P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner

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P-selectin drives complement attack on endothelium during intravascular hemolysis in

TLR-4/heme-dependent manner

Nicolas Merle, Romain Paule, Juliette Leon, Marie Daugan, Tania

Robe-Rybkine, Victoria Poillerat, Carine Torset, Veronique Fremeaux-Bacchi, Jordan Dimitrov, Lubka Roumenina

To cite this version:

Nicolas Merle, Romain Paule, Juliette Leon, Marie Daugan, Tania Robe-Rybkine, et al.. P-selectin

drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent

manner. Proceedings of the National Academy of Sciences of the United States of America , National

Academy of Sciences, 2019, 116 (13), pp.6280-6285. �10.1073/pnas.1814797116�. �hal-02124195�

!"

P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner

Nicolas S. Merle

, Romain Paule

, Juliette Léon

, Marie Daugan

, Tania Robe- Rybkine

, Victoria Poillerat

, Carine Torset

, Véronique Frémeaux-Bacchi

, Jordan D. Dimitrov

, Lubka T. Roumenina

1

INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France;

2

Sorbonne Universités, F-75006, Paris, France;

3

Université Paris Descartes, Sorbonne Paris Cité, F-75006, Paris, France

4

Service de médecine interne, Hôpital Foch, Suresnes

5

Assistance Publique – Hôpitaux de Paris, Service d’Immunologie Biologique, Hôpital Européen Georges Pompidou, F-75015, Paris, France

*

Correspondence to: Lubka T. Roumenina, Ph.D.

Cordeliers Research Center, INSERM UMRS 1138;

15 rue de l'Ecole de Medecine; 75006 Paris, France Phone: +33-1-44-27-90-96/ Fax: +33-1-40-51-04-20, e-mail: [email protected]

*

Correspondence to: Jordan D. Dimitrov, Ph.D.

Cordeliers Research Center, INSERM UMRS 1138;

15 rue de l'Ecole de Medecine; 75006 Paris, France Phone: +33-1-44-27-82-06

e-mail: [email protected]

#"

Abstract

Hemolytic diseases are frequently linked to multi-organ failure subsequent to vascular

damage. Deciphering the mechanisms leading to organ injury upon hemolytic event could

bring out novel therapeutic approaches. Complement system activation occurs in hemolytic

disorders, such as sickle cell disease, but the pathological relevance and the acquisition of a

complement-activating phenotype during hemolysis remain unclear. Here we found that

intravascular hemolysis, induced by injection of phenylhydrazine, resulted in increased ALT

plasma levels and NGAL expression. This liver damage was at least in part complement-

dependent, since it was attenuated in complement C3

mice and by injection of C5 blocking

antibody. We evidenced C3 activation fragments deposits on liver endothelium in mice with

intravascular hemolysis or injected with heme as well as on cultured human endothelial cells

(EC), exposed to heme. This process was mediated by TLR4 signaling, as revealed by

pharmacological blockade and TLR4 deficiency in mice. Mechanistically, TLR4-dependent

surface expression of P-selectin triggered an unconventional mechanism of complement

activation by non-covalent anchoring of C3 activation fragments, including the typical fluid

phase C3(H

O), measured by surface plasmon resonance and flow cytometry. P-selectin

blockade by an antibody prevented complement deposits and attenuated the liver stress

response, measured by NGAL expression, in the hemolytic mice. In conclusion, these results

revealed the critical impact of the triad TLR4/P-selectin/complement in the liver damage, and

its relevance for hemolytic diseases. We anticipate that blockade of TLR4, P-selectin or the

complement system could prevent liver injury in hemolytic diseases like sickle cell disease.

$"

Significance statement

Complement system activation occurs in sickle cell disease and other hemolytic disorders, but the pathological relevance and the acquisition of a complement-activating phenotype by host cells during hemolysis remain unclear. Here we demonstrated that intravascular hemolysis triggered liver damage, which was attenuated in C3-deficient mice or by C5 blockade, showing the importance of complement system for hemolysis-mediated tissue stress-response.

Heme-dependent complement deposits were detected on endothelium, appeared upon

activation of TLR4 and were mediated by P-selectin. This resulted in unconventional

complement activation, mediated by anchoring of C3(H

O) on endothelial surface. In

conclusion, our data suggest that complement system could be a novel therapeutic target in

hemolytic diseases, such as sickle cell disease.

%"

/body

Introduction

Intravascular hemolysis is a hallmark of a large spectrum of pathologies, including socially significant diseases as sickle cell disease (SCD) or malaria, characterized by the presence of a large amount of red blood cells (RBC) degradation products in the circulation(1, 2). Cell-free hemoglobin, heme and RBC microvesicles act by different mechanisms to promote vascular and tissue injury(3). Heme is a danger-associated molecular pattern molecule, which recruits and activates neutrophils(4–6) and macrophages(7, 8), induces vasoconstriction(9) and activates endothelial cells (EC)(10, 11). Moreover, heme modulates the activity of different plasma systems(12), including the innate immune complement cascade(11, 13–17).

In hemolytic diseases, signs of complement system(18, 19) activation are detected in circulation, on RBC and on endothelium, but the mechanisms and the pathological relevance are still not well understood(13, 20, 21). Heme triggers Toll-like receptor 4 (TLR4) signaling(7, 10) on EC, but the contribution of this innate immune receptor in transforming resting endothelium to a complement-activating surface is unclear. The liver is highly vascularized and impacted by hemolysis, being the second site of RBC fragments clearance and hemoglobin and heme detoxification. Furthermore, hepatic involvement is observed in 10%-40% cases of SCD crisis(22) and in models of hemolytic diseases(17, 23), but the contribution of complement system remains unexplored.

We describe a non-conventional mechanism of complement activation in hemolytic conditions. In it, the fluid phase component C3(H

O) (or a C3(H

O)-like) binds to the EC membranes via P-selectin (P-sel), expressed after heme-triggered TLR4 signaling.

Deficiency/blockade of complement, TLR4, P-sel and heme attenuated the liver stress

response in mice with intravascular hemolysis. These revealed the critical impact of the triad

&"

TLR4/P-sel/complement in the liver injury under hemolytic conditions, and its relevance for hemolytic diseases.

Results

C3 activation fragments are deposited on vascular endothelium in the liver of mouse models of intravascular hemolysis

Well-characterized models for heme injection and phenylhydrazine (PHZ)-induced intravascular hemolysis(20, 24) were used to determine to what extend hemolysis is responsible for complement deposits in the liver. We used an antibody (Ab), detecting the C3 activation fragments (C3b, iC3b, C3(H

O)-like) but not native C3. Treatment with PHZ or heme significantly increased C3 activation fragments deposition on endothelium in the liver, compared to PBS in WT mice (Fig. 1A and B). The double staining of C3 activation fragments/vWF was localized on macrovessels, mainly central hepatic veins, as well as on liver sinusoids (Fig. S1A: Fig. 1 from the SI Appendix), confirmed by co-localization with vascular CD31 (Fig. S1B). Bile ducts were not impacted by complement activation, as shown by the absence of co-localization with an epithelial marker cytokeratine (Fig. S1C). The C3 activation fragments staining in the liver sinusoids co-localized, though, with macrophages/Kupffer cells CD68 (Fig. S1D), granulocytes/neutrophils Ly6G (Fig. S1E) and with platelets’ marker CD41a (Fig. S1F). The presence of neutrophils and platelets here supports the concept that complement plays a critical role in the induction of platelet- neutrophil aggregates on endothelium(25).

As expected, no C3 activation fragments deposits were detected in C3

mice treated with

PHZ (Fig. S2). Importantly, in TLR4

mice, the complement deposits on vessels were

strongly reduced, compared to WT mice, despite treatments (Fig. 1A and B). These vascular

'"

deposits were attenuated by injection of heme scavenger hemopexin (Hx) in WT mice (Fig.

1C).

Complement activation and TLR4 signaling triggered by heme contribute to the hemolysis- induced liver stress response

We detected signs of liver injury in PHZ-treated mice by measuring plasmatic alanine aminotransferase (ALT) levels, which were significantly decreased in TLR4

and C3

hemolytic mice (Fig. 2A and B). Of note, basal ALT activity weakly but significantly increased in C3

compared to WT mice.

Further we studied hypoxic cellular stress response protein NGAL, a sensitive marker for acute liver injury(26–28), upregulated in the liver in models of intravascular hemolysis and hypoxia by hepatocytes, neutrophils, Kupffer cells and EC(29). Moreover, NGAL expression could be induced in a TLR4-dependent manner(26, 30). The expression of NGAL in WT mice injected with PHZ strongly increased at mRNA and protein level and was attenuated in TLR4

/-

and C3

hemolytic mice (Fig. 2C and D, Fig. S3A). NGAL upregulation was reproduced by injection of heme at different regiments (weak effect with 40 !mol/kg i.v. and strong one with two i.p. injections, Fig. 2C and D, Fig. S3A). Of note, i.v. injection of 100!mol/kg of heme was lethal. Therefore, the i.p. injection was selected for the rest of the study, since it was well tolerated by the mice(24, 31) and triggered complement deposits (Fig. 1A and B). The heme- mediated NGAL expression was attenuated by pretreatment with Hx (Fig. S3B). The NGAL staining in hemolytic mice was with punctiform appearance in the liver sinusoids (likely the EC and Kupffer cells) as well as on macrovessels walls in WT mice (ramification of hepatic arteries, portal veins and central hepatic veins), while in TLR4

, only a fraction of the sinusoidal staining was detected with no signal in the macrovessels.

Having shown the contribution of the C3 activation fragments in the liver injury, we studied

the role of C5a and C5b-9. No staining for C5b-9 was detected in the liver sections of PHZ-

("

injected mice. Nevertheless, C5a showed increased plasmatic levels 24h after PHZ injection (Fig. 2F). To find out if C5a contributes to the pathological process, we injected C5 blocking Ab BB5.1 (known to prevent mouse C5 cleavage(32)), which prevented the C5a generation (Fig. 2E). The treatment resulted in a partial decrease of the ALT levels (Fig. 2F). The NGAL liver staining by immunofluorescence (IF) showed significant decrease (Fig. 2G, Fig. S3C), despite that there was no significant difference in NGAL mRNA levels (Fig. S3D). As expected, the C3 activation fragment deposits were not modified by BB5.1 (Fig. S3E) Taken together, these results demonstrate the critical importance of the TLR4 signaling and complement activation at the level of C3 and to less extent - of C5 for the liver injury during intravascular hemolysis.

Heme triggers TLR4 activation on EC in vitro

Having shown TLR4-dependent EC activation and complement deposits in presence of heme

and hemolysis, we studied this process in vitro. TLR4 staining was positive on resting

macrovascular EC (primary human umbilical vein endothelial cells, HUVEC) by flow

cytometry (Fig. S4A), as described for multiple EC, including of liver origin(33). After 30

min incubation with heme, TLR4-inhibitor TAK-242 or TLR4-ligand LPS, TLR4 expression

remained stable (Fig. S4B-D). CD14 staining showed a double peak on resting state,

distributed at 50% of both dim (CD14

) and bright cells (CD14

) (Fig. S4E and F). Bright

population of CD14 increased up to 80% after heme treatment, only partially prevented by

TAK-242 (Fig. S4F). LPS had no effect (Fig. S4G and H). The absence of TLR4

internalization (occurring in macrophages(34)) despite the rapid CD14 upregulation from

intracellular stores (as azurophilic granules of neutrophils(35, 36)) could be explained by the

lack of TRAM in EC(37).

)"

TLR4 activation was studied by phosphorylation of its signaling pathway intermediates and was in agreement with previous data(10). Phosphorylation of p-38 increased within 6 h with

25 !M of heme in a time-dependent manner, measured by IF (Fig. S4I). Moreover,

translocation of P-p65 in the nucleus increased up to 6 h, measured by western blot and IF (Fig. S4J and K). Similarly, LPS induced an increase of the p38 and p65 phosphorylations.

These results indicate that heme triggers TLR4 signaling on EC.

Inhibition of TLR4 attenuates heme-induced complement deposits on EC.

Our in vivo experiments suggested a direct link between the heme-mediated TLR4 signaling and the EC complement deposits. Therefore, we used an in vitro model, where exposure of EC to heme and normal human serum (NHS) resulted in an increase of C3 activation fragments and C5b-9 deposition within 30 min (Fig. S5A and C). This was associated with increased Bb release (1,8-fold at 100!M heme, compared to medium), confirming the implication of the alternative pathway (AP)(11, 38). Inhibition of TLR4 by TAK-242 prevented about 50% of both C3 activation fragments and C5b-9 deposition (Fig. S5B and D).

However, LPS treatment did not induce complement deposits (SI Fig. S5E-H). Moreover, the effect of heme was not related to LPS contamination, since the deposits on heme-exposed EC were unaffected despite pre-incubation with LPS-inhibitor Polymixin B (Fig. S5I and J).

To find out why LPS did not promote complement deposits in the studied conditions, we incubated different doses of heme or LPS in NHS. At 2 !M, LPS did not trigger complement activation, as measured by release of Bb or sC5b-9 (Fig. S5K and L). In contrast, exposure to 100 !M resulted in release of Bb and sC5b-9 from both LPS and heme (Fig. S5K and L).

Inhibition of TLR4 does not impact the expression/binding of complement regulators and

receptors.

*"

A defect of complement regulation subsequent to TLR4 activation by heme could explain the partial protective effect of TAK-242 against complement deposition on EC. Thus, we studied the impact of heme-treatment on the factor H (FH) binding on EC and the MCP expression, the two major complement regulators, as well as the expression of C3a and C5a receptors (C3aR and C5aR, respectively) within 30 min.

Binding of FH increased on EC surface in presence of heme, and was not prevented by TAK- 242 (Fig. S6A and B). Heme caused a dose-dependent decrease of MCP (up to 50%), in agreement with Frimat et al.(11), which was not prevented by TAK-242 (Fig. S6C and D).

Treatment with LPS did not modulate FH binding or MCP expression (Fig. S7A-D).

To find out whether the downregulation of MCP contributes to the complement deposits on heme-exposed EC, we performed gene silencing, which reduced the expression of MCP to up to 80% (Fig. S6G and H). Upon exposure to heme, the EC with silenced MCP presented more C3 activation fragments deposition, compared with a control siRNA (Fig. S6G and H).

Expression of both C3aR and C5aR revealed a double peak on resting state, distributing 50%

of dim and bright cells. Heme-treatment rapidly increased both C3aR and C5aR expressions, enhancing the bright cells population (Fig. S6I-L). However, neither C3aR- nor C5aR- increased expression was prevented by TAK-242 (Fig. S6I-L). In comparison, LPS-treatment did not affect C3aR and C5aR (Fig. S7E-H). These data point towards a second mechanism, mediating complement activation on human EC, which is independent on TLR4 and related to the expression of complement regulators.

A rapid increase of P-sel expression is the causal link between TLR4 and complement

activation in presence of heme on EC in vitro.

!+"

P-sel expression induced by heme could participate to complement deposition on EC(39).

First, we observed that both LPS and heme induced Weibel Palade bodies (WPB) mobilization within 30 min, translated by the release of vWF and the rapid expression of P-sel (Fig. S8A-F). We did not detect properdin binding on EC (Fig. S8E and F).

Staining of C3b and P-sel by IF on HUVEC revealed a co-localization in the presence of heme (Fig. S9A). Investigation of the interaction between C3b and P-sel by SPR gave a Kd about 100nM (Fig. 3A). Moreover, C3(H

O) was also able to bind recombinant P-sel (Fig.

3B). Presence of heme did not affect the binding of both C3b and C3(H

O) on P-sel (Fig. S9B and C). C3(H

O) was detected non-covalently bound to heme-exposed EC, as acidic wash abolished C3a staining (Fig. 3C). The acidic wash reduced the C3 fragments binding to the same level as did the pre-incubation with TAK242, showing that the non-covalent binding was TLR-4 dependent (Fig. 3D).

In order to understand whether these interactions have functional consequences on EC, we tested the capacity of a blocking Ab against P-sel to prevent complement deposition. Blocking of P-sel prevented 50% of C3 fragments deposition, compared to cells treated with an irrelevant Ab. This inhibition was equivalent to TLR4 blocking by TAK-242, and no additive effects of TAK-242 and Ab against P-sel were observed (Fig. 3E and F).

P-sel blockade attenuates complement deposits and liver stress response in vivo

Finally, to test the hypothesis that P-sel is responsible for the complement deposits in vivo, we

pre-treated the WT mice with blocking anti-P-sel Ab, followed by heme or PHZ. This

treatment prevented liver stress response in the PHZ-treated mice, as measured by the

decrease of NGAL protein staining (Fig. S10A-C) and mRNA expression (Fig. 4A). This

indicates that the injected dose shows some efficiency, despite that it did not rescue the ALT

!!"

levels (Fig. S11). Importantly, this P-sel blockade prevented complement deposits on liver endothelium in PHZ- and heme-treated WT mice (Fig. 4B and C), both on large vessels (Fig.

4D left) and on liver sinusoids (Fig. 4D right). Together, the in vitro and in vivo data demonstrate that the P-sel expression is a causal link between TLR4 and complement activation during intravascular hemolysis.

Discussion

Here we demonstrate that intravascular hemolysis triggers complement-dependent liver injury. We found a direct link between heme-triggered TLR4 signaling on endothelium and complement system activation (Fig. S12). These complement deposits are mediated by P-sel expression, causing recruitment of C3b and C3(H

O) (or a heme-promoted C3(H

O)-like form) on the cell surface. TLR4 signaling-mediated complement activation triggers liver stress response in hemolytic conditions, relevant for SCD.

Despite the clear evidence of complement activation in hemolytic diseases, its pathological

relevance remains unclear. After hemolysis induction, C3 activation fragments deposits

occurred on liver endothelium and in the sinusoidal vessels. Moreover, the increase of the

ALT levels and the overexpression of the inflammation and cell damage marker NGAL were

largely prevented in C3

mice. The terminal pathway was also activated, as measured by

upregulation of plasmatic C5a, despite the lack of detectable C5b-9 deposits in the liver. At

least in part the liver injury was C5-dependent, since ALT and NGAL staining partially

decreased after blockade of C5. These results place complement, and especially the C3

activation fragments, as a key mediator of liver tissue damage in hemolytic conditions, such

as SCD.

!#"

The process behind the acquisition of complement-activating phenotype by the endothelium is not well understood. Although predominant in the kidney, this phenomenon is not restricted to glomerular microvasculature(20) and is detected on liver endothelium of heme-injected mice(31) and here in mice with PHZ-induced hemolysis. Here we establish that the complement deposits on endothelium are mediated by TLR4 and triggered by the TLR4 ligand heme. Furthermore, TLR4 deficiency partially prevented the liver stress response in our hemolysis model. Our results support the findings of Bozza and colleagues(7) for the involvement of TLR4 in heme sensing under hemolytic conditions, here in a system exempt from certain heme-related artefacts occurring in vitro(40). Further, we investigated the molecular and cellular mechanism explaining complement activation on EC under hemolytic conditions.

Belcher et al. demonstrated that heme triggers EC activation and WPB mobilization via

TLR4(10). Both P-sel(39, 41) and vWF(42, 43) modulate complement activation. P-sel

promotes anchoring of C3b to EC membrane(39, 41) and, indeed, here we detected C3b/P-sel

interaction. Together with the covalent and non-covalent binding of C3b to the cell surface,

we found a non-covalent attachment of C3(H

O) (or a heme-promoted C3(H

O)-like form) to

heme-exposed EC. C3(H

O) is the fluid phase activation product of C3, critical for the so

called “tick over” of the AP(44). Here we discovered a non-conventional mechanism of

complement deposits triggering the AP, where P-sel is expressed on heme-treated EC surface

via TLR4-mediated process and serves as a platform for C3(H

O) attachment. This explains

why C3(H

O), was found on heme-exposed EC membrane. Another platform molecule for

recruitment of C3 fragments is properdin(45, 46), but we did not detect it on heme-exposed

EC. Here, blocking P-sel/C3 activation fragments interaction drastically reduced complement

activation and the endothelium injury. Together, these results demonstrate a central role of P-

sel to tag endothelium as a target for complement activation in vivo, and provide the missing

!$"

link between TLR4 and complement activation. This phenomenon is of a particular relevance, since P-sel is a critical player in the vaso-occlusion process of SCD patients(47–50). Indeed, P-sel inhibitor crizanlizumab was associated with a lower frequency of sickle cell–related pain crises in patients(51). Complement activation is also observed in SCD(52–54) and its blockade prevents stasis in a mouse model(55). Therefore, we postulate that a P-sel blockade will reduce complement activation on endothelium of SCD patients, and therefore the complement-mediated endothelial lesions.

At the trace amount used, sufficient for signaling pathway activation and P-sel expression, as shown here and described for microvascular EC(56) and in mice(41), LPS failed to induce complement activation. Only very high doses of LPS were able to activate complement, in line with previous works performed in vitro (57, 58) and in vivo(59). In contrast, heme exposes more abilities to promote complement activation on EC. This disparity with LPS lies in the capacity of heme to bind C3 and to generate C3(H

O)(11). The concomitant expression of P-sel and C3(H

O) generation by heme may explain its capacity to activate complement on endothelium in our model.

Our in vitro results indicate that TLR4 stimulation is not the only mechanism, contributing to complement-activating phenotype of heme-exposed human EC, while it is dominant in mice.

The loss of MCP contributes to the C3 activation fragments deposits in TLR4-independent

way, as seen on the MCP-silenced, heme-exposed EC. The decreased MCP levels will hamper

the inactivation of C3b to iC3b, promoting this generation of novel convertases. FH binds to

the heme-exposed cells to strengthen this regulatory capacity, but is not sufficient to

compensate the regulatory function in case of severe MCP loss. Of note, mice lack expression

of MCP on EC, pointing at the species differences in the mechanism of complement

regulation on endothelium(60) and might explain the dominant effect observed in mice. In the

mouse model, the complement activation by RBC microvesicles and the hypoxia-mediated

!%"

cell and tissue injury during hemolysis could account for the TLR-4 independent, complement-dependent part of the liver injury.

C3aR and C5aR were rapidly upregulated on EC in a TLR4-independent manner, most likely through exocytosis from still unidentified granules, different from WPB(61) and likely related to lysosomes, as in T-cells(20). Heme triggers C3a and C5a release in serum and on EC surface(11) and we detected it in the plasma of PHZ-injected mice. This could induce C3aR and C5aR signaling and the subsequent amplification of complement deposits(41, 61). C3a and C5a are potent pro-inflammatory mediators, playing a key role in vascular and tissue injury(19). Hemolysis, TLR4 signaling and the complement anaphylatoxins would synergize to trigger vascular stress response and tissue damage in hemolytic diseases. Moreover, the liver injury is a complex process. Additional hemolysis-derived factors or the ischemia/reperfusion injury are also well known for activating complement system and for involving TLR4. Heme could synergize also with other endogenous TLR4 ligands, which could be released during hemolysis such as HMGB1(62, 63), and could contribute to the liver injury and stress response. RBC microvesicles, activating complement and carrying adhesive C3b deposits, as well as hemoglobin-mediated effects through NO scavenging contribute to these hemolysis-mediated pathologic effects(64, 65).

In conclusion, our study demonstrated that complement triggers tissue injury during

intravascular hemolysis, by a novel mechanism of acquisition of a heme-dependent,

complement-sensitive activated phenotype by endothelium (Fig.S12). The sequence of

reactions involves: 1) heme-mediated signaling through TLR4, 2) P-sel expression and 3)

interaction of C3b and C3(H

O). This process triggers the amplification loop of the AP,

generating local inflammation. This mechanism could operate at any type of endothelium,

since EC generally express TLR4. On the basis of these results, targeted therapies on heme,

TLR4 pathway or P-sel could prevent organ dysfunction in hemolytic diseases, including by

!&"

limiting complement activation. Moreover, complement appears as a novel therapeutic target, at C3 and to some extend at C5 level, to prevent organ injury in hemolytic diseases.

Materials and methods

For fully detailed procedures, please refer to SI Appendix Materials and Methods.

Mouse treatment. All experiments were conducted in accordance with the recommendations for the care and use of laboratory animals and with the approval APAFIS#7135-"

2016100520465430v5 of the French Ministry of Agriculture. Eight week old C57Bl/6, C3

and TLR4

mice were injected i.p. with 100!L of PBS, freshly prepared heme (40

!mol/kg(10)), or with PHZ (900 !mol/kg). In a set of experiments, mice were pre-treated

with i.p. injection of 40 !mol/kg of human Hx 1 h before heme or PHZ injection. Blocking Ab against P-selectin, anti-C5 or their isotype controls were administered in a set of experiments.

mRNA level analyses. Snap-frozen liver sections were recovered in RLT buffer (Qiagen)+

1% !-mercaptoethanol and used for mRNA extraction and gene expression analyzed by

RTqPCR as described(24). NGAL expression was normalized by actin.

IF. Five !m thick frozen sections of mouse livers were stained for C3 activation fragments or NGAL. Co-localization with vWF staining was used to quantify the complement deposits on vascular endothelium in the liver as % of double positive area, expressed as FC.

Gene silencing. At 80% confluency, HUVEC were transfected in Opti-MEM medium

supplemented with lipotransfectamine and 4 nM of MCP siRNA for 20 min at RT.

!'"

ALT activity quantification. ALT activity in mouse plasma was quantified using a colorimetric assay, expressed in nmol/min/!L following the protocol provided by the manufacturer.

Surface Plasmon Resonance. Binding studies were performed using the ProteOn XPR36.

Recombinant human P-selectin was covalently coupled to a sensor chip. Interaction were measured with increased concentration of C3b, and C3(H

O).

Acknowledgements

This work was supported by grants from: Agence Nationale de la Recherche (ANR JCJC - INFLACOMP 2015-2018 ANR-15-CE15-0001) to LTR; European Research Council (ERC Starting Grant, ERC-StG-2015-678905 CoBABATI) to JDD; by a grant from CSL Behring France to LTR, and by INSERM. The cytometric and microsocpy analysis were performed at the «" Centre d'Histologie, d'Imagerie et de Cytométrie», (CHIC), Centre de Recherche des Cordeliers UMRS1138, (Paris, France), UPMC Flow Cytometry network (RECYF). We are grateful to the CHIC and CEF teams and to Amélie DiGiovanni for the excellent technical assistance.

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Figure legends

Figure 1: Pattern of staining for C3 activation fragments in the liver of mice with intravascular hemolysis and their dependence on TLR4 and heme. A-B) C3 fragments deposition on vascular endothelium. A) Double staining for vWF (green) and C3b/iC3b (red) of WT and TLR4

mice liver frozen sections treated with PBS, heme or PHZ. Co-localization is in orange. Focus on macrovessels, in particular central hepatic veins. White arrows point to C3 activation fragments deposition along endothelium. B) Staining quantification, presented as fold change (FC) of the double positive C3 activation fragments (C3 act fr)/vWF area of each liver section, normalized to the staining of the PBS-injected group of the WT mice.

Comparison of WT and TLR4

(n"3) mice. *: p<0.05; **: p<0.005, Two-way ANOVA with Tukey’s test for multiple comparisons. C) Frozen liver sections of WT mice, treated with PHZ +/- Hx, were stained for vWF (green) and C3 activation fragments (red).

Figure 2: Hemolysis triggers liver injury in a TLR4- and complement-dependent manner. WT, TLR4

and C3

mice were injected with PBS, heme or PHZ. Livers were recovered after 24 hours. A) ALT activity was measured in WT and TLR4

mice, presented as fold change (FC), compared to the PBS group (n"4). B) ALT activity was measured in WT and C3

mice (n"3). C) Gene expression of NGAL in livers from WT and TLR4

mice, FC (n"4). D) Gene expression of NGAL in livers from WT and C3

mice, FC (n"3). E-G) Efficacy of C5 blockade to prevent liver injury. F) C5a levels in the plasma of mice, injected with PHZ or pre-treated with irrelevant IgG (Irr IgG) or anti-C5 blocking Ab BB5.1 ("-C5).

The levels of C5a are compared at day -3 (D-3) and day 1 (D-1) post –PHZ injection. The prevention of C5a release indicated that the Ab exerted its blocking effect (n"4). F) ALT activity levels (FC) or G) quantification of the % of NGAL positive area in the liver (FC) in the mice, injected with PBS or PHZ, pretreated with Irr IgG or anti-C5. *: p<0.05; **:

p<0.005, ***: p<0.001, ****: p<0.0001; Two-way ANOVA with Tukey’s test for multiple comparisons. Values are box plots with median and Min/Max points in (A), (B), (E), (F), (G), and mean +/- SEM in (C) and (D). White arrowheads point to NGAL staining in parenchyma, white arrows point to NGAL staining along endothelium.

Figure 3: P-selectin is induced secondary to heme-mediated TLR4 activation and serves

as an anchoring platform for C3 activation fragments. A,B) Interaction between C3b/P-

selectin and C3(H

O)/P-selectin were studied by surface plasmon resonance by injecting

##"

increased concentration of C3b (A) or freeze/thaw native C3 (B) on P-selectin-coated chip.

C,D) HUVEC were treated with 100 !M of heme and exposed to 33% NHS for 30 min at 37

°C. Cells were detached, washed 3x at pH 7,2 or pH 2,5 and stained for C3a (C) or C3 activation fragments (C3 act fr) (D) deposition. E,F) HUVEC were treated with 100 !M of heme +/- 400 nM of TAK-242 +/- irrelevant Ab (irr Ig) or anti-P-selectin Ab ("P-sel) for 30 min at 37 °C and exposed to 33% NHS (with 10 mM EGTA and 2 mM MgCl2) for 30 min at 37 °C. Cells were detached and stained for C3 activation fragments deposition (n>5, flow cytometry). *: p<0.05; **: p<0.005, ***: p<0.001, ****: p<0.0001; Two-way ANOVA with Tukey’s test for multiple comparisons. Values are box plots with median and Min/Max points. FC: fold change, compared to basal level.

Figure 4: P-selectin blockade prevents complement deposits and liver stress response in

mice with intravascular hemolysis. Anti-P-selectin administration prevents complement

activation on endothelium and increases of liver injury marker NGAL in hemolytic

conditions. WT mice were injected with PBS, heme or PHZ after first administration of an

irrelevant (Irr Ig) or blocking Ab against P-selectin ("P-sel). A) Gene expression of NGAL in

livers. B,C) Staining quantification (n"4) for vascular C3 activation fragments deposits (C3

act fr). B) after injection of heme or C) after induction of hemolysis by PHZ. D) Examples of

double staining for vWF (green) and C3 activation fragments (red) of liver frozen sections by

IF after PHZ injection. Focus on macrovessels, in particular central hepatic veins (left) and on

sinusoidal capillaries (right). White arrows point to colocalization between C3 activation

fragments and vWF (orange). *: p<0,05, **: p<0.005, ***: p<0.001. Two way ANOVA with

Tukey’s test for multiple comparisons. Values are represented as mean +/- SEM in (A) and

box plots with median and Min/Max points in (B,C). FC: fold change, compared to PBS-

injected mice.

#$"

Figure 1

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Figure 2

#&"

Figure 3

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Figure 4

1  

Suppl ementary Materials and methods Supplementary Figur es

P-selectin drives complement attack on endothelium during intravascular hemolysis in TLR-4/heme-dependent manner

Nicolas S. Merle

, Romain Paule

, Juliette Léon

, Marie Daugan

, Tania Robe- Rybkine

, Victoria Poillerat

, Carine Torset

, Véronique Frémeaux-Bacchi

, Jordan D. Dimitrov

, Lubka T. Roumenina

1

INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France;

2

Sorbonne Universités, F-75006, Paris, France;

3

Université Paris Descartes, Sorbonne Paris Cité, F-75006, Paris, France

4

Service de médecine interne, Hôpital Foch, Suresnes

5

Assistance Publique ± +{SLWDX[GH3DULV6HUYLFHG¶,PPXQRORJLH%LRORJLTXH+{SLWDO Européen Georges Pompidou, F-75015, Paris, France

*

Correspondence to: Lubka T. Roumenina, Ph.D.

Cordeliers Research Center, INSERM UMRS 1138;

15 rue de l'Ecole de Medecine; 75006 Paris, France Phone: 33-1-44-27-90-96/ Fax: 33-1-40-51-04-20, e-mail: [email protected]

M ethods

R eage nts

The Fe

form of heme (hemin [ferriprotoporphyrin IX], designated as heme, (Sigma-Aldrich))

was dissolved to 20 mM in 50 mM NaOH and 145 mM NaCl, and further diluted in an

appropriate vehicle just before use. Stock solution of PHZ of 25 mg/mL (Sigma-Aldrich) was

2   prepared in PBS immediately before use. Inhibition of TLR4 was performed using TAK-242 molecule, diluted in PBS to a final concentration of 400 nM (Calbiochem, CAS 243984-11-4).

Human plasma-derived Hx was provided by CSL Behring. To test contamination with LPS, heme solution was pre-incubated with 1µg/mL of LPS inhibitor Polymyxin B (Sigma-Aldrich, P4932).

Animal expe rimentation

Mouse t re atment. All experiments were conducted in accordance with the recommendations

for the care and use of laboratory animals and with the approval APAFIS#7135-   2016100520465430v5 of the French Ministry of Agriculture. C57Bl/6 mice (WT mice) were from Charles RiveU/DERUDWRULHV/¶$UEUHVOH)UDQFHDQG7/5-/-(1) and C3-/-(2) were from an in house colony. Eight week old C57Bl/6, C3-/- and TLR4-/- mice were injected i.p. with 100 µL of PBS (Gibco) or with freshly prepared heme (40 µmol/kg, corresponding to 28 µg/g body weight(3)) repeated 24 hours later (day 0 and day 1). In another set of experiments WT mice were injected intravenously with 20 µmol/kg, 40 µmol/kg or 100 µmol/kg heme and sacrificed 24h later. Alternatively, mice were only injected i.p. at day 1 with PHZ (900 µmol/kg, corresponding to 0.125 mg/g body weight) and organs were recovered at day 2. In a set of experiments, mice were pretreated with i.p. injection of 40 µmol/kg of human Hx 1 h before heme or PHZ injection. Blocking antibody against P-selectin (BD Pharmingen, 553742) or its isotype control (BD Pharmingen, 5553993) was administered by retro-orbital injection 5 min before injection of heme, PHZ or vehicle. Concentrations and route of administration were chosen as described(4, 5). At the endpoint, organs were recovered and frozen in nitric liquid and stock to -80°C. We used male and female C57Bl6/J and TLR4-/- or C3-/- mice; sex- matched animals were used for each experiment.

WT mice were pre-treated with blocking antibody against P-selectin or irrelevant IgG by i.v.

3   injection in the eye 15 min prior i.p. injection of PBS, free heme or PHZ. In another set of experiments, WT mice were injected i.p. with isotype control or the anti-C5 blocking antibody BB5.1 (Hycult, HM1073-10B) at 1mg/mouse. Two hours later PHZ was administrated i.p. as above and mice were sacrificed after 24h.

mRNA level analyses

Snap-frozen liver sections were recovered in RLT buffer (Qiagen)+ 1% E-mercaptoethanol (Gibco) and used for mRNA extraction and gene expression analyzed by RTqPCR as described(4) previously for kidney sections. NGAL expression was normalized on actin housekeeping gene expression.

Immunofluorescence on mouse tissue.

Five µm thick frozen sections of mouse livers were cut with Cryostat Leica AS-LMD and fixed

in acetone on ice for 10 min. Complement deposition was studied using rat anti-mouse C3

fragments (C3bi/C3b/C3c) (Hycult, HM1078), visualized with goat anti-rat AF555

(Thermoscientific, A-21434). NGAL expression was studied using goat anti-mouse NGAL

(R&D system, AF1857), revealed by donkey anti-goat AF555 or AF647 (Thermoscientific,

A21432 and A21447). Endothelial cells were visualized with two EC markers, vWF using

polyclonal sheep anti-human vWF (Abcam, ab11713) revealed by donkey anti-sheep AF647

(Thermoscientific, A21448), and CD31 using polyclonal rabbit anti-mouse CD31 (Abcam,

ab124432) revealed by goat anti-rabbit AF647 (Thermoscientific, A21245). Granulocytes and

macrophages were respectively labeled with rat monoclonal anti-mouse Ly6G-PE (BD

3KDUPLQJHQŒ and rat monoclonal anti-mouse CD68-3(H%LRVFLHQFHŒ-0681-

80, FA-11). Epithelial cells were marked by monoclonal rabbit anti-mouse cytokeratin 7

(Abcam, ab181598), revealed by goat anti-rabbit (Thermoscientific, A21245), and platelets,

4   with monoclonal rat anti-mouse CD41a-$3&H%LRVFLHQFHŒ-0411-82 MWReg30). Stained slides were scanned by slide scanner Axio Scan (Zeiss). Quantification of the staining was performed using Visiopharm software (Visiopharm A/S, Hørsholm, Denmark). The colocalization of vWF and the C3 activation fragments was used to quantify the complement deposits on vascular endothelium in the liver as % of double positive area. The results were presented as fold change (FC), compared to the results of the PBS-injected mice. This organ has high amount of easily identifiable macro-vessels to allow robust analyses of macro and microvasculature. The NGAL staining is quantified as % of positive area and presented as FC compared to the results of the PBS-injected mice

ALT a ct ivity quant ification. ALT activity in mouse plasma was quantified using a colorimetric assay (Sigma, MAK052-1KT), expressed in nmol/min/µL following the protocol provided by the manufacturer.

D etect ion of C5a in mouse plasma. C5a levels in mouse plasma was quantified using a ELISA assay, using Purified Rat Anti-Mouse C5a (BD Pharmingen, Clone I52-1486) as capture and Biotin Rat Anti-Mouse C5a (BD Pharmingen, Clone I52-278) as detection antibody. The levels of C5a were compared for every mice at basal level (day ±3) and 24h after injection of PHZ.

The prevention of the PHZ-mediated increase of C5a in the plasma after injection of the anti- C5 blocking antibody Bb5.1 was used as marker to validate the antibody efficacy.

Complement activation and complement receptors on E C

F low cytometry. HUVEC were cultured in 24-wells plates as previously described(6, 7),

exposed to heme at the indicated doses for 30 min at 37 °C in serum-free M199 cell culture

5   medium (Gibco) in presence or absence of TAK-242 (Calbiochem, 614316). For evaluation of complement activation, HUVEC either washed and further exposed to NHS diluted to 33% in M199 medium for 30 min at 37 °C. HUVEC were used for experiments until passage 4. Cells were washed, detached, labeled and fixed in 0.5% formaldehyde. For studying non-covalent binding of complement deposition, cells were briefly washed 3 times with either normal M199 2% FCS or with M199 2% FCS adjusted to pH 2.5 (acidic wash). Cells were analyzed by flow cytometry (BD LSR II), and further analyzed by FlowJo X. The following antibodies were used for staining: anti-human MCP-RPE (BIORAD, MCA2113PE), anti-human C3aR-PE (BioLegend, 345804), anti-human C5aR-APC (BioLegend, 344310), in addition to the corresponding isotype controls. After incubation with serum, staining was performed with anti- properdin mouse monoclonal IgG1 (Quidel, A233), anti-FH mouse monoclonal IgG1 (Quidel, A254), anti-C3c mouse monoclonal IgG1 antibody (Quidel, A205), anti-C3a rabbit polyclonal antibody (CompTech, A218), or with anti-C5b-9 mouse monoclonal IgG1 (kindly provided by Prof. Paul Morgan, Cardiff University) or an isotype control and revealed by anti-mouse-IgG1- PE secondary antibody (Beckman Coulter, IM0551) or goat anti-rabbit IgG-AF555 secondary antibody (ThermoFisher, A21429). Blocking antibody against P-selectin (R&D systems, AF137) was diluted in M199 FCS-free medium supplemented with 10mM EGTA and 2mM MgCl

to a final concentration of 25 µg/ml or with a polyclonal sheep antibody as an irrelevant antibody (R&D systems, 5-001). HUVEC were stained for C3 deposition and C5b-9 formation as described below.

G ene silencing. At 80% confluency, HUVEC have been cultured in F12K medium,

supplemented with 2 mM L-glutamine, 0.1 mg/mL heparin, 0.05 mg/mL ECGS (Merck, 02-

102) and 10% FCS. HUVEC were transfected in Opti-MEM medium (Gibco) supplemented

6   with lipotransfectamine (Thermo Fisher Scientific) and 4 nM of MCP siRNA (Qiagen, SI02654883) for 20 min at room temperature.

Immunofluoresence. HUVEC were cultured on slides in 24-well plates. Cells were fixed in paraformaldehyde 4% after staining of P-selectin, CD31, vWF, p38, p65. Respectively, staining was performed with anti-human P-selectin primary antibody (BIORAD, MCA796), rabbit anti- human vWF (DAKO, A0082), mouse anti-human CD31 (Ancell, 180-020), phospho-p38 (Thr180/Tyr182) (#9211, Cell Signaling), phospho-p65 (Ser536) (#3033, Cell Signaling) or matched isotype controls. Markers were revealed with rabbit anti-mouse AF-488 secondary antibody (ThermoFisher, A21204) or goat anti-rabbit AF555 (ThermoFisher, A21429) and DAPI (to stain nuclei). Staining was analyzed by Axiovert 200M microscope (Zeiss).

C ellular E LISA. HUVEC cells were cultured on slides in 24-well plates. HUVEC were exposed to 200 ng/mL LPS for 1 h. Cells were washed and stained for vWF HRP (DAKO, P022602) or P-selectin primary antibody (BIORAD, MCA796) and revealed with secondary anti-mouse HRP antibody (Santa Cruz, sc-2005).

Western Blot. Cells were treated with 25 µM of heme from 30 min to overnight, 100 ng/mL of

LPS as positive controls. Cells were washed, detached and incubated 30 min at 4°C in RIPA

lysis buffer. Phosphorylation of p65 was confirmed by WB using Phospho-65 (Ser536) (#3033,

Cell Signaling), controlled with total NF-ț%S'(&HOO6LJQDOLQJXVLQJWKH

SNAP technology (Merk Millipore), followed by anti-rabbit IgG, HRP-linked antibody (#7074,

Cell Signaling). The signal was revealed by chemiluminescence.