incubated for 1 hour at 37°C. After removal of the cells by centrifugation, the HBP content in the resulting supernatants was determined by capture enzyme-linked immunosorbent assay. The release induced by the bacterial cells or culture supernatants alone correspond to 100%. The bars represent the means ± standard deviations of 3 independent experiments performed in duplicates. P < .05.

Dow







Recombinant Leptospiral Proteins Induce Increased Endothelial

Permeability In Vitro

As HBP induces vascular leakage [14, 21, 33, 34], we sought to verify whether the leptospiral recombinant proteins can interfere with endothelial permeability indirectly by neutro-phil stimulation. We stimulated isolated human PMNs with the recombinant proteins and used the neutrophil exudates to treat endothelial monolayers. Significantly increased endothe-lial permeability was found for the neutrophil exudates treated with the 3 recombinant proteins, whereas treatment with PBS as a negative control rendered negligible FITC-dextran signal (the level was comparable to that of the control using cell culture medium only; Figure 5A). In parallel, we determined the HBP

levels in the exudates of the PMNs, which correlated well with levels after whole blood stimulation, with Lsa63 the most potent (Figure 5B). Interestingly, the endothelial permeability did not reflect the HBP levels, with LipL45-induced exudates being the most effective. As the leptospiral proteins alone did not induce increased permeability (data not shown), this effect could be due to another PMN factor induced by the recombinant pro-teins, which is currently under study.

Lsa63 Induces Increased Vascular Permeability and Leukocyte

Recruitment In Vivo

The effects of Lsa63 and LipL45 on the induction of inflamma-tory responses in vivo was evaluated by intravital microscopy

800

600

400

200

0

A

Perm

eabi

lity

(RFU

s)

Lsa63

LipL45

LipL21PBS

Contro

l

B

*

***

**

400

300

200

100

0

HB

P (n

g/m

L)

Lsa63

LipL45

LipL21PBS

** *

Figure 5. Recombinant leptospiral proteins Lsa63, LipL45, and LipL21 induce endothelial cell permeability. Isolated human polymorphonuclear neutrophils (PMNs; 1 × 106) were stimulated with recombinant proteins (5 µg/mL) or phosphate-buffered saline (PBS) in Roswell Park Memorial Institute 1640 medium supplemented with 10% plasma for 1 hour at 37°C. The neutrophil exudates were used to treat EA.hy926 cell monolayers for 24 hours at 37°C. A, The level of FITC-dextran that passed through the monolayer was measured by a fluorescence assay (excitation, 485 nm; emission, 535 nm). Addition of cell culture medium onto the cell monolayers was used as control. B, In parallel, the heparin-binding protein (HBP) levels in the exudates of the PMNs were determined by enzyme-linked immunosorbent assay. The bars represent the means ± standard deviations of 3 independent experiments using different blood donors. RFU, relative fluorescence unit. *P < .05, **P < .01, and ***P < .001.

120

100

80

60

40

20

0

AH

BP

leve

l (%

of

TX

-100

)

**

**

*

*

*

Lsa63

LipL45

LipL21 M

1LPS

PBS

TX-100

0.1μg/mL

1μg/mL

10μg/mL

150

100

50

0

B

HB

P le

vel (

% o

f co

ntro

l)

** *

*

Genist

ein

SB2035

80

AG1478

GF1092

03

PD9805

9H89

U-7312

2Lsa6

3

Wor

tman

ninPBS

150

100

50

0

C

HB

P le

vel (

% o

f co

ntro

l)

**

*

*

Genist

ein

SB2035

80

AG1478

GF1092

03

PD9805

9H89

U-7312

2

LipL45

Wor

tman

ninPBS

Figure 4. Ability of leptospiral recombinant proteins to induce heparin-binding protein (HBP) release. A, Different concentrations of the purified recombinant leptospiral proteins LipL21, LipL45, and Lsa63 (0.1, 1, and 10 µg/mL) were mixed with equal volumes of human whole blood and incubated for 1 hour at 37°C. Cells were removed by centrifugation, and the HBP content of the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Blood stimulated with lipopolysaccharide (LPS) or phosphate buffered saline (PBS) were used as negative controls, while M1 protein served as positive control for stimulation of HBP degranulation. The HBP concentrations were normalized according to the samples treated with Triton X-100 (for which there was 100% HBP release). The purified recombinant Lsa63 (B) and LipL45 (C) proteins (1 µg/mL) were mixed with human whole blood and incubated for 1 hour at 37°C in the presence of various signal transduction pathway inhibitors. Cells were removed by centrif-ugation, and the HBP content of the supernatants was determined by ELISA. Blood stimulated with PBS only was used as negative control. Values for samples stimulated with the recombinant proteins without additions were considered as 100% HBP release for graph normalization. The bars represent the means ± standard deviations of 3 independent experiments performed in duplicate. *P < .05.

Dow




after superfusion of the recombinant proteins onto the cre-master muscle of mice. Lsa63 induced significant recruitment (Figure 6A and 6C) and adherence of leukocytes, presumably neutrophils, in the microcirculation (Figure 6B and 6C), while LipL45 and PBS exerted no effect. Additionally, as observed in a modified Miles assay by Evans Blue dye injection and migration monitoring, the purified Lsa63 induced increased vascular leak-age (Supplementary Materials and Supplementary Figure 4). Altogether, the results indicate that Lsa63 is highly inflamma-tory in vivo.

HBP Levels Are Elevated in Serum Samples From Patients With

Leptospirosis

We measured the HBP level in sera from patients with a diag-nosis of leptospirosis to study its potential as a marker for

leptospirosis. Paired serum samples obtained from the same patients during the early (ie, MAT-negative) phase and con-valescent (ie, MAT-positive) phase of leptospirosis were used. Patients with leptospirosis exhibited high levels of HBP, espe-cially those with negative MAT results (Figure 7A). With the progression of leptospirosis to the nonhematogenous (ie, MAT-positive) phase, the HBP levels declined (Figure 7B). Sera from patients with other tropical diseases with a similar initial symp-tomatology (dengue, Chagas disease, and malaria) and bacterial infections (tuberculosis, hanseniasis, and syphilis) were evalu-ated for comparative purposes. On the basis of the proposed cutoff (ie, a mean normal HBP level ± 3 times the standard deviation), the calculated specificity was 0.895, and the sensi-tivity was 0.841 for MAT- negative patients and 0.443 for MAT-positive patients.

80

60

40

20

00 10 20 30

Minutes

AL

euko

cyte

rol

ling

(leuk

ocyt

es/m

in)

Lsa63 (150μg)

Lsa63 (75μg)

LipL45 (75μg)

PBS

50

40

30

20

10

00 10 20 30

Minutes

B

Adh

ered

leuk

ocyt

es (n

o.)

Lsa63 (150μg)

Lsa63 (75μg)

LipL45 (75μg)

PBS

C

Lsa63 - T0 Arteriole

Venule

Muscle tissue

20×

Lsa63 - T30 Venule

40×

LipL45 - T0

ArterioleVenule

Muscle tissue20×

LipL45 - T30 Venule

40×

Figure 6. Intravital microscopy of biological effects of purified recombinant Lsa63 and LipL45. The cremaster muscle of anesthetized Balb/c mice were exposed; mice were maintained in 37°C plates during the experiments. Recombinant proteins (150 or 75 µg in 30 µL of phosphate-buffered saline [PBS]) or PBS alone (as a control) were superfused onto the exposed cremaster muscle, and the dynamics in the microcirculatory network were monitored for up to 30 minutes. A and B, Rolling of leukocytes/minute (A) and the number of adhered leukocytes (B) in the venules were recorded. C, Representative micrographs displaying the rolling and adherence of leukocytes. Experiments were performed in triplicate. The bars represent the means ± standard deviations.

Dow





DISCUSSION

The exaggerated host responses against an infection can lead to an uncontrolled increase in vascular permeability and plasma leakage. This is a fundamental pathophysiological mechanism in bacterial infections and septic shock, resulting in hypoten-sion, disturbed microcirculation, hypoxia, and organ dysfunc-tion. Several investigations have demonstrated that activated neutrophils induce vascular leakage [29], and a recent study demonstrated that HBP is the most potent among the various substances released by those cells [7]. HBP was shown to induce increased vascular permeability and leakage in severe bacterial infections, in particular the ones caused by Streptococcus pyo-genes and Staphylococcus aureus [14, 17, 21, 33, 34].

PMN counts are frequently increased during the bacteremic phase of leptospirosis, and this is correlated with disease sever-ity [35–37]. However, patients with leptospirosis are likely to exhibit a unique neutrophil activation profile because canonical activation markers seem not to be expressed [37] and because PMN-mediated phagocytosis does not seem to play a major role in the disease [38]. Further supporting the role of neutrophils in leptospirosis independent of phagocytosis, recent studies suggest that Leptospira species trigger the formation of neutro-phil extracellular traps, which may influence the pathogenesis of the disease [39]. However, PMN immune responses, such as release of antimicrobial peptides and enzymes, oxidative stress, or formation of neutrophil extracellular traps, must be counter-acted by leptospires during infection. We have recently reported that leptospires trigger myeloperoxidase degranulation by

neutrophils but are resistant to its activity by inhibition, thus protecting the bacteria against the oxidative stress [26].

Bacterial products have been found as HBP degranulation mediators, including lipid leukotriene B4 [40], M protein [14, 33], streptolysin O [41], and phenol-soluble modulin α4 [17]. Here, we showed that leptospires and their secreted products stimulate neutrophils to release HBP through a controlled degranulation mechanism that is dependent on various cell signaling pathways and Ca2+ influx. Also, we characterized the leptospiral outer membrane proteins Lsa63 and LipL45 in terms of their ability to stimulate HBP degranulation. Both Lsa63 and LipL45 have been previously shown to be leptospiral membrane proteins that are expressed during human infection [27, 42]. We believe that leptospires express other proteins able to trigger HBP release, as Lsa63 and LipL45 did not stimulate the neutrophils HBP secre-tion via tyrosine kinase signaling pathways, as observed when L. interrogans cells and culture supernatants were used as stim-uli. Interestingly, also L. biflexa triggered HBP degranulation. Commonly, an inflammatory or pathophysiological host effect is not due to factors restricted to pathogenic/virulent strains. For example, saprophytic leptospires also bind host plasminogen, although less effectively than virulent strains [43–45].

The exudates of the PMNs treated with the recombinant lep-tospiral proteins can induce endothelial monolayer permeabil-ity in vitro, while the proteins alone exerted no effect. Although HBP is a potent vascular leakage inducer released by neutro-phils [7], there are also other factors, such as chemokines, pro-teases, and redox active enzymes, that are known to directly

A B C250200150

120110100908070605040302010

0

HB

P le

vel (

ng/m

L)

MAT neg

MAT po

s

Dengu

e

Chagas

disea

se

Bacter

ial in

fectio

ns

Normal

Malá

ria

Cut o�9,205

250200150

100

50

0

HB

P le

vel (

ng/m

L)

MAT neg

MAT po

s

1

1

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.10

0

Tru

e-po

sitiv

e ra

te (s

ensi

tivity

)

ROC

False-positive rate (1 − specificity)

Leptospirosis (MAT neg)

NonleptospirosisLeptospirosis (MAT pos)

Figure 7. Serum levels of heparin-binding protein (HBP) in patients with leptospirosis and other tropical infectious diseases. A, HBP levels in patients’ serum samples were quantified by enzyme-linked immunosorbent assay. Patients with negative results of microscopic agglutination test (MAT) were in the initial phase of leptospirosis. Patients with positive results of MAT were in the convalescent phase of the disease. Bacterial infections include 3 cases of tuberculosis, 4 cases of syphilis, and 2 cases of hanseniasis. Each dot represents the concentration in 1 individual sample. Horizontal lines represent the means of the values. The proposed cutoff based on values for normal serum samples is 9.205 (calculated as the normal mean ± 3 times the standard deviation). B, Progression of the HBP levels in the paired samples of the same individuals in the early and convalescent phases of leptospirosis. C, Receiver operating characteristic (ROC) curve calculated for samples from MAT-negative and MAT-positive patients, compared with samples from patients without leptospirosis, showing the sensitivity of the test for MAT-negative (0.841) and MAT-positive (0.443) patients, based on the proposed cutoff of 9.205.

Dow




or indirectly increase vascular permeability by weakening the endothelial cell junctional integrity [46]. The relationship of Lsa63, LipL45, and LipL21 and these factors will be further evaluated. Additionally, Lsa63 is a leptospiral protein likely to contribute to the pathophysiology of leptospirosis, as it is a potent inducer of inflammation.

Leptospirosis treatment by antibiotics can be effective when initiated early in the course of the disease [1]. However, leptospi-rosis has been underdiagnosed because of nonspecific symptoms and lack of sensitive and simple diagnostic tests. The reference method for the serological diagnosis of leptospirosis (ie, MAT) and other available assays have low sensitivities during the early phase of the disease [3, 47]. Several studies reported that during sepsis there is a significant increase in the HBP level in plasma. Thus, HBP has been exploited as a severe sepsis early marker [18–22]. Nonetheless, HBP is not a marker for all bacterial infections [20]. In this investigation, we found that patients with leptospi-rosis present with high levels of HBP in serum, especially in the early phase of the disease, when results of the reference method are negative. The mean HBP levels in leptospirosis samples are especially higher than those in patients for whom tropical dis-eases with similar initial symptoms and other bacterial diseases are diagnosed. However, levels in some samples from patients with malaria or Chagas disease were also above the cutoff. We thus suggest the use of HBP as an early marker of human lepto-spirosis screening, without excluding the use of definitive specific leptospirosis diagnostic methods, such as polymerase chain reac-tion analysis of blood samples, which can be useful in the MAT-negative phase. This could greatly enhance the early leptospirosis detection and the rate of antibiotics successful treatment.

In conclusion, our results suggest that the HBP release is an important mechanism for leptospiral dissemination that can play an important role in the pathophysiology of leptospirosis. We characterize Lsa63 as a new putative virulence factor able to induce pathological effects in vivo. The identification of this potentially important pathophysiological pathway can be help-ful in the development of new therapeutic strategies for lepto-spirosis. We also propose HBP as a new early diagnostic marker for human leptospirosis.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or com-ments should be addressed to the corresponding author.

Notes

Author contributions. M. L. V., S. P., M. L.-F., and H. H. con-ceived of and designed the experiments and analyzed the data. M. L. V., S. P., and M. L.-F. performed the experiments. M. L. V., M. L.-F., E. C. R., K. K., A. L. T. O. N., and H. H. contributed to

the preparation of reagents, materials, and analytical tools. M. L. V. wrote the manuscript. All authors revised the manuscript.

Acknowledgments. We thank Dr Mathieu Picardeau, for kindly providing the Leptospira strains; Pia Andersson, for excellent technical assistance; and Felipe José Passalia, for assis-tance with the MATLAB software.

Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo of Brazil (awards 2014/18337-4 and 2018/07054-2 to M. L. V. and award 2014/50981-0), the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil (award 150520/2017-4 to M. L. V.), the Alfred Österlund Foundation, the Knut and Alice Wallenberg Foundation (award KAW 2011.0037), the Ragnar Söderberg Foundation, the Medical Faculty of Lund University, the Swedish Foundation for Strategic Research (award K2014-56X-13413-15-3), and the Swedish Research Council (award 2013–2438).

Potential conflicts of interest. All authors: No reported con-flicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

1. Faine S, Adler B, Bolin C, Perolat P. Leptospira and Leptospirosis. 2nd ed. Melbourne, Australia: MediSci, 1999:259.

2. Levett PN. Leptospirosis. Clin Microbiol Rev 2001; 14:296–326.

3. Vinetz JM. Leptospirosis. Curr Opin Infect Dis 2001; 14:527–38.

4. Toyokawa T, Ohnishi M, Koizumi N. Diagnosis of acute leptospirosis. Expert Rev Anti Infect Ther 2011; 9:111–21.

5. Iversen LF, Kastrup JS, Bjørn SE, et al. Structure of HBP, a multifunctional protein with a serine proteinase fold. Nat Struct Biol 1997; 4:265–8.

6. Tapper H, Karlsson A, Mörgelin M, Flodgaard H, Herwald H. Secretion of heparin-binding protein from human neu-trophils is determined by its localization in azurophilic granules and secretory vesicles. Blood 2002; 99:1785–93.

7. Gautam N, Olofsson AM, Herwald H, et al. Heparin-binding protein (HBP/CAP37): a missing link in neutro-phil-evoked alteration of vascular permeability. Nat Med 2001; 7:1123–7.

8. Rasmussen PB, Bjørn S, Hastrup S, et al. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cyto-kine release from monocytes. FEBS Lett 1996; 390:109–12.

Dow




9. Pereira HA, Shafer WM, Pohl J, Martin LE, Spitznagel JK. CAP37, a human neutrophil-derived chemotactic fac-tor with monocyte specific activity. J Clin Invest 1990; 85:1468–76.

10. Linder A, Soehnlein O, Akesson P. Roles of heparin-bind-ing protein in bacterial infections. J Innate Immun 2010; 2:431–8.

11. Heinzelmann M, Platz A, Flodgaard H, Miller FN. Heparin binding protein (CAP37) is an opsonin for Staphylococcus aureus and increases phagocytosis in monocytes. Inflammation 1998; 22:493–507.

12. Chertov O, Michiel DF, Xu L, et al. Identification of defen-sin-1, defensin-2, and CAP37/azurocidin as T-cell chemo-attractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996; 271:2935–40.

13. Ostergaard E, Flodgaard H. A neutrophil-derived pro-teolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and throm-bospondin secretion. J Leukoc Biol 1992; 51:316–23.

14. Herwald H, Cramer H, Mörgelin M, et al. M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage. Cell 2004; 116:367–79.

15. Lundqvist K, Herwald H, Sonesson A, Schmidtchen A. Heparin binding protein is increased in chronic leg ulcer fluid and released from granulocytes by secreted prod-ucts of Pseudomonas aeruginosa. Thromb Haemost 2004; 92:281–7.

16. Snäll J, Linnér A, Uhlmann J, et al. Differential neutrophil responses to bacterial stimuli: Streptococcal strains are potent inducers of heparin-binding protein and resistin-re-lease. Sci Rep 2016; 6:21288.

17. Li L, Pian Y, Chen S, et al. Phenol-soluble modulin α4 medi-ates Staphylococcus aureus-associated vascular leakage by stimulating heparin-binding protein release from neutro-phils. Sci Rep 2016; 6:29373.

18. Linder A, Akesson P, Brink M, Studahl M, Björck L, Christensson B. Heparin-binding protein: a diagnostic marker of acute bacterial meningitis. Crit Care Med 2011; 39:812–7.

19. Linder A, Åkesson P, Inghammar M, Treutiger CJ, Linnér A, Sundén-Cullberg J. Elevated plasma levels of heparin-bind-ing protein in intensive care unit patients with severe sepsis and septic shock. Crit Care 2012; 16:R90.

20. Linder A, Christensson B, Herwald H, Björck L, Akesson P. Heparin-binding protein: an early marker of circulatory failure in sepsis. Clin Infect Dis 2009; 49:1044–50.

21. Linder A, Johansson L, Thulin P, et al. Erysipelas caused by group A streptococcus activates the contact system and induces the release of heparin-binding protein. J Invest Dermatol 2010; 130:1365–72.

22. Kjölvmark C, Akesson P, Linder A. Elevated urine levels of heparin-binding protein in children with urinary tract infection. Pediatr Nephrol 2012; 27:1301–8.

23. Fisher J, Linder A. Heparin-binding protein: a key player in the pathophysiology of organ dysfunction in sepsis. J Intern Med 2017; 281:562–74.

24. Lindmark A, Garwicz D, Rasmussen PB, Flodgaard H, Gullberg U. Characterization of the biosynthesis, pro-cessing, and sorting of human HBP/CAP37/azurocidin. J Leukoc Biol 1999; 66:634–43.

25. Vieira ML, Naudin C, Mörgelin M, Romero EC, Nascimento AL, Herwald H. Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp. PLoS Negl Trop Dis 2016; 10:e0004713.

26. Vieira ML, Teixeira AF, Pidde G, et al. Leptospira interro-gans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase. Virulence 2018; 9:414–25.

27. Vieira ML, de Morais ZM, Gonçales AP, Romero EC, Vasconcellos SA, Nascimento AL. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV. J Infect 2010; 60:52–64.

28. Ferreira MJ, Lima C, Lopes-Ferreira M. Anti-inflammatory effect of Natterins, the major toxins from the Thalassophryne nattereri fish venom is dependent on TLR4/MyD88/PI3K signaling pathway. Toxicon 2014; 87:54–67.

29. Edens HA, Parkos CA. Neutrophil transendothelial migra-tion and alteration in vascular permeability: focus on neutrophil-derived azurocidin. Curr Opin Hematol 2003; 10:25–30.

30. Cowland JB, Borregaard N. Molecular characterization and pattern of tissue expression of the gene for neutrophil gelatinase-associated lipocalin from humans. Genomics 1997; 45:17–23.

31. Burd JF, Usategui-Gomez M. A colorimetric assay for serum lactate dehydrogenase. Clin Chim Acta 1973; 46:223–7.

32. Lacy P. Mechanisms of degranulation in neutrophils. Allergy Asthma Clin Immunol 2006; 2:98–108.

33. Soehnlein O, Oehmcke S, Ma X, et al. Neutrophil degranu-lation mediates severe lung damage triggered by streptococ-cal M1 protein. Eur Respir J 2008; 32:405–12.

34. Påhlman LI, Mörgelin M, Eckert J, et al. Streptococcal M protein: a multipotent and powerful inducer of inflamma-tion. J Immunol 2006; 177:1221–8.

35. Craig SB, Collet TA, Wynwood SJ, Smythe LD, Weier SL, McKay DB. Neutrophil counts in leptospirosis patients infected with different serovars. Trop Biomed 2013; 30:579–83.

36. Craig SB, Graham GC, Burns MA, Dohnt MF, Smythe LD, McKay DB. Haematological and clinical-chemistry mark-ers in patients presenting with leptospirosis: a compar-ison of the findings from uncomplicated cases with those seen in the severe disease. Ann Trop Med Parasitol 2009; 103:333–41.

Dow




37. Raffray L, Giry C, Vandroux D, et al. Major neutrophilia observed in acute phase of human leptospirosis is not asso-ciated with increased expression of granulocyte cell activa-tion markers. PLoS One 2016; 11:e0165716.

38. Chen X, Li SJ, Ojcius DM, et al. Mononuclear-macrophages but not neutrophils act as major infiltrating anti-lepto-spiral phagocytes during leptospirosis. PLoS One 2017; 12:e0181014.

39. Scharrig E, Carestia A, Ferrer MF, et al. Neutrophil extra-cellular traps are involved in the innate immune response to infection with leptospira. PLoS Negl Trop Dis 2015; 9:e0003927.

40. Di Gennaro A, Kenne E, Wan M, Soehnlein O, Lindbom L, Haeggström JZ. Leukotriene B4-induced changes in vas-cular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin). FASEB J 2009; 23:1750–7.

41. Nilsson M, Sørensen OE, Mörgelin M, Weineisen M, Sjöbring U, Herwald H. Activation of human polymorpho-nuclear neutrophils by streptolysin O from Streptococcus pyogenes leads to the release of proinflammatory media-tors. Thromb Haemost 2006; 95:982–90.

42. Matsunaga J, Young TA, Barnett JK, Barnett D, Bolin CA, Haake DA. Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regu-lated peripheral membrane protein. Infect Immun 2002; 70:323–34.

43. Vieira ML, Vasconcellos SA, Gonçales AP, de Morais ZM, Nascimento AL. Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degrada-tion. Infect Immun 2009; 77:4092–101.

44. Vieira ML, Nascimento AL. Interaction of spirochetes with the host fibrinolytic system and potential roles in pathogen-esis. Crit Rev Microbiol 2016; 42:573–87.

45. Vieira ML, Atzingen MV, Oliveira R, et al. Plasminogen binding proteins and plasmin generation on the surface of Leptospira spp.: the contribution to the bacteria-host inter-actions. J Biomed Biotechnol 2012; 2012:758513.

46. DiStasi MR, Ley K. Opening the flood-gates: how neutro-phil-endothelial interactions regulate permeability. Trends Immunol 2009; 30:547–56.

47. Palaniappan RU, Ramanujam S, Chang YF. Leptospirosis: pathogenesis, immunity, and diagnosis. Curr Opin Infect Dis 2007; 20:284–92.

Dow



jiy589.pdf - Oxford Academic

Documents

Transcript of jiy589.pdf - Oxford Academic