Je Remercie Également Jean-Baptiste

Philippe Jean-Philippe Pascale

Merci À Caroline

Aline Katharina

Michel Luca

Les Sagag

Evelyne, Rabia, Rebekka, Yoann Romain Thibault

Notamment Rana

Lynda Rida

Anna Kévin

Anne-So Kaiyao

Anas, Wiktor, Laura, Quentin Elena Thibault

Romain Benoit

Elise Clarisse

Simon

Damien Baptiste

Ghadir J'

Julie Sarah

Sandrine

Marjo

Mes remerciements vont tout d'abord à mes directrices de thèse Véronique Rossi et Christine Gaboriaud. Un immense merci pour votre soutien, votre disponibilité, votre patience mais avant tout pour la confiance que vous m'avez accordée. Vous avez su me laisser réaliser ce travail avec beaucoup de liberté tout en gardant un oeil attentif sur l'avancement du projet.

Véronique, je te suis extrêmement reconnaissant de m'avoir accepté en stage lorsque je n'étais alors qu'un étudiant de L1, de m'avoir renouvelé ta confiance en master pour ensuite enchainer avec cette thèse. Grâce à toi, j'ai pu faire connaissance avec C1q et découvrir les joies de l'enzymo, des ELISA et de la SPR. Tu as grandement contribué à me donner l'envie d'embrasser la voie de la biochimie et d'aller jusqu'au bout avec cette thèse.

Christine, je te remercie pour tout le temps que tu m'as consacré durant ces trois année (et avant !). Je garderai un très bon souvenir de nos sessions synchrotron où tu m'as toujours accompagné dans la joie et la bonne humeur ! Tes photos de paysages et animaux sauvages vont me manquer mais j'espère en recevoir par mail régulièrement 😉😉.

Je remercie vivement Nicole Thielens pour sa bienveillance et toute l'attention qu'elle a pu porter sur mes travaux tout au long de ces trois années de thèse.

Merci à toutes les trois pour vos conseils avisés, votre bonne humeur et votre positivité qui ont été de précieux alliés pendant les trois années qui viennent de s'écouler.

Je remercie les membres de mon jury de thèse pour avoir accepté de consacrer de leur temps afin d'évaluer mon travail, Catherine Moali et Lubka Roumenina en tant que rapporteures et Yves Delneste et Franck Fieschi en tant qu'examinateurs.

Je tiens également à remercier affectueusement l'ensemble des membres du groupe IRPAS sans qui cette expérience n'aurait pas été aussi enrichissante.

Un immense merci à Isabelle pour tout ce qu'elle a fait pour moi, que ce soit pour me montrer et m'apprendre la culture cellulaire et la purification de C1q ou pour son aide pour réaliser les dernières manips pendant que j'étais coincé par la rédaction. J'ai passé de très bons moments au labo à tes côtés, merci pour ça.

II

De la même façon, je remercie Anne pour son aide au lancement du projet pour me montrer la production en bactéries. Merci pour ta bonne humeur permanente qui m'a permis de garder le sourire tout au long de ces trois années.

Merci à Samy, avec qui j'ai partagé le bureau pendant presque la totalité de ma thèse. Il s'en sera dit des choses dans ce bureau… Merci pour les moments que l'on a vécu ensembles (au labo et en dehors), qui m'ont permis de décompresser et relativiser quand les manips ne marchaient pas.

TABLE OF FIGURES

INTRODUCTION

I. The complement system 1

General overview

The complement system represents a major component of innate immunity and constitutes the fast and efficient first-line host defense against pathogens (Walport 2001a, b). Initially, the complement system was discovered in the 19 th century as a heat-labile serum component essential for antibody-mediated bactericidal activity. It is a very efficient part of innate immunity able to opsonize pathogens and apoptotic cells surfaces for their elimination through phagocytosis or lysis. This complex system consists in more than 40 soluble and membrane proteins. The main producer of the circulating soluble complement proteins is the liver (hepatocytes) but local production of complement proteins can be performed by epithelial, endothelial and immune cells [START_REF] Zhou | Hepatocytes: a key cell type for innate immunity[END_REF][START_REF] Lubbers | Production of complement components by cells of the immune system[END_REF]. Complement proteins are produced in an inactive form and require proteolytic cleavage for activation. After initial activation, they are able to associate and sequentially activate each other, triggering the local generation of fragments playing an important role in pathogens elimination through inflammatory signaling, opsonization and lysis (Merle et al. 2015b).

The complement cascade

The complement cascade can be initiated by three different pathways of activation: the classical, lectin and alternative pathways. Each activation pathway leads to the assembly of an enzymatic complex, the C3 convertase able to cleave the central component C3 that leads to the activation of a common amplification loop and terminal pathway (Figure 1).

3

The three complement activation pathways

The classical and lectin pathways

Both the classical and lectin activation pathways rely on the recognition of pathogens or alteredself components triggering the activation of proteases and initiating the complement cascade by the cleavage of C4 and C2 complement components, releasing C4a, C4b, C2a and C2b fragments. Association of C4b and C2a fragments generates the membrane-associated enzymatic assembly [C4bC2a] called C3 convertase that cleaves the C3 component into C3a and C3b (Figure 1). The classical and lectin pathways are initiated by homologous multimolecular complexes composed of a recognition molecule that belongs to the soluble defense collagens family associated with their cognate complement protease.

The soluble defense collagens are a family of proteins gathering complement C1q, mannosebinding lectin (MBL), the collectin CL-LK, ficolins and pulmonary surfactant proteins A and D (SP-A and SP-D). They all contain a C-terminal recognition domain preceded by a collagenlike triple-helical region [START_REF] Fraser | Directing an appropriate immune response: the role of defense collagens and other soluble pattern recognition molecules[END_REF][START_REF] Casals | Soluble defense collagens: Sweeping up immune threats[END_REF].

Soluble defense collagens are pattern recognition molecules of the innate immune system playing an important role in the sensing and elimination of pathogens and cell debris. Indeed, the defense collagens can recognize pathogens or apoptotic cells-associated molecular patterns through their C-terminal domain and trigger efficient immune reactions such as complement activation, phagocytosis stimulation or immune cells activation (Figure 2) [START_REF] Casals | Soluble defense collagens: Sweeping up immune threats[END_REF].

The classical pathway recognition molecule is the defense collagen C1q, which is able to interact with antibodies (IgG and IgM) complexed with antigens, pentraxins, pathogen associated molecular patterns (PAMPs) and apoptotic cell associated molecular patterns (ACAMPs). C1q interaction with its ligands triggers the subsequent activation of its associated proteases within the C1 complex: C1r and C1s. Activated C1s then cleaves C4 and C2 components to generate the classical pathway C3 convertase (C4b2a) (Figure 1). [START_REF] Casals | Soluble defense collagens: Sweeping up immune threats[END_REF].

The lectin pathway can be initiated by several defense collagens: MBL, CL-K1, CL-LK and ficolins 1, 2 and 3, able to recognize glycoproteins and glycolipids presents on pathogens surfaces (Walport 2001a;[START_REF] Fujita | Evolution of the lectin-complement pathway and its role in innate immunity[END_REF]. Each of these defense collagens is associated with MASP-1 and -2 (MBL-associated serine protease 1 and 2). As for the classical pathway, the defense collagen interaction with its ligands induces the activation of MASP-1 and MASP-2, leading to the C4 and C2 components cleavage and the same C3 convertase as for the classical pathway (Figure 1).

The alternative pathway and amplification loop

The alternative pathway activation does not rely on pathogen recognition and subsequent This mechanism of C3 cleavage generating C3b fragments associating with FB and promoting further C3 cleavage leads to a strong complement activity and is called the complement amplification loop (Figure 1).

The terminal pathway

Each of the above-described pathway of complement activation leads to the formation of an enzymatic complex called C3 convertase, able to cleave C3 into C3a and C3b fragments.

Thanks to the amplification loop, large amounts of C3 fragments can be produced even with a weak initial activation. Once the concentration of C3b deposition on the pathogen surface is high enough, the C3 convertase associates with additional C3b molecules and switches from C3 to C5 substrate specificity [START_REF] Pangburn | Structure and function of complement C5 convertase enzymes[END_REF][START_REF] Rawal | Formation of high affinity C5 convertase of the classical pathway of complement[END_REF]. The resulting C5 convertase then cleaves C5 into C5a and C5b fragments. C5b then sequentially associates with C6, C7, C8 and C9 on the target's membrane. Polymerization of C9 and association to the C5b-8 deposited complex leads to the formation of the final [C5b-9] membrane attack complex (MAC) which consists in a pore inserted in the target's membrane inducing cell lysis and death [START_REF] Podack | Molecular composition of the tubular structure of the membrane attack complex of complement[END_REF][START_REF] Tegla | Membrane attack by complement: the assembly and biology of terminal complement complexes[END_REF]) (Figure 1).

In addition to cell lysis through the MAC formation, the complement system exhibits diverse biological functions like opsonization and inflammation properties. Indeed, complement components fragments generated by the activation cascade are not exclusively involved in the terminal MAC formation. For example, C3a and C5a fragments, the by-products of C3 and C5 cleavage, are anaphylatoxins that interact with specific cell receptors, triggering important functions in immune cells recruitment, inflammatory signals generation and maturation of the adaptive immune system (Elsner et al. 1994a, b;[START_REF] Ehrengruber | Activation of human neutrophils by C3a and C5A. Comparison of the effects on shape changes, chemotaxis, secretion, and respiratory burst[END_REF][START_REF] Schraufstatter | Complement c3a and c5a induce different signal transduction cascades in endothelial cells[END_REF][START_REF] Peng | The role of anaphylatoxins C3a and C5a in regulating innate and adaptive immune responses[END_REF]Merle et al. 2015a). Moreover, complement component fragments deposited on a target surface can be directly recognized by immune cells receptors, thus enhancing targets clearance and phagocytosis. For example, after complement activation, target cells present both C3b and C4b fragments on their surface that can be recognized by several receptors on immune cells surfaces such as CR1, CR2, CR3 or CR4 [START_REF] Ricklin | Complement: a key system for immune surveillance and homeostasis[END_REF].

Regulation of the complement system

The complement system is a very efficient weapon of innate immunity against pathogens and damaged tissues elimination but it can be very harmful for neighboring cells if the activation cascade is uncontrolled. This highlights the need for a tight regulation of the complement cascade. Such control is performed by several soluble and membrane regulators that operate at different levels of the cascade [START_REF] Zipfel | Complement regulators and inhibitory proteins[END_REF].

The most relevant way of regulation of early complement activation is the inactive proenzyme state of the initiating proteases that are activated only after target recognition by the defense collagens of the initiation complexes. The consecutive activation of the proteases leads to the initiation of the classical and lectin pathways. In addition, the serine protease activity of C1r, C1s and the MASPs is controlled by a soluble inhibitor: C1-inhibitor (C1-inh; SERPING1).

C1-inh is a member of the serpin family of serine protease inhibitors that binds to the active site of its target protease and irreversibly blocks its enzymatic activity. This inhibitory process also induces the dissociation of the C1 complex [START_REF] Sim | Interaction of 125I-labelled complement subcomponents Cr and Cs with protease inhibitors in plasma[END_REF][START_REF] Davis | BIOLOGICAL ACTIVITIES OF C1 INHIBITOR[END_REF]. C1-inh is present in the serum at a 7-times higher concentration than the C1 complex, therefore preventing unsuitable activation of the C1-hosted proteases (137 µg/mL (C1-inh) versus 80 µg/mL (C1q); [START_REF] Schumaker | Activation of the First Component of Complement[END_REF]).

The next level of regulation of the complement cascade occurs at the level of the central C3 and C5 convertases through several membrane and soluble proteins that inhibit their association.

One of the principal regulators is factor I (FI), a soluble serine protease able to cleave C3b and C4b, thereby inducing the convertase inactivation. The activity of FI towards C3b and C4b

fragments is dependent on various cofactors such as the soluble factor H (FH), the C4b-binding protein (C4BP), the membrane complement receptor type 1 (CR1; CD35) or the membrane cofactor protein (MCP; CD46) [START_REF] Whaley | Modulation of the alternative complement pathways by beta 1 H globulin[END_REF][START_REF] Fujita | Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator[END_REF][START_REF] Masaki | Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46)[END_REF]).

In addition, FI and FH also inhibit the alternative C3 and C5 convertases formation by competing with C3b for the binding to FB and C5. Moreover, decay accelerating factor (DAF; CD55), a membrane-associated regulatory protein, inhibits the association and enhances the dissociation of both C3 and C5 convertases [START_REF] Hourcade | Decay-accelerating factor (DAF), complement receptor 1 (CR1), and factor H dissociate the complement AP C3 convertase (C3bBb) via sites on the type A domain of Bb[END_REF].

Conversely to these regulators aiming at dissociating the protein complexes forming the C3 and C5 convertases, the properdin (also known as factor P) is the only positive regulator of the complement cascade. Indeed, properdin stabilizes the alternative C3 and C5 convertases by bridging the C3b and Bb fragments [START_REF] Alcorlo | Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin[END_REF].

The terminal pathway and formation of the membrane attack complex are also tightly regulated at different levels of control. The complement component C8 plays an important role in the MAC formation. Indeed, if C8 binds to [C5b-7] in the absence of a cell membrane, conformational changes occur and inhibit the pore formation [START_REF] Nemerow | Restriction of complement-mediated membrane damage by the eighth component of complement: a dual role for C8 in the complement attack sequence[END_REF]). At another level, the MAC formation is regulated by the GPI-anchored CD59 which inhibits C9 binding to the [C5b-8] membrane complex, as well as C9 polymerization and final assembly of the membrane attack complex [START_REF] Rollins | The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9[END_REF][START_REF] Farkas | CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9[END_REF]. Finally, another regulation mechanism implies clusterin and vitronectin that capture the soluble [C5b-7], [C5b-8] and [C5b-9] complexes and prevent their binding to the membrane [START_REF] Poggi | Engagement of the leukocyte-associated Ig-like receptor-1 induces programmed cell death and prevents NF-kappaB nuclear translocation in human myeloid leukemias[END_REF][START_REF] Tschopp | Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9[END_REF][START_REF] Hadders | Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9[END_REF].

II. Focus on the C1q molecule (classical pathway) 1 Generalities

C1q is the molecule that activates the classical complement pathway after recognition of various molecular patterns associated with either pathogens or damaged tissues. As the majority of complement components, C1q is a circulating molecule and is present in the plasma at a concentration of around 80 µg/mL in physiological conditions [START_REF] Reid | Complement Component C1q: Historical Perspective of a Functionally Versatile, and Structurally Unusual, Serum Protein[END_REF]). However, C1q synthesis is not achieved by the hepatocytes but mostly by monocyte-derived cells and is under the control of various stimuli. For example, C1q production by macrophages and dendritic cells is upregulated by toll-like receptor (TLR) signaling induced by pathogen-associated lipopolysaccharide or lipoteichoic acid [START_REF] Walker | Expression and regulation of complement C1q by human THP-1-derived macrophages[END_REF][START_REF] Baruah | The tissue pentraxin PTX3 limits C1q-mediated complement activation and phagocytosis of apoptotic cells by dendritic cells[END_REF]). In addition to these exogenous stimuli, C1q synthesis can be regulated by cytokines such as IL-1, IL-6 or IFN-γ [START_REF] Walker | Expression and regulation of complement C1q by human THP-1-derived macrophages[END_REF][START_REF] Lu | The classical and regulatory functions of C1q in immunity and autoimmunity[END_REF]).

Structural and molecular organization of C1q

C1q is a multimeric protein and results from the assembly of 18 polypeptide chains of three O-glycosylation [START_REF] Reid | Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement[END_REF][START_REF] Pflieger | Analysis of Human C1q by Combined Bottomup and Top-down Mass Spectrometry[END_REF]. Six C1q ABC trimers assemble via their Nterminal CLR into a fiber bundle that splits up at mid-length (due to imperfect collagen repeats) into 6 collagen triple-helices with C-terminal globular heads at their extremity [START_REF] Reid | Complement Component C1q: Historical Perspective of a Functionally Versatile, and Structurally Unusual, Serum Protein[END_REF] (Figure 3). These two different C1q regions have been studied thanks to limited proteolysis of the whole C1q purified from human serum. Indeed, digestion of C1q with pepsin results in isolated CLR whereas collagenase digestion of C1q generates the GR (Figure 4) [START_REF] Reid | Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement[END_REF][START_REF] Brodsky-Doyle | Circular-dichroism and electron-microscopy studies of human subcomponent C1q before and after limited proteolysis by pepsin[END_REF][START_REF] Hughes-Jones | Reaction between the isolated globular sub-units of the complement component C1q and IgG-complexes[END_REF]. Studies on both isolated CLR and GR revealed that they host distinct functions. The globular regions of C1q are mostly responsible for the recognition of the multiple targets of C1q such as immunoglobulins, pathogens and apoptotic cells associated molecular patterns (reviewed in [START_REF] Kishore | Structural and functional anatomy of the globular domain of complement protein C1q[END_REF]) (Table 1).

Targets

C1q interacting region References

Endogenous ligands

IgG/IgM GR [START_REF] Gadjeva | Interaction of Human C1q with IgG and IgM: Revisited[END_REF]

Pathogen-associated ligands

Lipopolysaccharide (LPS) GR [START_REF] Loos | Interaction of bacterial endotoxin (LPS) with fluid phase and macrophage membrane associated C1Q. The FC-recognizing component of the complement system[END_REF][START_REF] Roumenina | Interaction of the globular domain of human C1q with Salmonella typhimurium lipopolysaccharide[END_REF] gp41 of HIV-1 gp21 of HTLV-1 GR [START_REF] Kojouharova | Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies[END_REF]) The C1q CLR are involved in immune mechanisms through their interaction with the tetramer of C1r and C1s proteases or membrane receptors [START_REF] Siegel | Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers[END_REF][START_REF] Eggleton | C1q receptors[END_REF] (Table 2).

Complement-associated functions of C1q

The initial and most studied function of C1q is its important role in the elimination of self-and non self-components via the activation of the classical complement pathway. In human serum, C1q is almost exclusively (90%) found associated with the C1r2s2 tetramer of C1r and C1s, forming the C1 complex.

In order to initiate the complement classical pathway, both C1r and C1s need to be activated by proteolytic cleavage of an arginine-isoleucine bond within the serine protease domain. Even if the exact mechanism underlying C1 activation still remains elusive, it is clearly established that

2016)

Siglec 3 (CD33) GR [START_REF] Son | Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression[END_REF] C1q binding to an activator surface induces C1r autolytic activation. Then, activated C1r cleaves and activates C1s that afterward cleaves C4 and C2, triggering formation of the classical C3 convertase and activation of downstream complement cascade [START_REF] Gaboriaud | Assembly of C1 and the MBL-and ficolin-MASP complexes: Structural insights[END_REF]).

Both C1r and C1s are serine proteases sharing the same modular architecture with the succession of CUB1-EGF-CUB2-CCP1-CCP2 modules followed by a C-terminal serine protease domain (Figure 5A) [START_REF] Gaboriaud | Deciphering the fine details of c1 assembly and activation mechanisms: "mission impossible[END_REF]). C1r and C1s proteases assemble into a heterodimer via their CUB1-EGF-CUB2 modules in a head to tail fashion (Figure 5A). The Ca 2+ -dependent assembly of C1r2s2 with C1q in the C1 complex is crucial for complement cascade initiation.

Site-directed mutational studies on recombinant C1r, C1s and C1q allowed the precise identification of the C1q binding sites on each protease and the C1q residues involved in the interaction. On the protease C1r CUB1 and CUB2 respectively, E49/Y56/D102 and D226/D228/Y235/D273 residues are involved in the C1 complex formation. On C1s CUB1 module the interaction implicates E45/D98 residues [START_REF] Bally | Identification of the C1q-binding Sites of Human C1r and C1s: a refined three-dimensional model of the C1 complex of complement[END_REF]. The crystal structure of C1r and C1s dimers revealed the association of two C1r/C1s dimers with the two C1r copies in the center (Figure 5B) [START_REF] Almitairi | Structure of the C1r-C1s interaction of the C1 complex of complement activation[END_REF]). On C1q side, the binding of C1r2s2 involved three lysine residues located in the collagen-like region of C1q: LysB61, LysC58 and LysA59, the latter in a lesser extent [START_REF] Bally | Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites[END_REF]). In addition, a C1q variant containing the substitution of its glycine residue at position 63 in the B chain into a serine, was identified in a patient suffering from lupus and was shown to be unable to interact with the C1r2s2 tetramer [START_REF] Roumenina | Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance[END_REF]. The identification of each partner binding sites coupled with the available structures of C1r and C1s led to the proposal of initial models for the C1 complex [START_REF] Bally | Identification of the C1q-binding Sites of Human C1r and C1s: a refined three-dimensional model of the C1 complex of complement[END_REF][START_REF] Venkatraman Girija | Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation[END_REF]. Due to the intrinsic flexibility of the C1q molecule, poor structural information is available for the whole C1 complex. However, a recent cryoelectron tomography study provided structural insight into C1 bound to an IgM (Figure 6) [START_REF] Sharp | Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b[END_REF]. Such model confirmed the arrangement of the C1r2s2 platform in the C1q collagen cone with the two C1r copies in the core of C1r2s2. However, the two C1r/C1s dimers are not as tightly packed as in the crystal structure (Figure 6). [START_REF] Sharp | Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b[END_REF].

Receptors and complement-independent functions of C1q

C1q interacts with multiple ligands and receptors on a large range of cell types, providing to this molecule a wide variety of functions independent of complement activation [START_REF] Nayak | Complement and non-complement activating functions of C1q: A prototypical innate immune molecule[END_REF]) (Tables 1 and2). It is now well established that C1q mostly interacts with danger and "eat-me" signals through its GR while receptors on immune cells recognize C1q-opsonized target via C1q CLR [START_REF] Eggleton | C1q receptors[END_REF][START_REF] Gaboriaud | The human c1q globular domain: structure and recognition of non-immune self ligands[END_REF]) (Table 1). The C1q-mediated immune effector mechanisms will therefore depend on which immune cell senses C1q and which immune receptors are involved in the C1q recognition.

Involvement of C1q in apoptotic cells phagocytosis

These really distinct functions of C1q GR and CLR are even more evident for the recognition of apoptotic cells by C1q that engages specifically its globular regions [START_REF] Navratil | The globular heads of C1q specifically recognize surface blebs of apoptotic vascular endothelial cells[END_REF][START_REF] Païdassi | C1q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition[END_REF]. Even if few immune receptors interact with the C1q GR, the major contribution is given by the C1q CLR, an interaction that generally results in the engulfment of C1q-labelled targets. C1q engagement in phagocytosis stimulation can be divided in three main mechanisms (Figure 7). First, C1q is indirectly involved in the phagocytosis of pathogens and apoptotic cells through complement activation after recognition of the Fc region of immunoglobulins associated with antigens (immune complexes, IC) at the surface of target cells. Indeed, C1q-mediated complement activation leads to the deposition of C3b and C4b fragments on target's surface and triggers the engulfment through CR1, CR3 and CR4 [START_REF] Fearon | Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte[END_REF][START_REF] Mevorach | Complement-dependent clearance of apoptotic cells by human macrophages[END_REF].

Second, Galvan and colleagues showed that C1q binding to the surface of macrophages positively regulates the expression of several genes coding anti-inflammatory proteins involved in the clearance of apoptotic cells such as the Mer tyrosine kinase (MERTK) and its ligands Growth arrest-specific 6 (Gas6) and protein S (Galvan et al. 2012a). However, the mechanism and the macrophage receptors involved in this genes upregulation still remain elusive. A recent study suggests that the C1q-dependent increase of MERTK expression engages the alarmin HMGB1 and is mediated through two immunoglobulin (Ig)-like receptors RAGE (receptor for advanced glycation end-products) and LAIR-1 (leukocyte-associated immunoglobulin-like receptor 1) [START_REF] Son | C1q and HMGB1 reciprocally regulate human macrophage polarization[END_REF].

Third, C1q globular regions are able to directly recognize pathogen-and apoptotic cellsassociated molecular patterns such as LPS, lipid A, DNA or phosphatidyl serine (PS) (Table1).

C1q can also interact through its CLR with several immune receptors involved in phagocytic functions such as CR1, LRP1 or SR-F1 (Table 2). Because of this dual recognition capacity, C1q was suggested to act as an opsonin on its own, bridging targets to be eliminated and phagocytic cells.

 Complement receptor 1 (CR1, CD35) was initially described for its complement regulatory functions alone or in association with factor I [START_REF] Fearon | Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane[END_REF][START_REF] Iida | Complement receptor is an inhibitor of the complement cascade[END_REF][START_REF] Masaki | Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46)[END_REF]. CR1 is a multimodular transmembrane receptor with an extracellular part composed of 30 CCP modules organized into 4 long homologous repeats (LHR) A, B, C and D. CR1 is involved in phagocytic mechanisms and is mainly engaged in the elimination of immune complexes and targets opsonized by the complement C3b and C4b fragments [START_REF] Fearon | Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte[END_REF][START_REF] Fearon | Immunoregulatory functions of complement: structural and functional studies of complement receptor type 1 (CR1; CD35) and type 2 (CR2; CD21)[END_REF]). Surface plasmon resonance experiments reported the in vitro interaction of CR1 with the collagen-like region of C1q [START_REF] Klickstein | Complement receptor type 1 (CR1, CD35) is a receptor for C1q[END_REF]. Further studies showed that C1q interaction with CR1 enhances erythrocytes adhesion and the uptake of apoptotic cells by dendritic cells and macrophages [START_REF] Tas | C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion[END_REF][START_REF] Nauta | Opsonization with C1q and Mannose-Binding Lectin Targets Apoptotic Cells to Dendritic Cells[END_REF]). During my PhD thesis, I was involved in the investigation of C1q interaction with CR1. Briefly, we demonstrated that C1q interacts with the CR1 CCP 24 and 25 modules in the long homologous repeat D, via its collagen-like regions (Article 3, [START_REF] Jacquet | C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules[END_REF].

 Low density lipoprotein receptor-related protein 1 (LRP1, CD91) is a multimodular transmembrane receptor composed of the succession of multiple modules of three different types: β-propeller domains, EGF and CR modules. LRP1 is a scavenger receptor involved in the clearance of apoptotic cells, especially in association with another known C1q receptor: calreticulin (CRT). C1q was described as a ligand for LRP1, suggesting its involvement in LRP1-dependent apoptotic cells removal. This was confirmed in different studies showing that C1q is required for apoptotic cells removal through LRP1/CRT [START_REF] Ogden | C1q and Mannose Binding Lectin Engagement of Cell Surface Calreticulin and Cd91 Initiates Macropinocytosis and Uptake of Apoptotic Cells[END_REF][START_REF] Vandivier | Role of Surfactant Proteins A, D, and C1q in the Clearance of Apoptotic Cells In Vivo and In Vitro: Calreticulin and CD91 as a Common Collectin Receptor Complex[END_REF]. However, another study pointed out that the phagocytosis enhancing capacity of C1q does not engage LRP1 [START_REF] Lillis | Murine low-density lipoprotein receptorrelated protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis[END_REF]. In order to address this question, I took part in the deciphering of C1q interaction with LRP1 that conducted to the identification of C1q collagen-like regions binding sites on LRP1 clusters II and IV (Article 4, Fouët et al. 2020).

 Class F scavenger receptor 1 (SR-F1, also known as SCARF1) is a multimodular transmembrane endocytic receptor composed of ten extracellular EGF repeats [START_REF] Adachi | Expression cloning of a novel scavenger receptor from human endothelial cells[END_REF]). SR-F1 was initially described as a receptor for multiple non-self and endogenous ligands, therefore mediating a large range of physiological functions [START_REF] Patten | SCARF1: a multifaceted, yet largely understudied, scavenger receptor[END_REF]. SR-F1 was reported to be an important actor of the C1q-dependent removal of apoptotic cells and as for LRP1, this process may require the formation of a multimolecular complex with CRT [START_REF] Berwin | SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin[END_REF][START_REF] Païdassi | Investigations on the C1q-Calreticulin-Phosphatidylserine Interactions Yield New Insights into Apoptotic Cell Recognition[END_REF][START_REF] Ramirez-Ortiz | The scavenger receptor SCARF1 mediates apoptotic cell clearance and prevents autoimmunity[END_REF][START_REF] Wicker-Planquart | Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells[END_REF].

Immune modulation

The elimination of apoptotic cells and immune complexes is a crucial process of immunity homeostasis and needs to be tightly controlled. Indeed, apoptotic cell phagocytosis has to be efficiently performed without inducing an excessive immune response against ingested autoantigens. Disruption of the silent immune complexes and apoptotic cells clearance is an important factor of chronic inflammation and autoimmune diseases onset such as systemic lupus erythematosus (SLE) [START_REF] Pieterse | Breaking Immunological Tolerance in Systemic Lupus Erythematosus[END_REF][START_REF] Tsai | The contribution of the programmed cell death machinery in innate immune cells to lupus nephritis[END_REF]). As discussed above, C1q appears to play a substantial role in the removal of apoptotic cells. A previous study showed that C1q-deficient mice are prone to lupus-like disease development and carry excess of apoptotic bodies [START_REF] Botto | Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies[END_REF]). Moreover, this hypothesis was supported by the strong association of genetic or acquired C1q deficiencies with SLE [START_REF] Walport | C1q and Systemic Lupus Erythematosus[END_REF][START_REF] Botto | C1q, Autoimmunity and Apoptosis[END_REF]). Botto's lab recently reported that aside from the C1q-mediated clearance of apoptotic cells, the main regulatory role of C1q is the modulation of CD8+ T-cells metabolism through its interaction with mitochondrial gC1qR [START_REF] Ling | C1q restrains autoimmunity and viral infection by regulating CD8+ T-cell metabolism[END_REF]).

In addition to its involvement in the enhancement of apoptotic cells phagocytosis, C1q was

shown to exhibit regulatory properties on immune cells which is consistent with the establishment of a safe removal of self-components. Indeed, several studies report the antiinflammatory influence of C1q on various immune cells such as macrophages, dendritic cells or microglial cells.

Modulation of cytokine synthesis

C1q alters the level and profile of cytokine synthesis as another way to reduce inflammation and maintain tolerance. Indeed, C1q fixation on peripheral blood monocytes was shown to inhibit the LPS-induced TLR activation signals with the suppression of the pro-inflammatory cytokines IL-1α and IL-1β expression and the stimulation of the anti-inflammatory IL-10 and the IL-1 receptor antagonist [START_REF] Fraser | C1q and MBL, components of the innate immune system, influence monocyte cytokine expression[END_REF]. Moreover, Nauta et al. showed that apoptotic cells opsonized with C1q induced a cytokine response of dendritic cells with the increase of IL-6, IL-10 and TNF-α expression whereas no effect was observed for apoptotic cells alone [START_REF] Nauta | Opsonization with C1q and Mannose-Binding Lectin Targets Apoptotic Cells to Dendritic Cells[END_REF]). This difference was suggested to be associated to different immune receptors such as LRP1, CR1 and calreticulin. Interestingly, C1q was shown to be able to modulate the cytokine profile secreted by endothelial cells. Namely, C1q binding to endothelial cells induces the increase of IL-8, IL-6 and MCP-1 expression (van den [START_REF] Van Den Berg | The first subcomponent of complement, C1q, triggers the production of IL-8, IL-6, and monocyte chemoattractant peptide-1 by human umbilical vein endothelial cells[END_REF]. This study can be related to the one of Kang and colleagues who showed the modulation of cytokine expression by the mannose-binding lectin (another defense collagen, homologous to C1q) on endothelial cells [START_REF] Kang | Mannose-binding lectin without the aid of its associated serine proteases alters lipopolysaccharide-mediated cytokine/chemokine secretion from human endothelial cells[END_REF].

Dendritic, T and B-cells maturation and activation

Cytokines are powerful molecules important for the recruitment and maturation of immune cells at the site of inflammation. It is therefore not surprising that C1q mediates immune tolerance through the modulation of the dendritic cells functions. First, C1q was shown to possess chemotactic properties toward dendritic cells in order to recruit them at the site of inflammation [START_REF] Vegh | Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR[END_REF]). Moreover, C1q was shown to induce dendritic cells with tolerogenic properties in several studies [START_REF] Yamada | Complement C1q regulates LPS-induced cytokine production in bone marrow-derived dendritic cells[END_REF][START_REF] Castellano | Maturation of dendritic cells abrogates C1q production in vivo and in vitro[END_REF]). However, C1q was also demonstrated to generate dendritic cells activation triggering IFNγ production by T cells [START_REF] Csomor | Complement protein C1q induces maturation of human dendritic cells[END_REF]. The latter study is in agreement with previous studies of Botto's lab who showed that LPS injection in C1q-/-mice induced less IFNγ production by T-cells than wild-type mice in a dendritic cells dependent manner [START_REF] Cutler | T cell-dependent immune response in C1qdeficient mice: defective interferon gamma production by antigen-specific T cells[END_REF][START_REF] Baruah | C1q enhances IFN-gamma production by antigen-specific T cells via the CD40 costimulatory pathway on dendritic cells[END_REF]).

Furthermore, C1q was shown to be involved in the biology of B-cell. Indeed, C1q is able to bind to inactive and activated B lymphocytes to either stimulate IgG or IgM production [START_REF] Young | Complement subcomponent C1q stimulates Ig production by human B lymphocytes[END_REF]. In line with this study, C1q-/-mice present a reduced tolerance of B cell to autoantigens displayed by apoptotic cells [START_REF] Ferry | Increased positive selection of B1 cells and reduced B cell tolerance to intracellular antigens in c1q-deficient mice[END_REF]).

However, the above mentioned tolerogenic activities of C1q toward immune cells may certainly engage other specific immune receptors, through unclear mechanisms that await further investigations. Son and colleagues recently proposed receptors from the immunoglobulin (Ig)like family to play a role in the maintenance of immune cells tolerance [START_REF] Son | C1q limits dendritic cell differentiation and activation by engaging LAIR-1[END_REF][START_REF] Son | C1q and HMGB1 reciprocally regulate human macrophage polarization[END_REF][START_REF] Son | Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression[END_REF]Son and Diamond 2015) (Figure 8). The interaction of C1q with these Ig-like receptors RAGE, CD33 and more specifically LAIR-1 is further described in the next section.

III. LAIR-1 1 Generalities

Leukocyte-associated immunoglobulin-like recptor-1 (LAIR-1; CD305) was first identified and characterized in 1997 on human natural killer (NK) cells for its efficient inhibition of NK cells cytotoxicity after cross-linking by the DX26 antibody, suggesting that LAIR-1 is an immune inhibitory receptor. Further investigations revealed that LAIR-1 is expressed on a wide range of human immune cells such as NK cells, T cells, B cells, monocytes, neutrophils, basophils and mast cells [START_REF] Meyaard | LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes[END_REF][START_REF] Meyaard | Leukocyte-associated Ig-like receptor-1 functions as an inhibitory receptor on cytotoxic T cells[END_REF][START_REF] Van Der | Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially expressed during human B cell differentiation and inhibits B cell receptor-mediated signaling[END_REF][START_REF] Saverino | Surface density expression of the leukocyteassociated Ig-like receptor-1 is directly related to inhibition of human T-cell functions[END_REF][START_REF] Verbrugge | Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation[END_REF][START_REF] Florian | Detection of novel leukocyte differentiation antigens on basophils and mast cells by HLDA8 antibodies[END_REF][START_REF] Jansen | Regulated expression of the inhibitory receptor LAIR-1 on human peripheral T cells during T cell activation and differentiation[END_REF][START_REF] Zhang | The role of LAIR-1 (CD305) in T cells and monocytes/macrophages in patients with rheumatoid arthritis[END_REF].

Molecular cloning and biochemical analyses demonstrated that LAIR-1 is a 32 kDa type I transmembrane receptor composed of an extracellular domain of 142 amino acids (aa), a transmembrane domain of 23 aa and an intracellular domain of 101 aa. LAIR-1 belongs to the immunoglobulin (Ig)-like superfamily with a single extracellular C2-type Ig-like domain containing one N-glycosylation site (Figure 9). LAIR-1 is structurally related to other Ig-like superfamily members localized to the leukocyte receptor complex (LRC) on human chromosome 19 [START_REF] Barrow | The extended human leukocyte receptor complex: diverse ways of modulating immune responses[END_REF]. Among these receptors, LAIR-2 (CD306) is a soluble homolog of LAIR-1 lacking the transmembrane and intracellular regions, with an Iglike domain sharing 84% of sequence homology with the Ig-like domain of LAIR-1.

The cytoplasmic region of LAIR-1 contains two amino acids sequences corresponding to immune receptor tyrosine-based inhibitory motifs (ITIMs), which explains the immune inhibitory activity of LAIR-1 (Figure 9) [START_REF] Meyaard | LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes[END_REF]. As for many receptors, intracellular ITIMs phosphorylation depends on LAIR-1 dimerization. The dimerization of LAIR-1 extracellular domains upon ligand binding results in the recruitment of intracellular Src homology 2-containing protein tyrosine phosphatases leading to a negative regulation of intracellular signaling associated with immune cell maturation, activation and differentiation [START_REF] Meyaard | LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes[END_REF][START_REF] Xu M J | Identification and characterization of leukocyte-associated Iglike receptor-1 as a major anchor protein of tyrosine phosphatase SHP-1 in hematopoietic cells[END_REF][START_REF] Sathish | Constitutive Association of SHP-1 with Leukocyte-Associated Ig-Like Receptor-1 in Human T Cells[END_REF][START_REF] Jin | LAIR-1 activation inhibits inflammatory macrophage phenotype in vitro[END_REF].

LAIR-1: a collagen receptor

Collagens are the most abundant protein family in the human body and constitute up to 30% of the whole animal's proteins [START_REF] Frantz | The extracellular matrix at a glance[END_REF]. Up to now, 28 different collagen family members have been identified and numbered collagens type I to XXVIII. All members of the collagen family are homo-or hetero-trimers composed of three identical or different polypeptide chains (named α chains) and are characterized by the presence of at least one triplehelical domain (Figure 10). This particular structural assembly relies on the presence of repeating Gly-X-Y triplet in the sequence of each collagen chain, X residue is frequently a proline and Y a 4-hydroxyproline generated after post-translational modifications (Ricard-Blum 2011). Within the collagen triple-helical structure, the three collagen chains are not in phase but they are shifted, leading to a registering order with leading, middle and trailing chains (Figure 10). Even if all collagens share a basic triple-helical feature, they can achieve diverse supramolecular assemblies, such as fibrils, hexagonal network or even beaded filaments (Figure 11) (Ricard-Blum 2011). Most of the collagen types assemble into fibrils in a heterotypic manner, meaning that the collagen fibrils are composed of more than one type of collagen. For example, collagen fibrils are made up of collagen types II and III or II, XI and IX in cartilage, I and III in skin or I and V in cornea [START_REF] Bruckner | Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules[END_REF][START_REF] Wu | Type III Collagen, a Fibril Network Modifier in Articular Cartilage[END_REF]. The proportion of triplehelical regions in collagens can vary from 10% in collagen XII to 96% in collagen I (Ricard-Blum 2011). Fibrillar collagens such as collagens I, II, III, V, XI, XXIV and XXVII have a triple helical-region that represents the major part of their sequence. In addition, a subfamily of collagens called fibril-associated collagens with interrupted triple helices (FACIT) gathers collagens that contain several collagen triple-helical regions separated by non-collagenous (nc)

regions. The FACITs do not form polymers alone but are associated with fibrils and are suggested to act as molecular bridges playing a major role in the organization and stability of extracellular matrices [START_REF] Shaw | FACIT collagens: diverse molecular bridges in extracellular matrices[END_REF].

LAIR-1 is a receptor for collagens. Indeed, collagens are able to cross-link LAIR-1 thus inhibiting the immune cell activation in vitro [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF]. LAIR-1 interaction with collagen depends on the presence of hydroxyproline residues. Indeed, surface plasmon resonance studies showed that LAIR-1 interacts well with an immobilized (GPO)10 peptide but exhibits low binding signal on an immobilized (GPP)10 peptide [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF]). Because LAIR-1 interacts with a common collagen feature, it was shown to interact with many collagen types such as collagens I, II, III, IV, XIII, XVII or XXIII [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF]Brondijk et al. 2010b). In addition, several LAIR-1 binding sites were identified on collagens II and III [START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF]).

Molecular and structural aspects of collagen/LAIR-1 interaction

Only few studies report collagen/LAIR-1 interaction from a molecular and structural point of view. In one of them, Tang and colleagues revealed the high importance of one LAIR-1 residue for the interaction with collagens: arginine 65. Indeed, the mutation of this single residue into a lysine (R65K) was tested both in vitro and on cell surface, revealing altered interaction properties with collagens and therefore inefficient collagen-mediated LAIR-1 phosphorylation and cellular signaling [START_REF] Tang | A single residue, arginine 65, is critical for the functional interaction of leukocyte associated inhibitory receptor (LAIR)-1 with collagens[END_REF]. The first structural information about LAIR-1 was given by Brondijk and colleagues with the crystal structure of LAIR-1 extracellular Ig-like domain (PDB code: 3KGR). This structure revealed that, as expected, LAIR-1 extracellular domain exhibits the characteristic Ig-like domain scaffold with two β-sheets connected by a disulfide bridge (Figure 9B). Nuclear magnetic resonance titration analysis of LAIR-1 interaction with a synthetic collagen III peptide identified the collagen binding region of LAIR-1 (Figure 12BC). Indeed, residues R59-E63, T67-N69, A84-F86, R100-I102, W109-S113, Y115-L116, and L118 displayed important chemical shift perturbations upon collagen interaction (Brondijk et al. 2010b). Some of these residues were mutated on membrane full-length LAIR-1 and tested for their ability to interact with collagen I, III and IV. Mutations of arginine 59 or glutamate 61 into an alanine residue drastically inhibit each collagen interaction with stably transfected cells. In addition, arginine 65 substitution into alanine showed an important decrease of collagens binding, also confirming arginine 65 major involvement in collagen/LAIR-1 interaction. Furthermore, arginine 62 and glutamate 111 mutation into alanine had a lesser impact on LAIR-1 interaction properties.

These identified residues are highly conserved in LAIR-1 and LAIR-2 proteins from several species, especially for the residues essential for collagen binding (Figure 12A).

LAIR-1 is structurally related to another Ig-like family member encoded in the LRC: the glycoprotein VI (GPVI). GPVI is an activating collagen receptor, expressed on platelets and megakaryocytes, playing a major role in thrombosis and hemostasis [START_REF] Farndale | The role of collagen in thrombosis and hemostasis[END_REF][START_REF] Horii | Structural basis for platelet collagen responses by the immune-type receptor glycoprotein VI[END_REF][START_REF] Leitinger | Mammalian collagen receptors[END_REF]. Interestingly, GPVI interaction with collagen is dependent on the presence of hydroxyproline residues, as it is the case for LAIR-1 [START_REF] Knight | Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen[END_REF][START_REF] Smethurst | Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets[END_REF]). This observation highly suggests that GPVI and LAIR-1 interactions with collagen rely on similar molecular mechanisms. Such hypothesis is supported by a study showing that LAIR-1 is able to inhibit collagen-induced GPVI signaling when coexpressed in the same cells [START_REF] Tomlinson | Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines[END_REF]). Moreover, the collagen binding sites of LAIR-1 (i.e., R59 and E61) are present in GPVI sequence and highly conserved in GPVI orthologs (Figure 13). As opposed to LAIR-1, GPVI structure in complex with a GPO synthetic collagen peptide is available in the protein data bank (PDB code 5OU8). The available crystal structure of GPVIcollagen complex does not support the GPVI collagen binding site previously suggested by computational docking and mutational analyses [START_REF] Smethurst | Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody[END_REF][START_REF] Horii | Structural basis for platelet collagen responses by the immune-type receptor glycoprotein VI[END_REF]). However, the GPVI-collagen complex structure supports a common collagen recognition mechanism with LAIR-1. Indeed, the structure shows that R38 and E40, the respective homologs of LAIR-1 R59 and E61 residues, are directly involved in the interaction of GPVI Ig-like domain with a GPO peptide (Figure 14B). All together, these observations are consistent with a common collagen binding mechanism for LAIR-1 and GPVI. Furthermore, one additional structural insight into collagen recognition by Ig-like domains could be obtained from the crystal structure of osteoclast associated receptor (OSCAR), another collagen Ig-like receptor encoded in the LRC and involved in osteoclast development, in complex with a collagen peptide (PDB code 5CJB) [START_REF] Haywood | Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors[END_REF]. In this particular case, collagen binding relies on three tyrosine (Y166, Y200 and Y208) and one proline (P204) residues (Figure 14A). Although collagen binding on OSCAR involves residues that are chemically different from the GPVI/LAIR-1 common collagen binding site (hydrophobic versus charged residues in GPVI/LAIR-1), the overall collagen binding region is basically the same on the Ig-like domain surface (Figure 14A).

Regulation of LAIR-1 inhibitory activity

LAIR-1 is broadly expressed on the surface of immune cells and because its ligands (collagens) are very abundant in the human body, the collagen/LAIR-1 interaction needs to be tightly regulated in order to obtain a suitable inhibitory signaling. This regulation can occur by different ways. Indeed, the level of LAIR-1 expression on cell surface can be modulated at different stages of immune cells differentiation or LAIR-1 can be also shed from the cell surface after cell activation, generating soluble LAIR-1 (van der Vuurst de [START_REF] Van Der | Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially expressed during human B cell differentiation and inhibits B cell receptor-mediated signaling[END_REF][START_REF] Ouyang | Establishment of an ELISA system for determining soluble LAIR-1 levels in sera of patients with HFRS and kidney transplant[END_REF][START_REF] Verbrugge | Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation[END_REF][START_REF] Jansen | Regulated expression of the inhibitory receptor LAIR-1 on human peripheral T cells during T cell activation and differentiation[END_REF][START_REF] Zhang | LAIR-1 shedding from human fibroblast-like synoviocytes in rheumatoid arthritis following TNF-α stimulation[END_REF]. Moreover, a recent study showed that LAIR-1 expression during the inflammation processes is tightly controlled.

Indeed, LAIR-1 is over expressed on monocytes and dendritic cells surface during inflammation and its expression comes back to a normal level during the inflammation resolution [START_REF] Carvalheiro | Leukocyte Associated Immunoglobulin Like Receptor 1 Regulation and Function on Monocytes and Dendritic Cells During Inflammation[END_REF]. The collagen/LAIR-1 interaction can also be regulated by the LAIR-1 soluble homolog: LAIR-2. Indeed, LAIR-2 was shown to interact with the same binding sites as LAIR-1 in collagen molecules. Therefore LAIR-2 acts as a soluble competitor for LAIR-1 interaction with collagens I and III [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen/LAIR-1 inhibitory immune interaction[END_REF]. This strongly suggests that LAIR-2 may operate as an immunoregulatory molecule by decreasing LAIR-1 inhibitory activity in vivo.

LAIR-1 involvement in pathologies

Due to its wide expression and important inhibitory activity, it is not surprising that LAIR-1 is involved in a large number of pathologies such as cancer or autoimmune diseases. This part describes selected diseases for which LAIR-1 positive or negative contribution has been reported.

LAIR-1 in cancer

Due to its wide range of expressing cell types, LAIR-1 has been reported to be involved in many cancer types. Indeed, it was shown that LAIR-1 expression is higher in cancer cells than in healthy cells. For example, LAIR-1 expression is increased in myeloid leukemia cells, epithelial ovarian cancer cells, cervical cancer cells, osteosarcoma and hepatocellular carcinoma cells [START_REF] Poggi | Engagement of the leukocyte-associated Ig-like receptor-1 induces programmed cell death and prevents NF-kappaB nuclear translocation in human myeloid leukemias[END_REF][START_REF] Zocchi | Leukocyte-associated Ig-like receptor-1 prevents granulocyte-monocyte colony stimulating factor-dependent proliferation and Akt1/PKB alpha activation in primary acute myeloid leukemia cells[END_REF]Cao et al. 2015;[START_REF] Wang | Clinical significance of leukocyte-associated immunoglobulin-like receptor-1 expression in human cervical cancer[END_REF][START_REF] Wu | Clinicopathologic significance of LAIR-1 expression in hepatocellular carcinoma[END_REF][START_REF] Zhang | LAIR-1 overexpression inhibits epithelialmesenchymal transition in osteosarcoma via GLUT1-related energy metabolism[END_REF]). However, the consequences of LAIR-1 activation on tumor development remain elusive. Indeed, LAIR-1 activation in myeloid and lymphocytic leukemia inhibits nuclear factor-κB (NF-κB) signaling, therefore preventing cancer cells proliferation [START_REF] Poggi | Engagement of the leukocyte-associated Ig-like receptor-1 induces programmed cell death and prevents NF-kappaB nuclear translocation in human myeloid leukemias[END_REF][START_REF] Poggi | Lack of the leukocyte-associated Ig-like receptor-1 expression in high-risk chronic lymphocytic leukaemia results in the absence of a negative signal regulating kinase activation and cell division[END_REF][START_REF] Zocchi | Leukocyte-associated Ig-like receptor-1 prevents granulocyte-monocyte colony stimulating factor-dependent proliferation and Akt1/PKB alpha activation in primary acute myeloid leukemia cells[END_REF]. This inhibitory activity of LAIR-1 upon tumor proliferation was also reported in the cases of ovarian cancer, cervical carcinoma and osteosarcoma (Cao et al. 2015;[START_REF] Wang | Clinical significance of leukocyte-associated immunoglobulin-like receptor-1 expression in human cervical cancer[END_REF][START_REF] Zhang | LAIR-1 overexpression inhibits epithelialmesenchymal transition in osteosarcoma via GLUT1-related energy metabolism[END_REF]. Only few studies reported a benefic role of LAIR-1 activation in the development of leukemia and hepatocellular carcinoma [START_REF] Kang | The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development[END_REF][START_REF] Wu | Clinicopathologic significance of LAIR-1 expression in hepatocellular carcinoma[END_REF].

Tumors are often associated with enhanced collagens expression that constitutes a suitable microenvironment for tumor growth and migration [START_REF] Kauppila | Aberrant type I and type III collagen gene expression in human breast cancer in vivo[END_REF][START_REF] Huijbers | A role for fibrillar collagen deposition and the collagen internalization receptor endo180 in glioma invasion[END_REF]).

These tumor-expressed collagens were shown to be ligands for soluble Fc-fused LAIR-1 and membrane LAIR-1 [START_REF] Rygiel | Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1[END_REF]). This suggests a role for LAIR-1 in tumor strategies to escape immune reactions. Indeed, LAIR-1 crosslinking through tumor-expressed collagens leads to the activation of the downstream suppressive LAIR-1 signaling in T and NK cells, thereby inhibiting their cytotoxic activities against tumor cells [START_REF] Rygiel | Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1[END_REF].

LAIR-1 in human malaria

LAIR-1 was recently reported to be involved in malaria. Indeed, a family of broadly reactive antibodies against malaria antigens was identified in individuals living in malaria-endemic regions and characterized [START_REF] Tan | A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens[END_REF][START_REF] Pieper | Public antibodies to malaria antigens generated by two LAIR1 insertion modalities[END_REF]. These antibodies present a unique feature with the insertion of an additional DNA sequence between the V and D/J segments, encoding a full Ig-like domain corresponding to the LAIR-1 extracellular domain (Figure 15A).

This supplementary domain does not totally fit with LAIR-1 Ig-like domain sequence and displays several mutations. Binding analysis of each mutation revealed that they enhance the binding to P. falciparum infected erythrocytes while reducing interaction with its native collagen ligands. These particular antibodies were shown to opsonize infected erythrocytes through interaction with RIFINs, surface-exposed antigens on P. falciparum infected erythrocytes (Figure 13B). Structure resolution of one of these antibodies Fab fragment revealed that the entire LAIR-1 Ig-like domain is well folded and exposed at the top of the heavy chain (Hsieh and Higgins 2017) (Figure 15A). This is the first and unique example where the antibody interaction properties with its cognate antigen are owned by an additional domain.

In this context, the antibody's Fab domain acts as a link between the antigen-recognizing additional Ig-like domain and the constant fragment of the antibody to induce subsequent immune responses (Figure 15B).

Independently of the broadly reactive antibody reported above, LAIR-1 is involved in the immune evasion of Plasmodium falciparum. In this case, wild-type LAIR-1 Ig-like domain interacts directly with a subset of RIFINs at the surface of infected erythrocytes. This finding raises the question of P. falciparum infected erythrocytes recognition by transmembrane LAIR-1 on immune cells surface. Indeed, leukocyte immunoglobulin-like receptor B1 (LILRB1), an Ig-like immune receptor structurally related to LAIR-1 was reported to interact with RIFINs, which mimic its natural ligand thus inducing LILRB1 inhibitory activity on immune cell function [START_REF] Saito | Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors[END_REF][START_REF] Harrison | Structural basis for RIFIN-mediated activation of LILRB1 in malaria[END_REF]. It is therefore reasonable to speculate that as for LILRB1, membrane LAIR-1 may be activated by RIFINs binding thereby allowing P.

falciparum immune evasion (Figure 15C).

LAIR-1 in autoimmune diseases

Appropriate immune responses rely on a delicate balance between activating and inhibitory signals. Disruption of the immune balance leads to the development of autoimmune diseases like systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Because of its broad expression and its important inhibitory role in immune cells phenotype, LAIR-1 is strongly suspected to be involved in the immune imbalance in the cases of SLE and RA.

SLE is a chronic autoimmune disease involving multiple organs and characterized by the presence of a wide variety of auto-antibodies produced by uncontrolled B cells [START_REF] Rahman | Systemic lupus erythematosus[END_REF][START_REF] Cava | Targeting B cells with biologics in SLE[END_REF]. SLE etiology remains unclear with multiple endogenous and environmental factors. However, one cytokine seems to have a central role in the development of SLE: interferon alpha (IFNα) [START_REF] Niewold | Interferon Alpha in Systemic Lupus Erythematosus[END_REF]). Indeed, SLE patients exhibit higher

IFNα serum levels and IFNα-inducible genes expression that correlate with disease activity [START_REF] Hooks | Immune interferon in the circulation of patients with autoimmune disease[END_REF][START_REF] Ytterberg | Serum interferon levels in patients with systemic lupus erythematosus[END_REF][START_REF] Bengtsson | Activation of type I interferon system in systemic lupus erythematosus correlates with disease activity but not with antiretroviral antibodies[END_REF][START_REF] Kirou | Coordinate overexpression of interferon-alphainduced genes in systemic lupus erythematosus[END_REF]).

Plasmacytoid dendritic cells (pDC) are a type of immune cells known to be important IFNα producers, especially after stimulation through TLR [START_REF] Kadowaki | Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c-type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN[END_REF][START_REF] Colonna | Plasmacytoid dendritic cells in immunity[END_REF]). Interestingly, crosslinking of LAIR-1 by anti-LAIR-1 antibodies or C1q strongly inhibits TLR-dependent IFNα production by pDC revealing a putative implication of LAIR-1 in SLE [START_REF] Bonaccorsi | The immune inhibitory receptor LAIR-1 is highly expressed by plasmacytoid dendritic cells and acts complementary with NKp44 to control IFNα production[END_REF]Son and Diamond 2015). This hypothesis is supported by studies reporting lower LAIR-1 expression on pDC in SLE patients than healthy controls [START_REF] Bonaccorsi | The immune inhibitory receptor LAIR-1 is highly expressed by plasmacytoid dendritic cells and acts complementary with NKp44 to control IFNα production[END_REF][START_REF] Kanakoudi-Tsakalidou | Simultaneous changes in serum HMGB1 and IFN-α levels and in LAIR-1 expression on plasmatoid dendritic cells of patients with juvenile SLE[END_REF].

RA is characterized by bone erosion in patients' joints as the consequence of excessive osteoclasts activation [START_REF] Takayanagi | A new mechanism of bone destruction in rheumatoid arthritis: synovial fibroblasts induce osteoclastogenesis[END_REF]. Osteoclasts express LAIR-1 on their surface and a study showed that LAIR-1 activation via anti-LAIR-1 antibodies inhibits osteoclastogenesis [START_REF] Zhang | Expression of leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1) on osteoclasts and its potential role in rheumatoid arthritis[END_REF]). These observations suggest that LAIR-1 may be involved in RA pathology. This hypothesis is supported by reports which demonstrate lower membrane LAIR-1 expression on osteoclasts surface and higher levels of soluble LAIR-1 and LAIR-2 in serum, urine and synovial fluids of RA patients in comparison with healthy controls [START_REF] Nordkamp | Enhanced secretion of leukocyteassociated immunoglobulin-like receptor 2 (LAIR-2) and soluble LAIR-1 in rheumatoid arthritis: LAIR-2 is a more efficient antagonist of the LAIR-1-collagen inhibitory interaction than is soluble LAIR-1[END_REF][START_REF] Zhang | Expression of leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1) on osteoclasts and its potential role in rheumatoid arthritis[END_REF]). These findings revealed that LAIR-1 inhibitory activity on osteoclasts is down-regulated in RA patients and may directly contribute to bone destruction.

Several studies demonstrate association of the above-mentioned pathologies with complement proteins and especially C1q or C1q-related receptors disturbance, therefore raising the question of a possible role of C1q interaction with LAIR-1 [START_REF] Botto | C1q, Autoimmunity and Apoptosis[END_REF][START_REF] Dembitzer | gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin[END_REF][START_REF] Tettey | Severity of malaria in relation to a complement receptor 1 polymorphism: a case-control study[END_REF]Son et al. 2015;[START_REF] Bulla | C1q acts in the tumour microenvironment as a cancerpromoting factor independently of complement activation[END_REF][START_REF] Mangogna | Is the Complement Protein C1q a Pro-or Anti-tumorigenic Factor? Bioinformatics Analysis Involving Human Carcinomas[END_REF].

C1q/LAIR-1 interaction

Collagenous regions are not an exclusive feature of the collagen family members and are present in various soluble proteins such as collectins, C1q, adiponectin and ficolins [START_REF] Myllyharju | Collagens, modifying enzymes and their mutations in humans, flies and worms[END_REF]) (Figure 2). Because LAIR-1 interacts with a generic collagenous scaffold, it would likely interact with these proteins containing collagen-like regions. Up to now, SP-D, MBL and C1q have been identified as LAIR-1 and LAIR-2 ligands in vitro, conferring them a putative role in the modulation of the immune system [START_REF] Son | C1q limits dendritic cell differentiation and activation by engaging LAIR-1[END_REF]Olde Nordkamp et al. 2014a, b).

Furthermore, functional studies of C1q interaction with LAIR-1 on immune cells surface revealed that C1q directly crosslinks LAIR-1, triggering its ITIMs phosphorylation which consequently inhibits monocyte to dendritic cell differentiation and suppresses INF-α production by plasmacytoid dendritic cells [START_REF] Son | C1q limits dendritic cell differentiation and activation by engaging LAIR-1[END_REF]). In addition, similar tolerogenic properties were identified for C1q/LAIR-1 interaction on monocytes surface. Indeed, this interaction inhibits toll-like receptor activating signals and maintains monocyte tolerance (Son and Diamond 2015) .

C1q interaction with LAIR-1 engages its collagen-like regions thus leaving its globular regions free to interact with other immune receptors [START_REF] Son | C1q limits dendritic cell differentiation and activation by engaging LAIR-1[END_REF]. In this context, initial studies identified and characterized two Ig-like immune receptors for their interaction with C1q globular regions: the receptor for advanced glycation end products (RAGE) and siglec-3 (CD33) [START_REF] Ma | RAGE binds C1q and enhances C1q-mediated phagocytosis[END_REF][START_REF] Son | C1q and HMGB1 reciprocally regulate human macrophage polarization[END_REF][START_REF] Son | Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression[END_REF].

RAGE is a 45 kDa activating transmembrane receptor expressed on monocytes, macrophages and dendritic cells surface with an extracellular region composed of three Ig-like domains: one V-type and two C2-types. This receptor triggers inflammatory reaction cascades when it interacts with several ligands such as AGEs (advanced glycation end products), HMGB1 (high mobility group box 1), LPS or DNA [START_REF] Sorci | RAGE in tissue homeostasis, repair and regeneration[END_REF][START_REF] Wang | RAGE Plays a Role in LPS-Induced NF-κB Activation and Endothelial Hyperpermeability[END_REF]) (Figure 16A). In addition, reports demonstrated RAGE interaction with phosphatidylserine, revealing its putative implication in apoptotic cells phagocytosis [START_REF] He | Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells[END_REF]. Son and colleagues showed that C1q is able to interact simultaneously with RAGE and LAIR-1, thus modulating the immune response. Indeed, in inflammatory conditions, RAGE pro inflammatory signals induced by high HMGB1 levels are suppressed by SHP-1 phosphatase recruited by activated LAIR-1 via C1q interaction [START_REF] Son | C1q and HMGB1 reciprocally regulate human macrophage polarization[END_REF]) (Figure 16BC).

CD33 receptor belongs to the siglecs receptor family (sialic acid immunoglobulin-like lectins), a family of receptors engaged in sialic acid dependent cell adhesion. CD33 is the shortest siglec member with two extracellular Ig-like domains: a V-type domain responsible for sialic acid interaction and a C2-type domain [START_REF] Crocker | Siglecs and their roles in the immune system[END_REF]). The cytoplasmic region of CD33 contains two ITIMs, conferring CD33 inhibitory properties regarding inflammation signals.

C1q was the first physiological ligand of CD33 identified inducing ITIMs phosphorylation [START_REF] Son | Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression[END_REF]. In addition, this study revealed that C1q crosslinks CD33 and LAIR-1 and generates a large CD33-C1q-LAIR-1 inhibitory complex on monocyte surface inhibiting dendritic cell differentiation and activation (Figure 16D). dimeric in solution (S92K and S92C mutations, respectively). These two LAIR-1 constructs will be of real benefit for that purpose (Results section, III).

In order to expand our project to the study of C1q interaction with LAIR-2 and CD33, we did preliminary trials to express their Ig-like domains (Results section, IV).

Finally, during my PhD, I contributed to the deciphering of C1q interaction with two other receptors: CR1 and LRP1 (Articles 3 and 4).

RESULTS

I. Article 1: Headless C1q: a new molecular tool to decipher its collagen-like functions

In order to better understand the challenge of controlling the collagen chain registering in the C1q collagen-like regions, I will introduce the specificities of collagens structure and assembly.

The critical role of trimerization domains

Every collagen triplet containing sequence has the intrinsic tendency to assemble in an uncontrolled manner leading to the formation of a gel called gelatin. In order to prevent such molecular aggregates, every collagen holds a C-terminal non-triple helical trimerization domain (Boudko et al. 2012). Four structural classes of trimerization domain have been identified so far: the nc1 domain of collagen IV, the C1q-type domain of collagens VIII and X, the multiplexin trimerization domain of collagens XV and XVIII and the C-propeptide of fibrillar collagens I, II, III, V and XI (Figure 17) [START_REF] Bogin | Insight into Schmid metaphyseal chondrodysplasia from the crystal structure of the collagen X NC1 domain trimer[END_REF][START_REF] Than | The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link[END_REF][START_REF] Sundaramoorthy | Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes[END_REF][START_REF] Kvansakul | Crystal structure of the collagen alpha1(VIII) NC1 trimer[END_REF][START_REF] Van Der | Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially expressed during human B cell differentiation and inhibits B cell receptor-mediated signaling[END_REF][START_REF] Boudko | Crystal structure of human collagen XVIII trimerization domain: A novel collagen trimerization Fold[END_REF][START_REF] Wirz | Crystal structure of the human collagen XV trimerization domain: a potent trimerizing unit common to multiplexin collagens[END_REF][START_REF] Bourhis | Structural Basis of Fibrillar Collagen Trimerization and Related Genetic Disorders[END_REF].

These trimerization domains were demonstrated to correctly align and register the three αchains at their C-terminal extremities, thus initiating collagen triple-helix folding that proceeds to the N-terminus in a zipper like fashion [START_REF] Engel | The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper[END_REF]. The presence of a trimerization domain to selectively align the three collagen α-chains is even more important in the case of heterotrimeric collagens because wrong chain registering into the triple-helix could induce the shedding of ligands binding sites, especially in the cases where the collagen ligand binding involves more than one collagen chain. For example, previous studies revealed that collagen I recognition by the von Willebrand factor (VWF) A3 domain is mediated through a binding site that depends on the relative position of the three collagen chains in the triple-helix.

Indeed, the VWF-A3 binding site on collagen I imposes the trailing position of the α2 chain and any other chain register induces a drastic reduction of the interaction [START_REF] Brondijk | Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex[END_REF][START_REF] Jalan | Chain alignment of collagen I deciphered using computationally designed heterotrimers[END_REF].

Type IX collagen belongs to the FACIT family and is encountered within collagens II and XI fibrils mostly found in cartilage. It is composed of three triple-helical collagen domains called COL1, COL2 and COL3 and four non-collagenous domains called nc1, nc2, nc3 and nc4

(Figure 18). In the case of collagen IX, as for most of the FACIT members, the correct trimerization of the triple helix is controlled by the second non-collagenous domain (nc2) (Boudko and Bächinger 2012). Historically, studies used C1q purified from human serum and derived fragments generated by limited proteolysis. The digestion of C1q with collagenase or pepsin results in GR or CLR fragments, respectively (Figure 4). Use of these serum-derived C1q CLR and GR allowed analysis of both C1q fragments functions [START_REF] Reid | Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement[END_REF]; Hughes-Jones and Gardner 1979). One drawback of the serum-derived C1q and fragments is that the CLR and GR fragments obtained after enzymatic digestion have C-terminal and N-terminal extremities that are directed by the pepsin and collagenase specificity. This is not fully convenient since after C1q digestion with collagenase, the resulting GR still have a short remaining N-terminal collagen sequence. The same problem is also encountered with serum-derived CLR that contains a short C-terminal non-collagen like sequence [START_REF] Thielens | Further characterization of the interaction between the C1q subcomponent of human C1 and the transmembrane envelope glycoprotein gp41 of HIV-1[END_REF]) (Figure 19).

Type
Therefore, serum-derived CLR and GR both share an overlapping sequence (residues 79-98, 81-100 and 78-97 of A, B and C chains respectively) that could hold a putative binding site for C1q partners, leading to difficult deconvolution of each fragment's contribution to the interaction. To avoid the presence of common remaining binding sites, recombinant production of C1q fragments would allow to restrict the sequence extremities to the region of interest.

Moreover, serum-derived C1q fragments only provide wild-type proteins. This problem has been solved with the development of recombinant production of the entire C1q molecule and the globular region. Such development allowed the production of C1q variants containing point mutations in a view to identify C1q residues involved in the interaction with ligands such as C1r2s2, pentraxin 3 or IgM [START_REF] Bally | Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites[END_REF][START_REF] Bally | Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants[END_REF][START_REF] Moreau | Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q[END_REF]). Up to now, only the globular regions of C1q were available in a recombinant form and the only tool to study the CLR was the pepsin-digested serum-derived CLR. This highlights the need of recombinant CLR for which the N and C-terminal extremities could be defined precisely.

As mentioned above, collagen triple-helix formation implies the proper alignment and positioning of the three collagen chains by a trimerization domain. In the case of C1q, this requirement is controlled by the C-terminal GR that specifically associate to initiate the collagen triple-helix formation [START_REF] Kishore | Modular organization of proteins containing C1q-like globular domain[END_REF]. The correct phasing of the C1q collagen chains is even more important because of its heterotrimeric nature. Indeed, the collagen-like region of C1q is composed of three different chains and if the chain order is not respected, some C1q partners' binding sites could be shed. Because of the intrinsic major structural role of the GR for collagen chains assembly and folding, their removal from the C1q sequence would result in the uncontrolled association of the CLR chains. Therefore, the GR cannot be simply removed but need to be replaced by another trimerization domain in order to specifically control the collagen chains registering.

We decided to use the collagen IX nc2 domain to control the CLR chains positioning and phasing. The nc2 domain was really suitable for GR substitution because it is a heterotrimeric domain with the unique structural function of collagen chains specific registering and alignment. The key step of the design of the CLR fusion with the nc2 domain (CLR_nc2) is the choice of which CLR and nc2 chains need to be fused together in order to achieve the same assembly as C1q. To do so, we used the structural information available in the literature and a model of a C1q collagen stem built from the GR crystal structure (PDB code 1PK6, Gaboriaud As shown in Figure 20, the GR A, B and C chains were superimposed on the nc2 α2, α1 and α3 chains, respectively. We thus decided to proceed to these chain combinations.

The following manuscript reports the production, purification and characterization of the resulting CLR_nc2 recombinant protein. LAIR-1 has been mostly described in the literature for its interaction with collagens and it was therefore reasonably suggested that its interaction with C1q was performed via its collagen-like regions. However, this question was addressed in only few studies and further investigations were needed. The use of the CLR_nc2 protein described in Article 1 unambiguously validated the CLR engagement into C1q interaction with LAIR-1. However, we had to slightly modify the recombinant CLR_nc2 protein before using it for the study with LAIR-1. Indeed, when we first designed the CLR_nc2 protein, we introduced three additional collagen GPP triplets between the CLR and nc2 sequences in order to facilitate the collagen triple-helix formation (Article 1). These GPP triplets are likely modified into GPO triplets during the CLR_nc2 processing. There is no doubt in the literature that GPO triplets are high affinity ligands for LAIR-1 and these additional triplets could have introduced artifactual supplementary binding sites in the molecule [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF]. Therefore, we had to remove these collagen triplets from the CLR_nc2 in order to avoid non C1q-specific binding of LAIR-1 to the CLR_nc2. Fortunately, the removal of these additional collagen triplets did not hamper the correct assembly and folding of the C1q collagen-like regions. In order to distinguish this new generation of the CLR_nc2 without the GPP triplets, we decided to rename it C1qCLR_nc2.

Moreover, we also generated the first recombinant C1q collagen stem by deleting the Nterminal bundle from the C1qCLR_nc2 that we called C1qstem_nc2. The three different CLR_nc2 fusion constructs used in the following manuscript are shown in Figure 21. Moreover, we investigated C1q/LAIR-1 interaction on the side of LAIR-1. To our knowledge, only one published study aimed at identifying the LAIR-1 amino acid residues involved in the interaction with collagen peptides (Brondijk et al. 2010b). In this study, Brondijk and colleagues performed nuclear magnetic resonance (NMR) experiments to map LAIR-1 residues involved in the interaction with a collagen III peptide. Binding assays using flow cytometry with cells transfected with wild-type or mutated membrane LAIR-1 allowed them to determine the essential role of the two highly conserved arginine 59 (R59) and glutamate 61 (E61) LAIR-1 residues. Interestingly, these two residues are also highly conserved in the glycoprotein VI, another Ig-like collagen receptor, homologous to LAIR-1. This strongly suggests that both LAIR-1 and GPVI interact with collagens using a common mechanism.

To our great surprise, we found a crystal structure of the GPVI Ig-like domain in complex with a collagen peptide deposited in the protein data bank, with no publication associated (PDB code 5OU8) (Figure 22). This structure revealed the central position of the GPVI R38 and E40 residues, homologous to the LAIR-1 R59 and E61, in the binding of the collagen peptide.

Moreover, analysis of this structure revealed that the collagen motif recognized by the GPVI mainly corresponds to the hydroxyproline residues of two consecutive GPO triplets (GPOGPO) (Figure 22). By homology, we made the assumption that this specific collagen motif is also likely recognized by LAIR-1 through it R59 and E61 residues in collagens in general and in our case in C1q CLR sequence.

Interestingly, the GPVI Ig-like domain contains one tryptophan residue (W76) that recognizes the neighboring collagen chain (Figure 22). This tryptophan residue is conserved in LAIR-1 sequence (W109). This particular residue may therefore be involved in the collagen recognition by LAIR-1.

III. Unpublished Data: LAIR-1 dimerization effects on C1q interaction 1 Scientific context

As for most of the transmembrane receptors, signal transduction via LAIR-1 relies on its dimerization on the cell surface. It has been previously reported that LAIR-1 intracellular ITIMs phosphorylation is induced by antibody-mediated LAIR-1 dimerization on immune cells, thus recruiting phosphatase and inhibiting positive immune signals induced by other receptors [START_REF] Meyaard | LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes[END_REF][START_REF] Xu M J | Identification and characterization of leukocyte-associated Iglike receptor-1 as a major anchor protein of tyrosine phosphatase SHP-1 in hematopoietic cells[END_REF]. Collagens, the natural ligands of LAIR-1 were shown to induce ITIMs phosphorylation upon binding to LAIR-1 [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF]. Based on the fact that LAIR-1 recognizes the collagen Gly-X-Y backbone and that collagen molecules contain multiple collagen triplets, it is likely that one collagen molecule could bind and aggregate several LAIR-1, thus transducing the inhibitory signal. In this context, several analyses of soluble LAIR-1 interaction with collagens used antibody Fc region fusions in order to induce the LAIR-1 Ig-like domain dimerization [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF][START_REF] Rygiel | Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1[END_REF]Olde Nordkamp et al. 2014a;[START_REF] Agashe | Leukocyte-Associated Ig-like Receptor 1 Inhibits T h 1 Responses but Is Required for Natural and Induced Monocyte-Dependent T h 17 Responses[END_REF][START_REF] Kumawat | LAIR-1 Limits Neutrophilic Airway Inflammation[END_REF]). In our particular case of the study of LAIR-1 interaction with C1q, such Fc fused LAIR-1 Ig-like domain cannot be considered because C1q is known to interact with the Fc region of antibodies through its globular regions [START_REF] Hughes-Jones | Reaction between the isolated globular sub-units of the complement component C1q and IgG-complexes[END_REF]. This rises the need for an alternative approach to obtain a dimeric LAIR-1 Ig-like domain.

Several crystal structures of the LAIR-1 Ig-like domain are available in the PDB and by analyzing them, we found one (PDB code: 3RP1) with two monomers presenting a possible dimeric-like association in the crystal packing. This association of two LAIR-1 Ig-like domains would be possible on the cell membrane. Indeed, the C-terminal extremities (membrane side) are oriented in the same direction, close to each other (12 Å distance) and the two collagen binding sites are exposed on the other side of the domain (Figure 23A). However, the interaction surface between the two LAIR-1 Ig-like domain is narrow and not favorable for the dimer formation, since PISA (proteins, interfaces, structures and assemblies, [START_REF] Krissinel | Inference of Macromolecular Assemblies from Crystalline State[END_REF] interface analysis gives a null score. This suggests that LAIR-1 Ig-like domain dimerization on the cell surface should be induced by ligand binding. We tried to detect the binding of LAIR-1 Ig-like domain on itself by SPR and did not detect any binding signal, therefore supporting the ligand-induced dimerization of LAIR-1. As illustrated in Figure 23B, this association of Ig-like domains could likely accommodate the binding of a collagen triple helix between the two monomers collagen binding surfaces which are perfectly aligned.

Interestingly, a remarkable serine residue (S92; in red in Figure 23) is located in the contact zone of the two LAIR-1 domains. We decided to mutate this serine into a cysteine residue (S92C) in order to block the dimer via disulfide bond for functional and structural studies. We first checked that the S92C mutation would properly position the two cysteines to accommodate an inter-monomer disulfide bridge by using two predictive softwares: modip and DdB2 [START_REF] Thangudu | Native and modeled disulfide bonds in proteins: knowledge-based approaches toward structure prediction of disulfide-rich polypeptides[END_REF][START_REF] Dombkowski | Protein disulfide engineering[END_REF]. This hypothesis was supported by the two theoretical approaches.

In addition, the serine 92 was also mutated into a bulkier lysine residue (S92K) in order to introduce steric hindrance and avoid formation of such a dimer.

Results

SEC-MALLS comparative analysis of LAIR-1 WT, S92K and S92C

In order to validate our strategy and investigate the oligomeric state of the two LAIR-1 S92K and S92C variants, we performed size-exclusion chromatography multi angle laser light scattering (SEC-MALLS) experiments on the purified samples. As shown in Figure 24A, the LAIR-1 WT Ig-like domain (blue curve) is mostly present in a monomeric form with a small portion of dimer. As opposed to LAIR-1 WT, the S92C variant presents a majority of dimers and SDS-PAGE analysis of the dimer fraction under non-reducing and reducing conditions revealed that the S92C dimer is covalently linked thanks to a disulfide bond between the two Ig-like domains (Figure 24B). As expected, the presence of a lysine residue at position 92 leads to a LAIR-1 variant exclusively monomeric in solution (Figure 24A, red curve).

We managed, with the mutations of the serine 92 of LAIR-1 Ig-like domain, to generate LAIR-1 variants that are exclusively monomeric (S92K) or dimeric (S92C) that will constitute useful tools for the study of LAIR-1 dimerization effects on C1q interaction.

Structural characterization of LAIR-1 S92C variant

In order to obtain information about the conformation of the LAIR-1 S92C covalent dimer, we performed X-ray crystallography to solve its structure (Appendix 2). As shown in Figure 25A, our crystal structure reveals that the covalent S92C dimer structure does not exhibit the expected conformation of the initial dimeric model and presented two Ig-like domains rotated around the disulfide bond to get a different orientation from the one presented in Figure 23A. In fact, it is not surprising that a unique disulfide bond at the molecular edge might not rigidly maintain the two monomers and likely allows free rotation around it. This highlights the fact that LAIR-1 extracellular Ig-like domains might not spontaneously dimerize at the cell surface. Thus, the dimerization of LAIR-1could possibly be dependent on the interaction of both monomers with the same collagen, acting as a bridging molecule. In order to get information about the conformation of the S92C dimer in solution, we performed small angle X-ray scattering (SAXS) experiments and compared the experimental data to the theoretical data generated by both the dimeric model and our solved crystal structure (Figure 25B). The experimental data do not fit with the simulated curves of either the model or the crystal structure (Figure 25B, red and blue curve, respectively). The intermediate conformation observed in the SAXS experiment can be explained by the dynamic rotation of the two monomers at each side of the disulfide bond. This again suggests that the parallel association shown in figure 27 is not stable and likely occurs only transiently.

Surface plasmon resonance analysis of LAIR-1 S92K and S92C interaction with C1q

The interaction of the LAIR-1 S92C dimer and S92K monomer with serum-derived C1q (sC1q) was investigated using surface plasmon resonance (SPR) and compared to the LAIR-1 WT. As shown in Figure 30A, LAIR-1 S92C dimer kinetic analysis of the interaction with immobilized sC1q reveals that the dissociation constant KD is 4 times lower than the one obtained with LAIR-1 WT (27 µM instead of 112 µM). Conversely, preliminary SPR experiments highlight that the LAIR-1 interaction is slightly decreased by the S92K mutations (20% decrease of the response, Figure 30B). However, this lower binding efficiency needs to be confirmed by kinetics experiments and KD value determination. These preliminary binding assays show the positive effect of LAIR-1 dimerization on the affinity of the interaction with C1q that could be attributed to an increase in avidity. In order to block the LAIR-1 S92C dimer in the conformation were the two Ig-like domains are oriented in the same orientation (Figure 23), we performed crystallization assays in the presence of the synthetic collagen peptide used in Article 2. As LAIR-1 S92C interacts with C1q, we also performed crystallization assays in the presence of the C1qstem_nc2 (Figure 21).

Unfortunately, we did not find conditions prone to crystal formation and further screenings of crystallogenesis conditions are needed.

IV. Preliminary data: expression of LAIR-2 and CD33 Ig-like domains 1 Design of the constructs

In order to extend the study with the two other Ig-like receptors LAIR-2 and CD33, we tried to produce their soluble Ig-like domains. To do so, we initially planned to use two different expression systems: a bacterial expression system (BL21(DE3)) for structural studies purposes and an eukaryotic expression system (HEK293F) for the functional interaction studies.

Therefore, we cloned the Ig-like domain sequences in two different expression vectors: pET28a(+) for expression in bacteria and pcDNA3.1(+) for eukaryotic expression. On one hand, for structural studies, we restricted the sequences cloned in pET28a to the Ig-like domain sequence (i.e., LAIR-2: 22-122 and CD33: 142-232; immature protein amino acids numbering).

One the other hand, the sequences cloned into the pcDNA3.1(+) vector were chosen to code for the entire extracellular region of each receptor (i.e. LAIR-2: 1-152 and CD33: 1-232; immature amino acids numbering) (Figure 27; Appendix 1).

Figure 27: Schematic representation of the Ig-like receptors fragments expressed in bacterial (A) and eukaryotic expression systems (B).

The N-and C-terminal residues are indicated with grey numbers (immature protein amino acids numbering). The additional C-terminal polyhistidine tags (Histag) are labelled in red.

Expression in bacteria: E. coli BL21(DE3)

We first tested the expression of the receptor constructs into E. coli BL21(DE3) cells. We tried the expression of LAIR-2 and CD33 using classical IPTG induction or an autoinduction medium (Appendix 1). As shown in Figure 16, none of the two tested induction procedures allowed the expression of the LAIR-2 Ig-like domain. Indeed, even if LAIR-1 and LAIR-2 Iglike domain sequences are highly homologous (84% of homology), no protein band appeared after induction (Figure 28A).

Regarding the Ig-like domain of CD33, the expression was induced in BL21(DE3) but the produce protein was only present in the non-soluble fraction (Figure 28B). This was confirmed by western blot analysis using an HRP-coupled monoclonal antibody directed against the polyhistidine tag (not shown). As for LAIR-2 the expression of CD33 Ig-like domain in BL21(DE3) could not be achieved in these conditions.

Therefore, the bacterial expression system did not seem suitable for the expression of LAIR-2 and CD33 Ig-like domains in the conditions tested.

Figure 28: Expression tests of LAIR-2 and CD33 Ig-like domains in E. coli BL21(DE3). SDS-PAGE analysis (14% acrylamide) of the different fractions of the expression tests of Ig-like domains of LAIR-2 (A) and CD33 (B). For each receptor

Expression in eukaryotic cells: HEK293F

In order to express the entire extracellular regions of LAIR-2 and CD33, we transfected HEK293F cells with the pcDNA3.1(+) vectors containing the corresponding coding sequences (Appendix 1). We then selected the stable transfectants using neomycin (400 µg/mL). The culture medium of stably expressing cells was analyzed by western blot using an HRP-coupled monoclonal antibody directed against the polyhistidine tag. The western blot analysis revealed that the stable cell lines expressed the three constructs in a soluble form. However, the expression levels are very different for the three receptor fragments: LAIR-2 was very poorly expressed whereas CD33 seemed to be expressed in reasonable quantities (Figure 29).

I. LAIR-1 and GPVI homologs share a common collagen backbone recognition mechanism

During this PhD thesis, we were interested in the deciphering of the immune Ig-like receptors (RAGE, CD33, LAIR-1 and LAIR-2) interaction with the defense collagen C1q. Due to limitations inherent to the protein production, we mainly focused on the receptor LAIR-1 for which we managed to produce a soluble form of the extracellular Ig-like domain. As expected, we showed that LAIR-1 Ig-like domain specifically interacts with the C1q CLR with the essential contribution of LAIR-1 R59 and E61 residues. These residues have already been described for their critical role for membrane LAIR-1 interaction with collagens I, III and IV peptides (Brondijk et al. 2010a). Therefore, this suggests that LAIR-1 recognition of the C1q CLR collagen backbone shares some specificity with other collagens through its R59 and E61 residues. Interestingly, these two residues are conserved in GPVI, a collagen receptor homologous to LAIR-1 (Figure 13). Therefore, LAIR-1 and GPVI interaction with collagens might engage similar mechanisms. This hypothesis was first suggested by Tomlinson et al., who showed that LAIR-1 co expression with GPVI inhibits the collagen-induced GPVI signaling [START_REF] Tomlinson | Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines[END_REF]). Unfortunately, there are no studies in the literature reporting the engagement of these two arginine and glutamate residues (R38 and E40, in GPVI sequence)

in the GPVI interaction with collagen. However, a crystal structure of the GPVI Ig-like domain in complex with a collagen peptide has been deposited in the PDB in 2017 with no associated publication (PDB code: 5OU8). This structure unambiguously demonstrates that the R38 and E40 residues of GPVI are critical for collagen recognition with a central position at the interaction surface (Figures 14B and22). As for GPVI, LAIR-1 interaction with collagen is dependent on the presence of hydroxyproline residues. Consistently, R38 and E40 residues in the GPVI/collagen crystal structure mainly interact with hydroxyproline residues and, extended to LAIR-1 by homology, this strongly suggests that LAIR-1 R59 and E61 residues recognize hydroxyproline residues (Figure 22). Based on that assumption, we identified putative LAIR-1 binding motifs on C1q and, thanks to additional data from our experiments, we proposed a model for LAIR-1 interaction with a C1q collagen stem (Article 2). This model allowed us to identify LAIR-1 residues involved in the specific recognition of C1q collagen chains. This highlights that the interaction of C1q with LAIR-1 isn't just based on generic collagen backbone recognition, but is more specific with the accurate recognition of C1q CLR amino acids by LAIR-1 distal residues in addition to the central R59 and E61.

II. LAIR-1 dimerization and C1q interaction

We wanted to explore the question about the LAIR-1 dimerization associated to its signaling function, and more precisely in the context of its interaction with C1q. Indeed, LAIR-1 dimerization is required in order to trigger signal transduction and inhibitory responses.

Generally, studies of receptors dimerization use Fc region fusion proteins and there are many examples in the literature of studies using soluble Fc-fused LAIR-1 Ig-like domains [START_REF] Lebbink | Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1[END_REF][START_REF] Lebbink | Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III[END_REF][START_REF] Rygiel | Tumor-expressed collagens can modulate immune cell function through the inhibitory collagen receptor LAIR-1[END_REF]Olde Nordkamp et al. 2014a;[START_REF] Agashe | Leukocyte-Associated Ig-like Receptor 1 Inhibits T h 1 Responses but Is Required for Natural and Induced Monocyte-Dependent T h 17 Responses[END_REF][START_REF] Kumawat | LAIR-1 Limits Neutrophilic Airway Inflammation[END_REF]. The use of such fusion proteins could be delicate in the case of C1q interaction study because it is now well established that Fc regions of immunoglobulins are high affinity ligands for the C1q globular regions [START_REF] Marqués | Arginine residues of the globular regions of human C1q involved in the interaction with immunoglobulin G[END_REF][START_REF] Ugurlar | Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement[END_REF]. Therefore, the use of Fc-fused proteins could likely introduce additional interaction properties independent of the LAIR-1 Ig-like domain. Based on available structural data, we managed to produce a dimeric variant of our LAIR-1 Ig-like domain construct by introducing an intermolecular disulfide bond instead of using Fc fusion (Results part, section III). First SPR experiments revealed that the S92C dimeric LAIR-1 variant interacts with higher affinity than wild-type LAIR-1 over immobilized C1q. Even if this result needs to be replicated, it indicates that we now have soluble LAIR-1 variants (exclusively monomeric or dimeric) to test the in vitro effects of LAIR-1 dimerization on C1q interaction.

Moreover, we used X-ray crystallography to solve the structure of LAIR-1 S92C dimer. This structure did not match with the one that we were expecting. Indeed, the two Ig-like domains were elongated in our crystal structure. We then performed SAXS experiments and showed that this covalently linked LAIR-1 dimeric variant displays a free rotation around the disulfide bond, likely introducing dynamic feature of the dimer in solution. These results support the idea that LAIR-1 dimerization on a cell surface should be induced by ligand interaction (Figure 30A).

In order to limit the free rotation of the two LAIR-1 Ig-like domains, CHO cells were transfected with full-length LAIR-1 containing the S92C mutation. Indeed, on the cell surface, the orientation of the C-terminal extremity of the Ig-like domain will also be constrained by the transmembrane domain, therefore possibly limiting the rotation around the disulfide bond (Figure 30B). This cellular aspect of the study is part of a collaboration with the team of Yves Delneste at the Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA). Furthermore, Olde Nordkamp and colleagues reported that the soluble Fc-fused Ig-like domain of LAIR-2 was able to efficiently inhibit both the classical and lectin complement pathways (Olde Nordkamp et al. 2014a). Therefore, it would be interesting to test if the dimeric LAIR-1 S92C Ig-like domain could also inhibit complement activation through the classical and lectin pathways.

III. CLR_nc2 fusion protein: a promising tool to investigate the functions associated to C1q collagen-like regions

In order to investigate the interaction of LAIR-1 with the collagen-like regions of C1q, we engineered the first recombinant C1q CLR (Article 1). The most delicate issue of the recombinant production of heterotrimeric C1q CLR is to preserve its specific trimerization and registering of the three different collagen chains. Indeed, this registering function is achieved by the C1q GR and their removal could result in random association of the C1q collagen chains.

We fixed this issue by replacing the C1q GR by the second non-collagenous trimerization domain of collagen IX (nc2). Such substitution results in the production of a chimeric C1q called CLR_nc2 consisting in an N-terminal C1q collagen-like region with 6 C-terminal nc2 domains instead of the GR. We showed that the CLR_nc2 protein is well assembled and folded using different biochemical and biophysical methods and SPR experiments revealed that this chimeric "headless C1q" retains its functional interaction properties towards C1q CLR ligands.

We used this newly developed tool to investigate the interaction of LAIR-1 with the collagenlike region of C1q, especially by taking advantage of the recombinant production to introduce point mutations into the CLR sequence. Therefore, based on our model of LAIR-1 interaction with C1q, we produced two C1qCLR_nc2 variants comprising the mutation of R72 or K65 residues of C1q A and B chains into a glutamate. We tested these two variants for their interaction with C1q using SPR and showed that the C1q A_R72 and B_K65 are not essential for LAIR-1 binding but are likely involved in the specificity of the interaction.

Before use, the CLR_nc2 variants were tested for their interaction with C1r2s2 in order to check their functionality. Interestingly, one CLR_nc2 variant containing the mutation of lysine 65 on CLR B chain into a glutamate residue (B_K65E) exhibited a reduced binding capacity for C1r2s2 compared to the wild type (Appendix 3). Therefore, we unexpectedly revealed the importance of this particular residue, in the interaction with the C1r2s2 tetramer. This highlights the potential use of the CLR_nc2 protein to investigate C1q interaction with C1r2s2 and therefore may help in the understanding of complement classical pathway activation.

One additional interesting application of the newly generated CLR_nc2 protein lies in the development of an ELISA for detection of autoantibodies directed against C1q in patients suffering from autoimmune diseases. Indeed, the anti-C1q autoantibodies found in patients suffering from SLE are mainly directed against its collagen-like regions [START_REF] Uwatoko | Characterization of C1q-binding IgG complexes in systemic lupus erythematosus[END_REF][START_REF] Uwatoko | C1q solid-phase radioimmunoassay: evidence for detection of antibody directed against the collagen-like region of C1q in sera from patients with systemic lupus erythematosus[END_REF]. Moreover, the principal issue in the development of such ELISA development is the intrinsic ability of C1q to interact with the serum immune complexes present in patients with SLE, thus perturbating the detection process. Therefore, to prevent this nonspecific interaction, two solutions are reported in the literature: the use of high ionic strength (0.5-1 M NaCl) or the replacement of C1q by the CLR [START_REF] Antes | Evidence for the presence of autoantibodies to the collagenlike portion of C1q in systemic lupus erythematosus[END_REF][START_REF] Wener | Antibodies to the collagen-like region of C1q in sera of patients with autoimmune rheumatic diseases[END_REF][START_REF] Kohro-Kawata | The effect of high salt concentration on detection of serum immune complexes and autoantibodies to C1q in patients with systemic lupus erythematosus[END_REF]. However, the production of C1q CLR by enzymatic digestion is a hard and expensive process. Therefore, the recombinant CLR_nc2 protein provides an easier way to rapidly generate high quality antigen for ELISA purpose.

In addition, the CLR_nc2 protein could constitute an important tool to deconvolute the contribution of C1q functional regions (i.e., CLR and GR) in the interaction with its receptors.

For example, we showed that CR1 and LRP1 interaction with C1q mostly involves its CLR but a contribution of the GR was also observed (Articles 3 and 4). This suggests that the possible shared sequence between the C1q CLR and GR fragments (Figure 19), could contain a binding site for these receptors or that secondary binding to C1q GR may take place. Therefore, the use of the CLR_nc2 protein, for which the extremities can be user-defined, should unambiguously answer this question.

Aside from in vitro interaction studies the CLR_nc2 protein could be used in cellular investigations. In this context, we plan to study the effects of CLR_nc2 binding on the cytokines expression profile by macrophages. This work was initially proposed as a part of the collaboration with the team of Yves Delneste at the Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA). Such analysis could provide insights into the contribution of the CLR in the C1q-mediated macrophage polarization.

Moreover, we generated a truncated fragment of the CLR_nc2 protein by deleting the Nterminal sequence corresponding to the collagen bundle until the hinge region of each C1q chain. This results in the generation of a single heterotrimeric collagen stem with a C-terminal nc2 domain. The interaction of LAIR-1 with this fragment was similar to the one with C1q, supporting the presence of the LAIR-1 binding site in the collagen cone.

Furthermore, this smaller fragment of the CLR_nc2 (C1qstem_nc2) was initially developed for structural studies purpose. The recombinant production of user-defined small heterotrimeric C1q CLR fragments will likely open the way towards the structural characterization of C1q interaction with its CLR ligands/receptors. For instance, a previous study reports the crystal structure of the CUB1-EGF-CUB2 domains of C1s in complex with a synthetic collagen peptide [START_REF] Venkatraman Girija | Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation[END_REF]. In this study, the authors used a homotrimeric collagen peptide corresponding to a MBL-like sequence. The MBL and C1q sequences in the binding region of C1r2s2 and MASPs share similar amino acids and allowed the authors to extrapolate and discuss about C1s interaction with C1q. The C1qstem_nc2 fragment containing the "real" heterotrimeric collagen helix of C1q could be used to solve the structure of C1r and/or C1s in complex with a C1q CLR fragment. In the context of my PhD, and especially the investigation of LAIR-1 interaction with C1q, we performed crystallization trials of the C1qstem_nc2 alone and with the Ig-like domain of LAIR-1. We tested several conditions using the Classics Suite and PEGs I (Qiagen) screens available at the HTX lab platform of EMBL Grenoble. None of the tested conditions gave promising results with mostly clear drops. These unsuccessful trials could be explained by the certainly too long and too flexible C1qstem_nc2 protein. Indeed, the C1qstem_nc2 contains 16 Gly-X-Y collagen triplets in addition to the nc2 domain, which results in a quite long molecule certainly presenting intrinsic flexibility. Therefore, the collagen part should be reduced in size and optimized to help crystallization. For example, the structural study of Wallis laboratory used a 9-triplets long collagen peptide and the collagen I guest fragment used for the nc2 domain crystal structure contains only 11 collagen triplets [START_REF] Venkatraman Girija | Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation[END_REF][START_REF] Boudko | Structural insight for chain selection and stagger control in collagen[END_REF]. Therefore, the C1qstem_nc2 protein needs to be shortened in order to have an optimal collagen triple helix size, containing the LAIR-1 binding site. In addition, the many post-translational modifications undergone by the collagen region constrains the use of eukaryotic expression systems (in our case, 293EXPI cells) to produce the C1qstem_nc2. Unfortunately, only weak purification yields have been obtained (around 300 µg per liter of cell culture). These low purification yields limited the crystallization assays and further expression/purification steps optimization would be required to improve the purification yields and try higher protein concentrations.

IV. The duality of C1q complement-dependent and independent functions

As mentioned above, the molecular dissection of C1q showed that LAIR-1 interaction with C1q exclusively involves its CLR. To more precisely identify the LAIR-1 binding site on the C1q CLR, we performed a competition assay with C1r2s2, the canonical C1q CLR ligand and showed that LAIR-1 is no more able to interact with C1q when it is associated with C1r2s2 in the C1 complex. This inhibitory effect of C1r2s2 on C1q recognition by LAIR-1 demonstrates that the LAIR-1 binding site is not located on the N-terminal collagen bundle of C1q as previously suggested [START_REF] Liu | HMGB1-C1q complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis[END_REF]) but in the collagen cone, close to the binding site of the C1r2s2 tetramer. The location of LAIR-1 binding site into the collagen cone of C1q instead of its Nterminal collagen bundle is somehow consistent with the better accessibility of the isolated collagen stems. Importantly, the C-terminal half of the C1q collagen stem of the cone is rich in basic residues that could constitute an environment prone to receptors binding. Moreover, we also reported the ability of C1r2s2 to prevent the binding of two C1q CLR receptors: CR1 and LRP1 (Articles 3 and 4). Interestingly, we showed that none of LAIR-1, CR1 or LRP1 binding to C1q involved the same binding site as C1r2s2, namely C1q A_K59, B_K61 and C_K58 residues [START_REF] Bally | Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites[END_REF]. Exclusive interaction of C1q with the C1r2s2 tetramer or its CLR receptors highlights the intrinsic distinction of C1q CLR functions. On one hand, when associated with the C1r2s2 tetramer, C1q triggers pathogens and damage-self elimination through complement classical pathway activation. On the other hand, when C1q is devoid of its cognate proteases, it is able to interact with several membrane receptors through its collagenlike region (LAIR-1, CR1 and LRP1) and trigger complement-independent functions such as phagocytosis enhancement or immune response modulation.

This PhD thesis aimed at deciphering the molecular mechanisms underlying the immune tolerance and modulation functions of C1q, especially, through its interaction with the immune Ig-like receptor LAIR-1. We provided the first detailed investigation of C1q interaction with LAIR-1 Ig-like domain from a structural and molecular point of view. This work will contribute to the understanding of the C1q immune tolerogenic properties mediated through LAIR-1

interaction. However, it is important here to keep in mind that immune cells also possess receptors for the globular regions of C1q at their surface such as the two Ig-like receptors CD33

and RAGE that have distinct activities regarding inflammatory signals. Therefore, the characterization of C1q GR interaction with these receptors is needed in order to further decipher the mechanisms underlying C1q-mediated immune modulation.

APPENDICES

I. Appendix 1: Material and methods for protein expression 1 Molecular biology: Design of the expression vectors

The DNA coding the full-length sequences of human LAIR-1, LAIR-2 and CD33 were purchased from OriGene (product numbers RG206439, RC210108 and SC122608, respectively). The sequences of interest were amplified by polymerase chain reaction (PCR) using oligonucleotides designed in order to insert specific restriction enzymes cleavage sites for cloning purposes. When needed, additional polyhistidine tag (Histag) and a stop codon were also inserted at the 3'end.

Design of the pET28a vectors for expression of LAIR-1, LAIR-2 or CD33 in bacteria

The coding sequences of the Ig-like domain of , andCD33 (142-232) (immature protein amino acids numbering for all constructs) were amplified using the oligonucleotides listed in Table 3. The oligonucleotides were designed in order to insert flanking NcoI and XhoI restriction sites at the 5' and 3' extremities, respectively. The pET28a vector (Novagen, product number 69864-3) contains a polyhistidine tag (6 His) followed by a stop codon, we therefore inserted a XhoI restriction site just after the coding sequence of the protein so that we did not have to insert also the tag by PCR (Figure 31). The PCR experiments were performed with the Phusion High-fidelity PCR Master mix following the manufacturer's protocol (ThermoFisher Scientific, product number F532S) (Table 4).

Name Sequence (5'3') Modification

PCR REACTION MIX

Template DNA 1 ng (2 µL at 0.5 ng/µL) The PCR products were loaded on a 1% agarose gel and purified using the NucleoSpin gel and PCR clean-up kit following the manufacturer's protocol (Macherey-Nagel, product number 740609). Then the PCR products and the pET28a vector were digested with both NcoI and XhoI restriction enzymes (New England Biolabs, product numbers FD0573 and FD0694, respectively) and the digested products were purified using the NucleoSpin gel and PCR cleanup kit.

The digested vector and inserts were ligated using the T4 DNA ligase using the manufacturer's protocol (ThermoFisher Scientific, product number EL0011) (Table 5). DH5α competent cells were transformed using the ligation products and selected on kanamycin (50 µg/mL) LB-Agar petri dishes following the manufacturer's protocol (ThermoFisher Scientific, product number 18265017).

LIGATION REACTION MIX

Plasmid DNA of growing colonies was extracted and purified using the NucleoSpin plasmid mini kit (Macherey-Nagel, product number 740588). The sequence of the purified DNA was controlled by Sanger sequencing (Eurofins Genomics).

Design of the pcDNA3.1(+) vectors for expression of LAIR-1, LAIR-2 or CD33 in mammalian cells

The coding sequences of the entire extracellular regions with the N-terminal signal peptide of LAIR-1 (1-163), LAIR-2 (1-152) or CD33 (1-232), (immature protein amino acids numbering for all constructs) were amplified by PCR using the oligonucleotides listed in Table 6. The oligonucleotides were designed in order to amplify the sequence coding the desired protein and insert the coding sequence for a polyhistidine tag followed by a stop codon at the 3' extremity and two flanking NheI and XhoI restriction sites at the 5' and 3'end, respectively. The PCR experiments were performed in the same conditions as described in Table 4. The rest of the cloning procedure was similar to the one used for the design of the pET28a vectors with the differences that NcoI restriction enzyme was replaced by NheI (New England Biolabs, product number FD0973) and the insert was cloned in a pcDNA3.1(+) vector (ThermoFisher Scientific, product number V79020) (Figure 32).

Name Sequence (5'3') Modification

Expression tests 2.1 Bacterial expression system: BL21(DE3)

The expression tests in E. coli BL21(DE3) were performed with two different induction methods: (i) IPTG (1 mM) or (ii) auto induction medium.

IPTG induction

BL21(DE3) competent cells (Sigma-Aldrich, product number CMC0014) were transformed with the pET28a vector containing the coding sequence of LAIR-1, LAIR-2 and CD33 Ig-like domain and selected on kanamycin (50 µg/mL) LB-Agar petri dishes following the manufacturer's recommendations. Positive colonies were cultured overnight in 10 mL of LB medium containing kanamycin (50 µg/mL) at 37°C, 180 rpm agitation speed.

Then, 100 mL of LB medium containing kanamycin (50 µg/mL) were inoculated at 0.1 of OD600 with the overnight preculture and incubated at 37°C, 180 rpm. Once the bacterial culture raised an OD600 value of 0.8, IPTG at a final concentration of 1 mM was added to the culture before incubation for 3 hours, at 37°C, 180 rpm. To get a control of expression before IPTG addition, a 1 mL sample of culture was taken and centrifuged at 11,000 g for 5 minutes. After centrifugation, the supernatant was removed and the pellet was resuspended in 4X Laemmli reducing buffer (Homemade: Tris 125 mM, β-mercaptoethanol 1.4 M, glycerol 20%, bromophenol blue 0.1%, pH 6.8) for SDS-PAGE analysis (NI lanes; Figure 28). For the control after induction, a 500 µL sample of culture was taken and treated as described for the NI control (I lanes; Figures 28). The analyses of the supernatant and pellet fractions of the cells after lysis were performed as follows. 2 mL of the 100 mL culture were centrifuged 30 minutes at 4,000 g, 4°C. The supernatant was removed and the cells were resuspended in buffer A (Tris 25 mM, NaCL 150 mM, pH 8.2) before lysis by sonication (2 min, 10 sec ON, 20 sec OFF). The lysate was then centrifuged 30 minutes at 11,000 g, 4°C. 75 µL of supernatant were sampled and mixed with 25 µL of 4X Laemmli reducing buffer for SDS-PAGE analysis (Figure 28). The pellet was resuspended in 2 mL of Buffer A and 75 µL were sampled and mixed with 25 µL of 4X Laemmli reducing buffer for SDS-PAGE analysis (Figure 28).

Autoinduction medium

The transformed BL21(DE3) cells were cultured in 30 mL of LB containing kanamycin (50 µg/mL) for 8 hours at 37°C, 180 rpm. After incubation, a 250 µL sample was taken and cells were harvested (11,000 g, 5 min) and resuspended in 4X Laemmli reducing buffer for SDS-PAGE analysis (NI lanes; Figure 28). One liter of autoinduction medium (Table 7) was then inoculated with 25 mL of preculture and submitted to incubation at 37°C, 180 rpm. After 2 hours of incubation, the temperature was decreased to 27°C and the culture was grown overnight.

Solutions Preparation

MgSO4 1M MgSO4-7H2O: 24. After the overnight incubation, a 250 µL sample was taken and the cells were harvested (11,000 g, 5 min) and resuspended in 4X Laemmli reducing buffer for SDS-PAGE analysis (I lanes;

Figure 28).

The analyses of the supernatant and pellet fractions of the cells after lysis were performed as follows. Two mL of the 1 L culture were centrifuged 30 minutes at 4,000 g, 4°C. The supernatant was removed and the cells were resuspended in buffer A (Tris 25 mM, NaCL 150 mM, pH 8.2) before lysis by sonication (2 min, 10 sec ON, 20 sec OFF). The lysate was then centrifuged for 30 minutes at 11,000 g, 4°C. 75 µL of supernatant were sampled and mixed with 25 µL of 4X Laemmli reducing buffer for SDS-PAGE analysis (Figure 28). The pellet was resuspended in 2 mL of Buffer A and 75 µL were sampled and mixed with 25 µL of 4X

Laemmli reducing buffer for SDS-PAGE analysis (Figure 28).

Eukaryotic expression system: HEK293F

The production of LAIR-1, LAIR-2 and CD33 extracellular domains was performed using the HEK293F expression system. HEK293F cells were transfected with the pcDNA3.1(+) vectors containing the coding sequence of LAIR-1, LAIR-2 or CD33 extracellular region following the manufacturer's protocol (ThermoFisher Scientific, product number R79007). After transfection, the cells expressing the protein of interest were selected using neomycin (also known as geneticin or G418) at a concentration of 400 µg/mL (ThermoFisher Scientific, product number 10131027). The culture supernatant was harvested and replaced every 72 or 96 h of growth during the selection process to get stable cell lines. The mortality (number of dead cells over total number of cells) was determined at each cells dilution and is shown in Figure 33. After the selection, the culture supernatant was tested for the presence of the constructs of interest by western blot using an HRP-coupled monoclonal anti-Histag antibody according to the manufacturer's recommendations (Sigma-Aldrich, product number A7058).

One mL of cell culture was centrifuged at 1,000 g for 5 minutes and the supernatant was harvested and mixed with 1 mL of trichloroacetic acid (TCA) 50% and kept at 4°C to induce proteins precipitation. After 1 night at 4°C, the sample was centrifuged 30 minutes at 11,000 g, 4°C, the supernatant was discarded and the pellet was washed with 1 mL of acetone 100% and centrifuged 30 minutes at 11,000 g, 4°C. The supernatant was removed and the pellet was dried and resuspended in 2X Laemmli reducing buffer. Samples were loaded on a 14% acrylamide gel for SDS-PAGE and western blot analyses (Figure 29). at 5 mg/mL (WT), 4 mg/mL (S92K) or 4.5 mg/mL (S92C) were injected with a constant flow rate of 0.5 mL/min. Online MALLS detection was performed with a DAWN-HELEOS II detector (Wyatt Technology Corp.) using a laser emitting at 690 nm. Protein concentration was determined by measuring the differential refractive index online using an Optilab T-rEX detector (Wyatt Technology Corp.) with a refractive increment dn/dc of 0.185 mL -1 g -1 . Weightaveraged molecular weight determination was done with the ASTRA6 software (Wyatt Technologies).

3

Crystallization and structure determination of LAIR-1 S92C dimer

Crystallization

A first automated screening of the crystallization conditions of LAIR-1 S92C was performed by vapor diffusion using the sitting drop method at 293 K (HTXLab, CRIMS©). The protein, at a concentration of 23 mg/mL was tested with 4 screens: Classics-Suite (Qiagen), PACT (Molecular Dimensions), Wizard_I+II (Rigaku Reagents) and Morpheus (Molecular Dimensions). For each screen, we used a 1:1 ratio for the mixed protein and reservoir solution volumes, respectively.

Based on these 4 screens, we refined the crystallization condition for which the best crystals were obtained (i.e. 0.1 M MES, 0.2 M NaCl, 20 % (w/v) PEG 6000, pH 6). To do so, we varied the PEG 6000 concentrations, the salt and the pH of the reservoir solutions (Table 9). For this crystallization assay, we used the hanging drop method by mixing 1 µL of protein sample concentrated at 21.5 mg/mL with 0.9 µL of reservoir solution. The crystals were harvested directly from the drops (B1, C1, C2 and D2) and flash-frozen in liquid nitrogen. The crystal structure presented in the results section was solved using the diffraction data obtained from the crystals grown in C1 conditions (MES 0.1 M, LiCl2 0.

Data collection and structure determination

LAIR-1 S92C data were collected at the European Synchrotron radiation Facility (ESRF) on the ID30-A3 beamline. The collected data were automatically processed using the EDNA autoprocessing available at the ESRF. Molecular replacement was then performed with the CCP4 suite using Molrep. The model used for molecular replacement was the structure of the monomeric LAIR-1 Ig-like domain (PDB code: 3KGR). The structure was refined with multiple cycles of manual building using COOT [START_REF] Emsley | Features and development of Coot[END_REF]) and of refinement using Refmac5 (CCP4 suite). The refinement statistics (Table 10) were obtained using the last refmac5 refinement cycle.

Diffraction data collected on ID30-A3 beamline

Other diffraction data have been collected for the S92C LAIR-1 obtained in different conditions, but the same crystal packing as above has always been obtained.

Small angle X-ray scattering (SAXS)

SAXS experiments on LAIR-1 S92C (3.5 mg/mL) were performed at the European Synchrotron radiation Facility (ESRF) on the BM29 "BioSAXS" beamline using an X-ray energy of 12.5 keV. To prevent radiation damage, the measurement was performed 10 times and then averaged. Samples scattering curves were obtained after subtraction of the average buffer signals using standard protocols with PRIMUS [START_REF] Konarev | PRIMUS: A Windows PC-based system for small-angle scattering data analysis[END_REF]. The theoretical SAXS curve was generated from the PDB files of our model and solved structures using the FoXS online server [START_REF] Schneidman-Duhovny | FoXS: a web server for rapid computation and fitting of SAXS profiles[END_REF].

Surface plasmon resonance (SPR)

The SPR experiments were performed at 25°C on a BIAcore T200 instrument (GE Healthcare).

Serum-derived C1q (35 µg/mL in 10 mM sodium acetate, pH5) was immobilized on a CM5

sensor chip using the amine coupling chemistry at a flowrate of 10 µL/min in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20, pH 7.4. The interaction experiments were performed at a flowrate of 30 µL/min in 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 0.05% surfactant P20 (pH 7.5). All data were analyzed using the T200 Biaevaluation 3.1 software (GE Healthcare).

LAIR-1 S92C was injected at increasing concentration (1-32 µM) over immobilized serumderived C1q (14,000 RU) with association and dissociation steps of 360 s. The equilibrium dissociation constant (KD) values were calculated from measured responses at equilibrium (Req) by fitting plots Req versus concentration using steady state analysis (Biaevaluation software).

LAIR-1 WT and S92K were injected at a concentration of 10 µM over immobilized serumderived C1q (14,000 RU) with association and dissociation steps of 180 s.

6

Crystallization assays of LAIR-1 S92C with a synthetic collagen peptide or the C1qstem_nc2 construct Automated screenings of the crystallization conditions of LAIR-1 S92C in the presence of a synthetic collagen peptide (Ac-GPOGPOGPOGPKGEQGPOGPO-NH2) or the C1qstem_nc2 at a molar ratio of 1:1 (LAIR-1 S92C:collagen) were performed in each case by vapor diffusion using the sitting drop method at 293 K (HTXLab, CRIMS©).

For the assays with the synthetic collagen peptide, LAIR-1 S92C was at a concentration of 15 mg/mL and four screens were tested: Classics-Suite (Qiagen), PACT (Molecular Dimensions), Midas-plus (Molecular Dimensions) and Morpheus (Molecular Dimensions).

For the assays with the C1stem_nc2 construct, LAIR-1 S92C was at a concentration of 5 mg/mL and two screens were tested: Classics-Suite (Qiagen) and PEGs-I (Qiagen).

Résumé

La molécule C1q a longtemps été étudiée pour son rôle majeur dans l'activation de la voie classique du complément. C1q peut reconnaître de nombreux motifs à la surface des pathogènes ou du soi altéré, via ses six régions globulaires C-terminales (GR). L'autre partie de la protéine, les régions collagéniques (CLR) de C1q, sont généralement associées au tétramère de protéases C1r et C1s (C1r2s2), responsable de l'initiation de la cascade du complément. Ces dernières années, des interactions de C1q avec de nombreux récepteurs membranaires ont été mises en évidence. Parmi ces fonctions indépendantes du complément, un rôle de modulation de la réponse immune par C1q est médié par son interaction avec des récepteurs à domaines de type immunoglobuline (Ig-like) : RAGE (receptor for advanced glycation end-products), CD33, LAIR-1 (leukocyte-associated Ig-like receptor 1) et LAIR-2. Ces récepteurs ont des activités opposées sur les signaux inflammatoires : RAGE induit des signaux pro-inflammatoires alors que CD33 et LAIR-1 sont des récepteurs anti-inflammatoires. Les signaux induits par l'interaction de C1q dépendent donc des récepteurs mis en jeu. Des études récentes suggèrent que RAGE et CD33 interagissent avec les GR alors que LAIR-1 reconnaît les CLR de C1q, ce qui génère un réseau d'interactions complexe qui module la réponse immune en regroupant des signaux pro-et anti-inflammatoires. Cette thèse a pour but d'élucider les détails moléculaires de l'interaction de C1q avec ces récepteurs afin d'aider à la compréhension de la modulation de la réponse immune par C1q. Nous nous sommes principalement intéressés à l'interaction de C1q avec LAIR-1. Une stratégie de dissection moléculaire de C1q, couplée à des expériences de compétition ainsi que l'utilisation de variants de C1q nous ont permis de localiser le site d'interaction de LAIR-1 proche mais différent de celui de C1r2s2 sur les CLR de C1q. Du côté de LAIR-1, une analyse par mutagénèse dirigée nous a permis d'identifier les acides aminés impliqués dans l'interaction avec C1q. Ces résultats suggèrent un modèle d'interaction de C1q avec le domaine Ig-like de LAIR-1. De plus, l'interaction spécifique de LAIR-1 avec les CLR de C1q nous a incité à développer la première production recombinante de cette région de C1q (CLR_nc2). Le remplacement des GR de C1q par le deuxième domaine non collagénique du collagène IX nous a permis de contrôler la mise en registre spécifique des chaînes collagéniques de C1q. La protéine CLR_nc2 a constitué un outil important dans le cadre de cette thèse et le sera certainement également pour l'étude future de l'interaction de C1q avec ses nombreux récepteurs.

Abstract

C1q has long been studied and described for its central role in the activation of the complement classical pathway. This C1q molecule is able to recognize and interact with a large number of molecular patterns on pathogens or altered-self surfaces through its six C-terminal globular regions (GR). On the other side of the protein, the collagenlike regions (CLR) of C1q are generally associated with the soluble tetramer of C1r and C1s proteases (C1r2s2) that is responsible for the initiation of the complement cascade. Over the last years, several complementindependent functions were shown to be held by C1q through its interaction with various membrane receptors present on cell surfaces. Among these functions, the immune modulation properties of C1q have been recently described and were shown to engage immune receptors from the immunoglobulin-like (Ig-like) family: receptor for advanced glycation end-products (RAGE), CD33, leukocyte-associated Ig-like receptor 1 (LAIR-1) and LAIR-2. These receptors have opposite activities towards inflammation: RAGE triggers pro-inflammatory pathways whereas LAIR-1 and CD33 are immune inhibitory receptors. The C1q-mediated signaling differs whether C1q interacts with one or several of these receptors simultaneously. Recent studies suggest that RAGE and CD33 interact with C1q GR while LAIR-1 binds the C1q CLR, leading to a complex interaction network that congregates pro and anti-inflammatory signals, thus modulating the immune response. This PhD thesis aims at deciphering the fine details of C1q interaction with these receptors in order to provide fundamental insights into the C1q-mediated immune modulation. We mainly focused on the interaction of C1q with the collagen receptor LAIR-1. Thanks to the molecular dissection of C1q associated with competition assays and use of recombinant C1q variants, we showed that LAIR-1 binding site is located in close proximity to but is different from the one of C1r2s2 on C1q CLR. On the LAIR-1 side, we performed a mutational analysis based on literature and structural data to identify the key residues involved in C1q binding. These results led us to propose a model of C1q CLR interaction with the Ig-like domain of LAIR-1. Moreover, the specific recognition of C1q CLR by LAIR-1 led us to develop and generate the first recombinant production of C1q CLR (CLR_nc2). The replacement of the C1q GR by the second non-collagenous domain of collagen IX allowed the preservation of the specific C1q collagen chains registering. The CLR_nc2 was an important tool in the context of this PhD thesis and it will certainly also be useful for the future studies on C1q interaction with its many receptors.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 : 21 Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :Figure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 26 :Figure 27 :Figure 28 : 97 Figure 29 :Figure 30 :Figure 31 :Figure 32 :Figure 33 :Figure 34 :Table 2 :

 1234567892110111213141516171819202122232425262728972930313233342 Figure 1: Overview of the complement cascade. ..................................................................... 3 Figure 2: Mechanisms used by soluble defense collagens to respond to infection. .................. 4 Figure 3: Schematic representation of C1q assembly. .............................................................. 9 Figure 4: Generation of C1q GR and CLR fragments by limited proteolysis with collagenase and pepsin. .................................................................................................................................. 9 Figure 5: The C1r2s2 complex. .............................................................................................. 13Figure 6: C1 binding to IgM platforms. .................................................................................. 14 Figure 7: C1q involvement in apoptotic cell clearance. .......................................................... 15 Figure 8: C1q interaction network with the immunoglobulin-like receptors. ......................... 19 Figure 9: Structural organization of LAIR-1. ......................................................................... 21 Figure 10: Crystal structure of a [(Pro-Pro-Gly)10]3 collagen triple-helix. ............................. 22 Figure 11: Supramolecular assemblies formed by collagen. .................................................. 23 Figure 12: LAIR-1 residues involved in collagen binding. ..................................................... 25 Figure 13: LAIR-1 essential collagen binding residues are conserved in the GPVI. ............. 26 Figure 14: OSCAR (A) and GPVI (B) crystal structures in complex with a collagen peptide. .................................................................................................................................................. 27 Figure 15: LAIR-1/RIFIN interaction and immune response. ................................................ 30 Figure 16: Modulation of immune signals by C1q interaction with the Ig-like receptors RAGE, LAIR-1 and CD33. ................................................................................................................... 33 Figure 17: The four structural classes of collagen C-terminal trimerization domains. ........... 37 Figure 18: Domain organization of collagen IX. .................................................................... 38 Figure 19: Location of the sites of limited cleavage by collagenase and pepsin in C1q A, B and C chains. ................................................................................................................................... 39 Figure 20: Choice of the CLR_nc2 chains combination. ........................................................ 41 Figure 21: Schematic representation of the CLR_nc2 (left), C1qCLR_nc2 (middle) and C1qstem_nc2 (right). ................................................................................................................ 54 Figure 22: Molecular and structural determinants of collagen recognition by the glycoprotein VI. ............................................................................................................................................. 56 Figure 23: Parallel LAIR-1 subunits association observed in the crystal packing of LAIR-1 Iglike domain structure. ............................................................................................................... 92

Figure 1 :

 1 Figure 1: Overview of the complement cascade. The complement activation can be initiated by the three different classical, lectin or alternative pathways. Each pathway results in the formation of the C3 convertase. The C3 convertase then cleaves the C3 component, generating the C3b that associates with the C3 convertase and forms the C5 convertase, responsible for C5 cleavage. The C5b fragment initiates the formation of the membrane attack complex (MAC). Multiple steps of the complement cascade are under the regulation of soluble or membrane proteins. The C1r/C1s/MASPs proteases are inhibited by the C1inhibitor (C1-INH) serpin. The convertases are regulated by the factor I (FI) and its cofactors: C4b-binding protein (C4BP), factor H (FH), decay accelerating factor (DAF), complement receptor 1 (CR1), membrane cofactor protein (MCP). Assembly of the MAC is prevented by CD59. Enzymatic activities are represented with green arrows. (Adapted from Defendi et al. 2020).

Figure 2 :

 2 Figure 2: Mechanisms used by soluble defense collagens to respond to infection. (Taken from[START_REF] Casals | Soluble defense collagens: Sweeping up immune threats[END_REF].

  proteolytic activity. Indeed, in the plasma, C3 component can spontaneously hydrolyze and generate the C3(H2O) component which can bind to complement factor B (FB), a structural and functional homolog of C2. FB is then cleaved by factor D (FD), leading to the formation of the alternative fluid phase C3 convertase [C3(H2O)Bb], able to cleave C3 into C3a and C3b fragments. In addition to the spontaneously produced C3b-like molecule, FB can bind to deposited C3b fragments on pathogens and generate the [C3bBb] alternative C3 convertase.

  different types: A, B and C. C1q chains assemble by 3 into 6 ABC heterotrimers, each containing an N-terminal collagen-like triple helical region of around 81 amino acids (CLR) and a C-terminal globular region of 135 residues (GR). These heterotrimers are maintained together through interchain non-covalent interactions and disulfide bonds between chains A-B and C-C. The N-terminal collagen-like region of each C1q chain contains the characteristic collagen Gly-X-Y repetitive sequence pattern where X is often a proline residue and Y a hydroxyproline. As for every collagen, C1q collagen region undergoes several posttranslational modifications such as proline and lysine residues hydroxylation and hydroxylysine

Figure 3 :

 3 Figure 3: Schematic representation of C1q assembly. The C1q molecule is assembled from 18 polypeptide chains of three different types (A, B and C) each containing an N-terminal collagen-like sequence and a C-terminal globular domain (gC1q). Six C1q chains assemble into the (ABC)2 sub-unit that then trimerizes into the full-length (ABC)6 C1q exhibiting its characteristic bouquet-like shape with six C-terminal globular regions (GR) linked together through six collagen-stems (CLR) assembled into an N-terminal bundle. The right panel shows the crystal structure of C1q GR (PDB code: 1PK6). (Taken from Fouët et al. 2020a; Article 1).

Figure 4 :

 4 Figure 4: Generation of C1q GR and CLR fragments by limited proteolysis with collagenase and pepsin.

Figure 5 :

 5 Figure 5: The C1r2s2 complex. A. Schematic representation of the C1r and C1s modular organization. B. Crystal structure of the C1r and C1s CUB1-EGF-CUB2 domains forming the core of the C1r2s2 tetramer (PDB code: 6F1C).

Figure 6 :

 6 Figure 6: C1 binding to IgM platforms. Model of C1q and C1r2s2 bound to an hexameric IgM showing the orientations of C1r and C1s within the C1 complex. Cryoelectron tomography snapshot of C1-mediated cleavage of C4 (Taken from[START_REF] Sharp | Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b[END_REF].

Figure 7 :

 7 Figure 7: C1q involvement in apoptotic cell clearance. C1q is involved in apoptotic cells (in pink) engulfment by macrophages (in blue) through three mechanisms. Left: C1q contributes to C3b opsonization generated by the classical complement pathway activation. Middle: C1q directly opsonizes the apoptotic cell for macrophage recognition. Right: C1q interaction with macrophage receptors increases phagocytic Mer tyrosine kinase (MERTK) and Gas6 expressions (Adapted from Galvan et al. 2012b).

Figure 8 :

 8 Figure 8: C1q interaction network with the immunoglobulin-like receptors. C1q interaction with the immunoglobulin-like receptors RAGE, LAIR-1/2 and CD33 induces different inflammatory signals to modulate the immune response.

Figure 9 :

 9 Figure 9: Structural organization of LAIR-1. A. Amino acids sequence of human LAIR-1. Signal peptide, extracellular region, transmembrane domain and intracellular region sequences are labeled with blue, green, orange and grey rectangles, respectively. Phosporylated ITIM tyrosines are labeled with star icons and the extracellular Ig-like domain sequence is indicated in bold. B. Schematic representation of LAIR-1 with the crystal structure of LAIR-1 Ig-like domain (PDB code 3KGR) cartoon representation is shown in green. The stars represent intracellular phosphorylated tyrosine residues.

Figure 10 :

 10 Figure 10: Crystal structure of a [(Pro-Pro-Gly)10]3 collagen triple-helix (PDB code 1K6F,[START_REF] Berisio | Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)(10)](3)[END_REF]. The three αchains are colored in red, blue and green.

Figure 11 :

 11 Figure 11: Supramolecular assemblies formed by collagen (Taken from Ricard-Blum 2011). FACITs: fibril-associated collagens with interrupted triple helices.

Figure 12 :

 12 Figure 12: LAIR-1 residues involved in collagen binding. A. Alignment of LAIR-1 sequences from several species. Key residues for human LAIR-1 interaction with collagen III are in red and residues showing NMR chemical shift perturbation in bold. The most conserved residues in LAIR-1 orthologs are labelled with red triangles. Cartoon (B) and surface (C) representations of LAIR-1 Ig-like domain (PDB code 3KGR). Key residues for human LAIR-1 interaction with collagen III are in red and labelled in black and residues showing NMR chemical shift perturbation in white.

Figure 13 :

 13 Figure 13: LAIR-1 essential collagen binding residues are conserved in the GPVI. A. Sequence alignment of GPVI from several species. Conserved residues R38 and E40 homologs to LAIR-1 collagen-interacting residues (R59, E61) are shown in red (Taken from Brondijk et al., 2010b). B. Cartoon representation of LAIR-1, in green, and GPVI, in blue (PDB codes 3KGR and 5OU9, respectively) structure alignment. The homologous collagen-interacting arginine and glutamate residues are labelled with black arrows.

Figure 14 :

 14 Figure 14: OSCAR (A) and GPVI (B) crystal structures in complex with a collagen peptide (PDB codes 5CJB and 5OU9, respectively).Receptors Ig-like domains are represented in blue and the collagen peptides in purple. Side chains of the key residues for the interaction are shown and labelled in black.

Figure 15 :

 15 Figure 15: LAIR-1/RIFIN interaction and immune response. A. schematic representation of LAIR-1 containing antibody. The cartoon representation of the crystal structure of the Fab fragment (PDB code 5NST) is shown in the insert. B. LAIR-1 Iglike domain insertion in immunoglobulins gives them interaction properties with RIFIN, triggering the Ig-mediated cytotoxic activities. C. When exposed on immune cells surface, membrane LAIR-1 interaction with RIFIN induces intracellular tolerogenic signals that inhibit T, B and NK-cells cytotoxicity (Adapted from Lanzavecchia 2018).

  These studies of Son and colleagues put C1q-mediated immune regulation in a whole new perspective. Indeed, thanks to its multivalence and different functional regions, C1q could likely simultaneously interact with LAIR-1/2, RAGE and CD33, thus regulating the immune response via a complex local aggregation of pro and anti-inflammatory signals(Figures 8 and 16).

Figure 16 :

 16 Figure 16: Modulation of immune signals by C1q interaction with the Ig-like receptors RAGE, LAIR-1 and CD33. Schematic representation of RAGE activation by HMGB1 (A) and C1q-mediated immune modulation through RAGE and LAIR-1 in normal (B) and inflammatory (C) conditions. D. Schematic representation of C1q crosslinking of LAIR-1 and CD33. The intracellular phosphorylations are represented with red asterisks.

Figure 17 :

 17 Figure 17: The four structural classes of collagen C-terminal trimerization domains. A. crystal structure of collagen IV nc1 domain (PDB code 1M3D). B. crystal structure of the collagen X C1q-type domain (PDB code 1GR3). C. crystal structure of the collagen XVIII multiplexin trimerization domain (PDB code 3HSH). D. Crystal structure of the collagen III C-propeptide (PDB code 4AK3). Each crystal structure is shown as a cartoon representation with one chain subunit colored in green andthe two others in grey. The four crystal structures are oriented in the same direction with the N-terminal extremities on the left of the structure.

  IX collagen is composed of three different α-chains and is the only heterotrimeric FACIT member. It is demonstrated that the collagen IX nc2 domain, beyond playing a role in collagen chains trimerization, is the leading domain for the selective registering of the three different α-chains[START_REF] Boudko | The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization[END_REF][START_REF] Boudko | Structural insight for chain selection and stagger control in collagen[END_REF].Indeed, the nc2 domain consists in three different alpha helices that selectively assemble in a heterotrimeric manner, thus registering the upstream and downstream neighboring collagen sequences. Structural study of the nc2 domain of collagen IX revealed that α2, α1 and α3 chains correspond to the leading, middle and trailing chains, respectively[START_REF] Boudko | Structural insight for chain selection and stagger control in collagen[END_REF].

Figure 18 :

 18 Figure 18: Domain organization of collagen IX. The four non-collagenous domains (NC) are labeled in black. Three different collagen chains are colored in magenta, cyan and orange. (Taken from Boudko and Bächinger 2016a)

Figure 19 :

 19 Figure 19: Location of the sites of limited cleavage by collagenase and pepsin in C1q A, B and C chains. (Taken from[START_REF] Thielens | Further characterization of the interaction between the C1q subcomponent of human C1 and the transmembrane envelope glycoprotein gp41 of HIV-1[END_REF].

  et al. 2003) was aligned with the crystal structure of the collagen IX nc2 domain with a collagen I fragment on their C-terminal Gly-Pro motifs (PDB code 5CTI, Boudko and Bächinger 2016a).

Figure 20 :II. Article 2 :1

 202 Figure 20: Choice of the CLR_nc2 chains combination. Side (A) and top (B) views of the alignment of the crystal structure of the collagen IX nc2 domain with an N-terminal collagen I guest sequence (PDB code 5CTI) with the C1q model built from GR crystal structure (PDB code 1PK6). The nc2 domain is represented in cartoon and superimposed to the GR surface representation. The collagen chains are shown with a cartoon representation. C1q CLR and GR A, B and C chains are colored in cyan, magenta and orange, respectively. The nc2 α1, α2 and α3 chains are colored in magenta, cyan and orange, respectively.

Figure 21 :

 21 Figure 21: Schematic representation of the CLR_nc2 (left), C1qCLR_nc2 (middle) and C1qstem_nc2 (right). The nc2 domains are represented as red, orange and purple cylinders, the additional GPP triplets inserted into the CLR_nc2 are colored in black and the C1q collagen chains are shown in red, blue and green.

Figure 22 :

 22 Figure 22: Molecular and structural determinants of collagen recognition by the glycoprotein VI. Cartoon representation of the crystal structure of the glycoprotein VI (GPVI) (in green) in complex with a GPO collagen peptide (in yellow) (PDB code 5OU8). The two central arginine 38 (R38) and glutamate 40 (E40) as well as the tryptophan 76 (W76) are labeled in red. The inserts show a close view of the GPVI-collagen interface. The side chains of the residues involved in the interaction are shown with a stick representation. The hydrogen bonds between the GPVI and collagen amino acids are represented with black dashed lines.By cross-checking these literature-based and structural data with our own experiments, we proposed a model for LAIR-1 Ig-like domain interaction with the C1q collagen-like region. The following manuscript (in preparation) reports our work on the detailed molecular deciphering of C1q interaction with LAIR-1 Ig-like domain.

  Figure 1

Figure 23 :

 23 Figure 23: Parallel LAIR-1 subunits association observed in the crystal packing of LAIR-1 Ig-like domain structure. A.Side view (upper part) and top view (lower part) of A and C subunits in the crystal packing of one LAIR-1 Ig-like domain structure (PDB code: 3RP1). The two essential LAIR-1 residues (R59 and E61) for the collagen interaction and the remarkable serine 92 (S92) residue are labelled in red. The C-terminal and N-terminal extremities are labelled in black. B. Model illustrating that the collagen binding-sites in these two subunits (in blue and green) are perfectly aligned, and thus possibly offering an extended binding site for a GPO collagen peptide (in brown, extracted from PDB 5OU8). The side chains of the hydroxyproline residues at the interaction surface are shown as sticks.

Figure 24 :

 24 Figure 24: Oligomeric states of LAIR-1 WT, S92C and S92K. A. SEC-MALLS analysis of LAIR-1 WT, S92C and S92K injected on Superdex 75-10/300 column at concentrations of 5, 4.5 and 4 mg/ml respectively. B. SDS-PAGE (14% acrylamide) analysis of LAIR-1 S92C covalent dimer under non-reducing (NR) and reducing (R) conditions.

Figure 25 :

 25 Figure 25: Structural characterization of LAIR-1 S92C covalent dimer. A. Crystal structure of LAIR-1 covalent dimer. B. SAXS analysis of LAIR-1 S92C covalent dimer. Experimental data (black dots) are compared to theoretical scattering profiles of the model of parallel association (red curve) or our crystal structure (blue curve).

Figure 26 :

 26 Figure 26: SPR analysis of LAIR-1 S92C and S92K interaction with C1q. A. LAIR-1 S92C was injected at increasing concentrations (1-32 µM) over immobilized sC1q (14,000 RU). Fits obtained by a global fitting of the data to a steady-state model are shown in the top right corner. B. LAIR-1 WT (green curve) and S92K (red curve) were injected at a concentration of 10 µM on immobilized sC1q (14,000 RU).

  Figure 28: Expression tests of LAIR-2 and CD33 Ig-like domains in E. coli BL21(DE3). SDS-PAGE analysis (14% acrylamide) of the different fractions of the expression tests of Ig-like domains of LAIR-2 (A) and CD33 (B). For each receptorIg-like domain, the transformed cells were analyzed before (NI) and after (I) induction. The analyses of the supernatant and pellet fractions of the cells after lysis are also shown. The induction procedure (i.e. IPTG induction or autoinduction medium) is indicated at the bottom of the corresponding lanes. The theoretical molecular weight of each fragment is indicated in black at the top of each gel. The expected Mw of the Ig-like domains are indicated with its schematic representation on the right side of the gel. The molecular weights of the ladder bands (in kDa) are indicated in black on the left side of each gel.

Figure 29 :

 29 Figure 29: Expression of the extracellular domains of LAIR-1, LAIR-2 and CD33 in HEK293F cells. Western blot analysis of the cell culture supernatants (1 mL of supernatant precipitated with trichloroacetic acid) of HEK293F transfected cells using an anti-polyhistidine antibody coupled with horseradish peroxidase. The schematic representation of the three constructs corresponding to the protein bands are shown on the right side of the membrane. The molecular weights of the ladder bands (in kDa) are indicated in black on the left side of the membrane.

Figure 30 :

 30 Figure 30: Schematic representation of LAIR-1 WT and S92C dimerization on cell surface. A. LAIR-1 WT dimerization of the cell surface requires the binding of a ligand. B. LAIR-1 S92C dimer on the cell surface is blocked by the formation of the disulfide bond between the two monomers. The Ig-like domains are represented in green, the transmembrane domain in yellow and the intracellular ITIMs are represented with red asterisks. The disulfide bond between the two Ig-like domains of LAIR-1 S92C is shown in black.

  List of the oligonucleotides used for the design of the pET28a vectors. All primers were purchased from Eurogentec.

Figure 31 :

 31 Figure 31: Design of pET28a(+) vectors containing the coding sequence of LAIR-1, LAIR-2 and CD33 Ig-like domains. A. Schematic representation of the cloning procedure. B. Amino acids sequences of the LAIR-1, LAIR-2 and CD33 constructs cloned in pET28a(+) for bacterial expression. The additional leucine and glutamate residues due to the presence of the XhoI restriction site are indicated in black. The polyhistidine tags (Histag) of pET28a(+) vectors are shown in orange.

  of the oligonucleotides used for the design of the pcDNA3.1(+) vectors. All primers were purchased from Eurogentec.

Figure 32 :

 32 Figure 32: Design of pcDNA3.1(+) vectors containing the coding sequence of LAIR-1, LAIR-2 and CD33 extracellular regions. A. Schematic representation of the cloning procedure. B. Amino acid sequences of LAIR-1 (green), LAIR-2 (blue) and CD33 (red) Ig-like domains. The native signal peptides and the polyhistidine tags (Histag) are shown in yellow and orange, respectively.

Figure 33 :

 33 Figure 33: Selection of HEK293F cells after transfection with pcDNA3.1(+) vectors containing the coding sequences of LAIR-1, LAIR-2 or CD33 extracellular region.

Table 1 : List of C1q targets (non-exhaustive

 1

). The functional region of C1q involved in the target interaction and the associated references are indicated.

Table 2 : List of C1q receptors and soluble effectors (non-exhaustive). The functional region of C1q involved in the receptor interaction and the associated references are indicated. Soluble effectors and membrane receptors C1q interacting region References

 2

	C1r2s2	CLR	(Siegel and Schumaker 1983)
	gC1qR (P33)	GR	(Ghebrehiwet et al. 1994)
	cC1qR (calreticulin)	CLR/GR	(Païdassi et al. 2011)
	CR1 (CD35)	CLR/GR	(Klickstein et al. 1997; Jacquet et al. 2018)
	LRP1 (CD91)	CLR/GR	(Fouët et al. 2020b)
	SR-F1	CLR	(Wicker-Planquart et al. 2020)
	Integrin α2β1	CLR	(Zutter and Edelson 2007)
	DC-SIGN	GR	(Pednekar et al. 2016)
	LAIR-1 (CD305)	CLR	(Son et al. 2012)
	LAIR-2 (CD306)	CLR	(Son et al. 2012)
	RAGE	GR	(Ma et al. 2012; Son et al.

Table 4 : Conditions of the PCR experiments.

 4

	Phusion Master Mix 2X		25 µL
	Forward primer (final concentration)	0.5 µM (2.5 µL at 10 µM)
	Reverse primer (final concentration)	0.5 µM (2.5 µL at 10 µM)
	Sterile H2O	18 µL (add to 50 µL)
		PCR CYCLES
	Steps	Temperature	Time	Number of cycles
	Initial denaturation	98 °C	60 s	1
	Denaturation	98 °C	30 s
	Annealing	55 °C	30 s	10
	Extension	72 °C	30 s
	Denaturation	98 °C	30 s
	Annealing	60 °C	30 s	20
	Extension	72 °C	30 s
	Final extension	72 °C	5 min	1
	Hold	15°C	∞

Table 5 : Conditions of the ligation experiments.

 5

Table 7 : Composition of the auto induction medium.

 7

  2 M, PEG 6000 15 %, pH 6).

	PEG 6000	15% 17% 19% 20% 21% 23%
	Glycerol	8%
	0.1 M MES,
	pH6
	0.2 M NaCl
	PEG 6000	15% 17% 19% 20% 21% 23%
	Glycerol	8%
	0.1 M MES,
	pH6.5
	0.2 M NaCl
	PEG 6000	15% 17% 19% 20% 21% 23%
	Glycerol	8%
	0.1 M MES,
	pH6
	0.2 M LiCl2
	PEG 6000	15% 17% 19% 20% 21% 23%
	Glycerol	8%
	0.1 M MES,
	pH6.5
	0.2 M LiCl2

Table 9 : Plan of the crystallization plate used for the refinement of the LAIR-1 S92C crystallization conditions.

 9

  Outer resolution shell values are indicated in parentheses.

	Outer shell (Å)	2.70 -2.746
	Rmerge	0.033 (0.417)
	Total observations	44680 (2343)
	Unique observations	14887 (748)
	Completeness (%)	98.0 (99.6)
	Multiplicity	3.0 (3.1)
	CC (1/2)	0.999 (0.882)
	Structure refinement statistics
	Resolution limits	2.7-72
	Rwork	0.193
	Rfree	0.257
	RMSD bond lengths (Å)	0.009
	RMSD bond angles (°)	1.705
	Space group	H3
	Cell parameters	a=b=143.133Å c=72.404 Å, α=β=90° γ=120°
	Wavelength (Å)	0.96770
	Resolution limits (Å)	2.7-47

Table 10 : LAIR-1 S92C diffraction data and structure refinement statistics.

 10

DISCUSSION & PERSPECTIVES

IV. Aim of the project

The defense collagen C1q plays a critical role in various immune mechanisms and their control.

The impressive versatility of C1q functions relies on its wide range of ligands but above all, on a constantly increasing number of newly discovered C1q receptors triggering different signals upon C1q recognition. Thanks to its multivalence, C1q is likely able to interact with several copies of the same or different receptors and therefore modulates the immune response depending on the receptors engaged. However, how C1q is implicated in these processes remains unclear and awaits further investigations in order to understand the C1q-mediated immune modulation and tolerance mechanisms.

Recently, the first elements clarifying this question were provided by Son and colleagues who reported that C1q-mediated immune modulation was dependent on its interaction with immune receptors from the immunoglobulin-like family: RAGE, CD33 and LAIR-1/2 [START_REF] Son | C1q limits dendritic cell differentiation and activation by engaging LAIR-1[END_REF][START_REF] Son | C1q and HMGB1 reciprocally regulate human macrophage polarization[END_REF][START_REF] Son | Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression[END_REF] (Figure 8). These first studies suggest that LAIR-1 and LAIR-2 interact with the C1q CLR while RAGE and CD33 interaction with C1q engages its GR. These different binding regions on the C1q molecule enable the bridging of the receptors through C1q to congregate pro-and anti-inflammatory signals, thus modulating the immune response (Figure 16). However, the fine details dictating these interactions are fundamental for the understanding of C1q-mediated immune modulation and remain to be identified. In this line, my PhD project initially aimed at identifying the molecular and structural determinants underlying the C1q interaction with its recently identified Ig-like receptors RAGE, CD33 and LAIR-1/2.

We first focused on the analysis of C1q interaction with the Ig-like receptor LAIR-1. As a collagen receptor, LAIR-1 was previously described to interact with the collagen-like regions of C1q. In order to investigate the interaction properties of LAIR-1 with C1q CLR, we took up the challenge of C1q collagen chains registering and developed the first reported recombinant production of C1q collagen-like regions (Article 1).

Then, we combined molecular dissection and mutagenesis strategies to identify the LAIR-1 binding region on C1q and vice versa. This approach allowed us to propose a model for C1q interaction with the inhibitory receptor LAIR-1 (Article 2).

We attempted to investigate the dimerization effect of LAIR-1 on its interaction with C1q. For that part of the project, we also developed two LAIR-1 variants exclusively monomeric or Supplementary 1 Supplementary 2 Supplementary 3 Supplementary 4

II. Appendix 2: Material and methods for unpublished data: LAIR-1 dimerization effects on C1q interaction 1 Production of LAIR-1 S92K variant and S92C covalent dimer

The pET28a vectors containing the coding sequences of LAIR-1 Ig-like domain S92K and S92C variants were generated by site directed mutagenesis of the pET28a_LAIR-1_22-122His vector (Appendix 1). The mutagenesis was performed using the Quikchange II XL kit (Agilent, product number 200522) and the oligonucleotides used are listed in Table 8 The two LAIR-1 variants were expressed and purified as described for the wild-type. In the particular case of the S92C variant, it was purified in a monomeric form and concentrated up to 5 mg/mL. The concentrated sample was kept at 4°C for at least 1 week in order to trigger the spontaneous formation of the disulfide bond formation between two LAIR-1 S92C monomers.

Then, the sample was loaded back on a Superdex 75 column in 25 mM Tris, 150 mM NaCl, pH 8.2 in order to separate the dimers from the remaining monomers (generally, the dimeric portion represents 60% of the total proteins). The dimeric fraction was collected and concentrated up to 2 mg/mL. BIBLIOGRAPHY