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SARS-CoV-2 infects cells by attachment to its receptor – the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation- and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affect the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism, and capacity to evade immune responses during evolution.

Recently, numerous studies report bats as reservoirs of emerging pathogens with little to no signs of infections. This is thought to be connected to the unique immune system of bats, which remains poorly characterized. Despite the physiological importance of the Neonatal Fc receptor (FcRn) in the homeostasis of IgG antibodies, it is unclear how its functional activity is evolutionary conservative among mammals, and so is the case for bats. Using surface plasmon resonance-based technology, we tested the interactions of IgG antibodies isolated from three bat species with recombinant human and mouse FcRn. Our data show that IgG from the studied bat species binds to both human and mouse FcRn, albeit with distinct affinities. Importantly, the binding pattern of bat IgG is similar to human IgG. This confirms the conservative nature of IgG-FcRn interaction and highlights the importance of FcRn IgG salvaging system in bats.

International audience; Decrypting the B cell ontogeny of HIV-1 broadly neutralizing antibodies (bNAbs) is paramount for vaccine design. Here, we characterized IgA and IgG bNAbs of three distinct B cell lineages in a viremic controller, two of which comprised only IgG+ or IgA+ blood memory B cells; the third combined both IgG and IgA clonal variants. 7-269 bNAb in the IgA-only lineage displayed the highest neutralizing capacity despite limited somatic mutation, and delayed viral rebound in humanized mice. bNAbs in all three lineages targeted the N332 glycan supersite. The 2.8-Å resolution cryo-EM structure of 7-269-BG505 SOSIP.664 complex showed a similar pose as 2G12, on an epitope mainly composed of sugar residues comprising the N332 and N295 glycans. Binding and cryo-EM structural analyses showed that antibodies from the two other lineages interact mostly with glycans N332 and N386. Hence, multiple B cell lineages of IgG and IgA bNAbs focused on a unique HIV-1 site of vulnerability can codevelop in HIV-1 viremic controllers.

International audience; Therapeutic antibodies should cover particular physicochemical and functional requirements for successful entry into clinical practice. Numerous experimental and computational approaches have been developed for early identification of different unfavourable features of antibodies. Immune repertoires of healthy humans contain a fraction of antibodies that recognize nitroarenes. These antibodies have been demonstrated to manifest antigen-binding polyreactivity. Here we observed that >20 % of 112 clinical stage therapeutic antibodies show pronounced binding to 2,4-dinitrophenol conjugated to albumin. This interaction predicts a number of unfavourable functional and physicochemical features of antibodies such as polyreactivity, tendency for self-association, stability and expression yields. Based on these findings we proposed a simple approach that may add to the armamentarium of assays for early identification of developability liabilities of antibodies intended for therapeutic use.

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

International audience; Therapeutic antibodies have transformed the clinical practice. Not surprisingly, development of antibody thera-peutics is currently the main focus of the biotechnology industry. Nonetheless, the development process is complex , and many antibodies do not reach the clinic. Reasons for the failures include, undesired binding behavior (polyreactivity), low stability, poor expression yields, unfavorable pharmacokinetics etc. Numerous studies have proposed different analytical methods for assessment of physicochemical parameters of antibodies and identification of problematic molecules at early stages of the development process. These studies, however, have not addressed the basic mechanistic question of how sequence features of variable regions determinate the different biophysical characteristics and the binding behavior of the antibodies. In a recent study, Jain et al assessed 12 biophysical qualities of 137 monoclonal therapeutic antibodies. We used the raw data from this comprehensive study to perform correlation analyses of different biophysical measurables with various sequence features of variable regions of the antibodies-number of mutations, length of hypervariable loops, and frequency of amino acid residue types. The obtained data reveled significant relationships between the sequence characteristics of the variable domains and different physiochemical properties of antibodies. The data from this study can assist in design of a set of criteria for early identification of antibodies with developability issues. Moreover, our findings provide novel fundamental insights into the sequence-function relationship of antibodies.

Antibodies are essential components of adaptive immunity. A typical antibody repertoire comprises an enormous diversity of antigen-binding specificities, which are generated by the genetic processes of recombination and mutation. Accumulating evidence suggests that the immune system can exploit additional strategies to diversify the repertoire of antigen specificities. These unconventional mechanisms exclusively target the antigen-binding sites of immunoglobulins and include the insertion of large amino acid sequences, post-translational modifications, conformational heterogeneity and use of nonprotein cofactor molecules. Here, we describe the different unconventional routes for diversification of antibody specificities. Furthermore, we highlight how the immune system has a much greater level of adaptability and plasticity than previously anticipated, which goes far beyond that encoded in the genome or generated by the acquisition of somatic mutations. Diversification of the antibody repertoire is well known to be driven by genetic recombination and mutation. However, it is becoming apparent that other processes can also diversify antibody specificities. Here, Dimitrov and colleagues discuss these unconventional strategies for antibody diversification and consider why these extra strategies have evolved.

The study of kinetics and thermodynamics of protein-protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein-protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Immunoglobulin repertoires contain a fraction of antibodies that recognize low molecular weight compounds, including some enzymes’ cofactors, such as heme. Here, by using a set of 113 samples with variable region sequences matching clinical-stage antibodies, we demonstrated that a considerable number of these antibodies interact with heme. Antibodies that interact with heme possess specific sequence traits of their antigen-binding regions. Moreover they manifest particular physicochemical and functional qualities i.e. increased hydrophobicity, higher propensity of self-binding, higher intrinsic polyreactivity and reduced expression yields. Thus, interaction with heme is a strong predictor of different molecular and functional qualities of antibodies. Notably, these qualities are of high importance for therapeutic antibodies, as their presence was associated with failure of drug candidates to reach clinic. Our study reveled an important facet of information about relationship sequence-function in antibodies. It also offers a convenient tool for detection of liabilities of therapeutic antibodies., Lecerf et al. examine over 100 samples with variable region sequences matching clinical-stage immunoglobulin antibodies, to demonstrate that many interact with heme. They find that those that do interact with heme possess specific sequence traits that manifest qualities such as increased hydrophobicity, self-binding and intrinsic polyreactivity, thus suggesting that heme interaction is a predictor for therapeutically important qualities.

The study of kinetics and thermodynamics of protein-protein interactions can contribute to assessment of the mechanism of molecular recognition process. These analyses can provide information about conformational changes and noncovalent forces that influence the initial recognition between proteins and stabilization of the complex. Studying these aspects may lead to a better comprehension of functions of proteins in biological environment and can become useful for the rational modification of some interactions by engineering of one of the implicated partners. Real-time biosensor assays based on surface plasmon resonance have been widely applied for the label-free evaluation of protein-protein interactions, allowing their characterization in term of binding affinity and kinetics. In the present chapter, we provide a protocol for the assessment of interactions involving complement proteins or antibodies, the protagonists of the immune system. We reported guidelines and indications concerning the analysis of the experimental data for the estimation of the kinetic parameters and for the evaluation of activation and equilibrium binding thermodynamics.

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

Heme (Fe protoporphyrin IX) serves as a prosthetic group of numerous proteins implicated in oxidative metabolism. This molecule is abundantly present in the red blood cells where it serves as a cofactor of hemoglobin. As consequence of various pathological conditions, the membrane of red blood cells can be damaged and therefore large quantities of hemoglobin and subsequently heme released in the extracellular space. Since heme is a highly reactive compound, when released extracelluarly it can influence the functional activity of different plasma components. Thus, previous investigations have demonstrated that heme can interact with components of complement system and immunoglobulins, profoundly affecting their functions. Here we propose two basic protocols that can be used for characterization of interaction of free heme with complement proteins and immunoglobulins. The first technique is based on UV-Vis absorbance spectroscopy. It allows general characterization of the heme binding to the protein and estimation of the number of heme binding sites. The second protocol consists in the use of biosensor assay based on surface plasmon resonance. This protocol would be useful for evaluation of heme binding kinetics and equilibrium affinity. Besides for complement components and immunoglobulins, the presented protocols can be utilized for characterization of the interaction of heme with other proteins.

Heme's interaction with Toll-like receptor 4 (TLR4) does not fully explain the proinflammatory properties of this hemoglobin-derived molecule during intravascular hemolysis. The receptor for advanced glycation end products (RAGE) shares many features with TLR4 such as common ligands and proinflammatory, prothrombotic, and pro-oxidative signaling pathways, prompting us to study its involvement as a heme sensor. Stable RAGE-heme complexes with micromolar affinity were detected as heme-mediated RAGE oligomerization. The heme-binding site was located in the V domain of RAGE. This interaction was Fe3+ -dependent and competitive with carboxymethyllysine, another RAGE ligand. We confirmed a strong basal gene expression of RAGE in mouse lungs. After intraperitoneal heme injection, pulmonary TNF-α, IL1β, and tissue factor gene expression levels increased in WT mice but were significantly lower in their RAGE-/- littermates. This may be related to the lower activation of ERK1/2 and Akt observed in the lungs of heme-treated, RAGE-/- mice. Overall, heme binds to RAGE with micromolar affinity and could promote proinflammatory and prothrombotic signaling in vivo, suggesting that this interaction could be implicated in heme-overload conditions.

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFβ, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naive mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4+ T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties.

International audience; Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans.

Neonatal Fc receptor (FcRn) has a key role in the homeostasis of IgG. Despite its physiological and clinical importance, the interaction of IgG and FcRn remains not completely comprehended. Thus, IgG molecules with identical constant portions but with minor differences in their V regions have been demonstrated to interact with FcRn with a considerable heterogeneity in the binding affinity. To understand this discrepancy, we dissected the physicochemical mechanism of the interaction of 10 human IgG1 to human FcRn. The interactions of two Abs in the presence of their cognate Ags were also examined. Data from activation and equilibrium thermodynamics analyses as well as pH dependence of the kinetics revealed that the V region of IgG could modulate a degree of conformational changes and binding energy of noncovalent contacts at the FcRn binding interface. These results suggest that the V domains modulate FcRn binding site in Fc by allosteric effects. These findings contribute for a deeper understanding of the mechanism of IgG–FcRn interaction. They might also be of relevance for rational engineering of Abs for optimizing their pharmacokinetic properties.

International audience; In humans, maternal IgGs are transferred to the fetus from the second trimester of pregnancy onwards. The transplacental delivery of maternal IgG is mediated by its binding to the neonatal Fc receptor (FcRn) after endocytosis by the syncytiotrophoblast. IgGs present in the maternal milk are also transferred to the newborn through the digestive epithelium upon binding to the FcRn. Importantly, the binding of IgGs to the FcRn is also responsible for the recycling of circulating IgGs that confers them with a long half-life. Maternally delivered IgG provides passive immunity to the newborn, for instance by conferring protective anti-flu or anti-pertussis toxin IgGs. It may, however, lead to the development of autoimmune manifestations when pathological autoantibodies from the mother cross the placenta and reach the circulation of the fetus. In recent years, strategies that exploit the transplacental delivery of antigen/IgG complexes or of Fc-fused proteins have been validated in mouse models of human diseases to impose antigen-specific tolerance, particularly in the case of Fc-fused factor VIII (FVIII) domains in hemophilia A mice or pre-pro-insulin (PPI) in the case of preclinical models of type 1 diabetes (T1D). The present review summarizes the mechanisms underlying the FcRn-mediated transcytosis of IgGs, the physiopathological relevance of this phenomenon, and the repercussion for drug delivery and shaping of the immune system during its ontogeny.

The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 μg). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins.

International audience; The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.

International audience; The canonical functions of antibodies are based on joining the process of antigen recognition with initiation of innate immune reactions. With the introduction of modern research approaches, and the use of sophisticated model systems, the recent years have witnessed the discovery of numbers of non-canonical functions of antibodies. These functions encompass either untypical strategies for neutralization of pathogens or exertion of activities by antibodies that are typically characteristic for other proteins (cytokines, chaperons or enzymes). Here we provide an overview of non-canonical functions of antibodies and discuss their mechanisms and implications for immune regulation and immune defence. A better comprehension of these functions will enrich our knowledge about the adaptive immune response and shall inspire the development of novel therapeutics.

International audience; Natural antibodies (nAbs) are most commonly defined as immunoglobulins present in the absence of pathological conditions or deliberate immunizations. Occurrence of nAbs in germ- and antigen-free mice suggest that their production is driven, at least in part, by self-antigens. Accordingly, nAbs are constituted of natural autoantibodies (nAAbs), and can belong to the IgM, IgG, or IgA subclasses. These nAbs provide immediate protection against infection while the adaptive arm of the immune system mounts a specific and long-term response. Beyond immediate protection from infection, nAbs have been shown to play various functional roles in the immune system, which include clearance of apoptotic debris, suppression of autoimmune and inflammatory responses, regulation of B cell responses, selection of the B cell repertoires, and regulation of B cell development. These various functions of nAbs are afforded by their reactivity, which is broad, cross-reactive, and shown to recognize evolutionarily fixed epitopes shared between foreign and self-antigens. Furthermore, nAbs have unique characteristics that also contribute to their functional roles and set them apart from antigen-specific antibodies. In further support for the role of nAbs in the protection against infections and in the maintenance of immune homeostasis, the therapeutic preparation of polyclonal immunoglobulins, intravenous immunoglobulin (IVIG), rich in nAbs is commonly used in the replacement therapy of primary and secondary immunodeficiencies and in the immunotherapy of a large number of autoimmune and inflammatory diseases. Here, we review several topics on nAbs features and functions, and therapeutic applications in human diseases.

In a healthy immune repertoire, there exists a fraction of polyreactive antibodies that can bind to a variety of unrelated self- and foreign antigens. Apart from naturally polyreactive antibodies, in every healthy individual, there is a fraction of antibody that can gain polyreactivity upon exposure to porphyrin cofactor heme. Molecular mechanisms and biological significance of the appearance of cryptic polyreactivity are not well understood. It is believed that heme acts as an interfacial cofactor between the antibody and the newly recognized antigens. To further test this claim and gain insight into the types of interactions involved in heme binding, we herein investigated the influence of a group of aromatic guanylhydrazone molecules on the heme-induced antibody polyreactivity. From the analysis of SAR and the results of UV-vis absorbance spectroscopy, it was concluded that the most probable mechanism by which the studied molecules inhibit heme-mediated polyreactivity of the antibody is the direct binding to heme, thus preventing heme from binding to antibody and/or antigen. The inhibitory capacity of the most potent compounds was substantially higher than that of chloroquine, a well-known heme binder. Some of the guanylhydrazone molecules were able to induce polyreactivity of the studied antibody themselves, possibly by a mechanism similar to heme. Results described here point to the conclusion that heme indeed must bind to an antibody to induce its polyreactivity, and that both π-stacking interactions and iron coordination contribute to the binding affinity, while certain structures, such as guanylhydrazones, can interfere with these processes. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3794]

International audience; Therapeutic intravenous immunoglobulin preparations (IVIg) are used for treatment of wide range of auto-immune and inflammatory diseases. Versatile mechanisms have been reported to contribute to the im-munomodulatory effects of IVIg. Here we demonstrate that IVIg has a strong potential to inhibit pro-in-flammatory effect of extracellular heme. Indeed, the presence of immunoglobulins reduced the potential of heme to activate the complement system on the surface of human endothelial cells. Since extracellular heme is considered as one of the principal pathogenic factors in hemolytic disorders, its therapeutic scavenging by IVIg may have significant clinical repercussions.

International audience

Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal.

Introduction Le syndrome hemolytique et uremique atypique (SHUa) est une microangiopathie thrombotique renale caracterisee par une hemolyse mecanique et une suractivation du complement. Il est demontre que l’heme, derive de l’hemolyse, lie C3a, amplifiant ainsi l’activation du complement. Les mecanismes d’action du complexe heme-C3a sont inconnus. Une interaction du C3a avec le Recepteur aux produits de glycation avancee (RAGE) est rapportee. RAGE, recepteur multi-ligands ubiquitaire, est implique dans differentes pathologies a tropisme renal (nephropathie diabetique, lupique, polykystose) mais aucun role dans le SHUa n’est decrit a ce jour. Notre objectif etait ainsi d’analyser l’interaction C3a-RAGE en conditions hemolytiques. Patients/Materiels et methodes Les interactions entre RAGE, l’heme et C3a etaient etudiees en resonance plasmonique de surface (SPR), spectrophotometrie et chromatographie d’exclusion serique. Pour analyser in vivo l’impact de RAGE en conditions hemolytiques, des souris C57Bl/6 RAGE−/− et leurs controles +/ + littermates recevaient ± une injection intraperitoneale d’heme (50, 100, 200 μmol/kg) avant sacrifice a 16 h. L’expression renale des genes de RAGE et de marqueurs inflammatoires (IL1β, IL6, TNFα) etait etudiee par RTqPCR. Observation/Resultats En SPR, le RAGE recombinant liait la carboxymethyllysine, son principal ligand connu, mais l’interaction RAGE-C3a restait indetectable. A contrario, C3a expose a l’heme liait le RAGE avec une haute affinite. Cette interaction etait donc mediee par l’heme, qui liait RAGE de facon dose-dependante. L’immobilisation des differents domaines de RAGE permettait d’identifier le site de liaison de l’heme sur le domaine V. L’interaction domaine V-heme etait tres affine et specifique, alors que l’heme ne se liait pas aux domaines C1 et C2. Cette liaison etait confirmee en spectroscopie et chromatographie d’exclusion. In vivo, l’injection d’heme a des souris induisait une augmentation dose-dependante de l’expression de RAGE ainsi que l’activation du complement au niveau renal, menant au clivage du C3 et la generation de fragments d’activation du C3 sur l’endothelium glomerulaire. Nous observions egalement une augmentation dose-dependante de l’expression renale des marqueurs de l’inflammation, quantifiee a la condition heme 200 μM/kg autour de × 4, × 200 et × 3,5 pour IL1β, IL6 et TNFα respectivement par rapport au controle (PBS). Les analyses preliminaires montraient une elevation moindre de l’expression de ces genes chez les souris RAGE−/−, dans les memes conditions : × 1,5, × 50 et × 2 respectivement. Discussion/Conclusion Ce travail revele RAGE comme un nouveau recepteur de l’heme. En conditions hemolytiques, les souris RAGE−/− semblent en partie protegees de l’inflammation au niveau renal, ouvrant des perspectives therapeutiques dans les maladies hemolytiques via les molecules anti-RAGE.