cysteine in FVIII-deficient mice reduces immune responses to therapeutic FVIII (in preparation)

Hemophilia A is a rare X-linked hemorrhagic disease consecutive to the lack of functional pro-coagulant factor VIII (FVIII). In patients with the severe form of hemophilia A, uncontrolled hemorrhages increase the morbidity and affect the quality of life. To prevent or treat bleeding episodes, therapeutic FVIII is administered intravenously. However, an anti-FVIII immune response develops in 5 to 30% of the patients. The anti-FVIII immune response is characterized by the development of anti-FVIII IgG antibodies that inhibit FVIII activity. These antibodies, referred to as "FVIII inhibitors" represent the major complication in the treatment of hemophilia A patients.

Several risk factors have been suggested or proposed to predispose to the development of FVIII inhibitors. Amongst them, inspired by the "danger" theory, the presence of inflammatory mediators released upon bleeding episodes is thought to adjuvant the anti-FVIII immune response. Bleeding episodes trigger several pro and anti-inflammatory mechanisms. Thus, damage to the endothelium triggers the recruitment and activation of effector cells from the innate and adaptive compartments of the immune system.

During my PhD, I investigated the role of bleedings in the development of the anti-FVIII immune response. Thus, I discovered an association between a polymorphism in the promoter region of a gene that is induced in response to bleedings, and the presence of FVIII inhibitors in a cohort of patients with severe hemophilia A. I followed the development of the immune response to therapeutic FVIII in a mouse model that mimics the localized bleedings found in patients with severe hemophilia A. Ultimately, I characterized the effects of reactive oxygen species (ROS) that are potentially released at the bleeding site in view of the structure, function and immunogenicity of the FVIII molecule.

RÉSUMÉ

L"hémophilie A est une maladie hémorragique rare liée au chromosome X qui se traduit par un déficit en facteur VIII (FVIII) fonctionnel. Chez les patients atteints de la forme sévère de l"hémophilie A, l"apparition d"hémorragies incontrôlées augmente la morbidité et affecte leur qualité de vie. Le traitement de choix permettant de prévenir ou de traiter les saignements consiste en l"injection intraveineuse de FVIII thérapeutique. Cependant, chez 5 à 30% des patients, une réponse immunitaire dirigée contre le FVIII se développe. La réponse anti-FVIII est caractérisée par l"apparition d"anticorps qui inhibent l"activité pro-coagulante du FVIII. Ainsi l"apparition de ces anticorps, appelés « inhibiteurs » représente-t-elle la complication majeure du traitement des patients hémophiles A.

Plusieurs facteurs de risque ont été suggérés ou proposés comme affectant le développement de la réponse anti-FVIII. Parmi eux, supporté par la théorie du « danger », la présence de médiateurs pro-inflammatoires générés par les épisodes de saignement est considérée comme adjuvant dans la réponse immunitaire anti-FVIII. Cependant, les saignements induisent de nombreux mécanismes pro mais aussi anti-inflammatoires. Ainsi, l"activation de l"endothélium vasculaire induit-elle le recrutement et l"activation des cellules effectrices appartenant aux compartiments innés et adaptatifs du système immunitaire.

Au cours de ma thèse, je me suis concentré sur le rôle des saignements dans le développement de la réponse immunitaire dirigée contre le FVIII. Ainsi, ai-je découvert une association entre la présence d"un polymorphisme dans un gène induit par les saignements et la présence d"inhibiteurs du FVIII dans une cohorte de patients hémophiles A sévères. J"ai également étudié le développement de la réponse immunitaire dirigée contre le FVIII thérapeutique dans un modèle murin qui mime les saignements locaux observés chez les patients hémophiles A sévères. Finalement, j"ai caractérisé l"effet des formes réactives de l"oxygène (FRO), qui sont générées au niveau du site de saignement, sur la structure, la fonction et l"immunogénicité du FVIII.

Les résultats obtenus au cours de ma thèse démontrent que les saignements ne sont pas associés à un risque plus élevé de développer des inhibiteurs dans le modèle murin largement utilisé d"hémophilie A sévère, contrairement à ce qui est communément admis. De plus, j"ai démontré une association entre un polymorphisme présent dans le promoteur du gène HMOX1 et la survenue d"inhibiteurs dans une cohorte de patients atteints d"hémophilie A sévère. En parallèle, je décris pour la première fois une altération du statut oxydatif chez les souris déficientes en FVIII et je démontre que le contrôle du statut oxydatif in vivo permet de moduler la réponse immunitaire anti-FVIII. Au contraire, l"exposition du FVIII aux FRO augmente son immunogénicité.

De fait, mes résultats suggèrent que, bien que les molécules pro-inflammatoires libérées suite aux saignements puissent affecter positivement la réponse immunitaire dirigée contre le FVIII, de puissants médiateurs anti-inflammatoires sont générés in vivo et amenuisent l"effet potentiellement adjuvant des saignements. Également, ces résultats soulignent la balance complexe entre les médiateurs pro-et anti-inflammatoires qui sont générés consécutivement aux saignements ainsi que leurs effets sur la réponse immunitaire dirigée contre le FVIII thérapeutique.

The results I obtained during my PhD demonstrate that bleedings are not associated with a higher risk for FVIII inhibitor development in the commonly used mouse model of severe FVIII deficiency. Additionally, I demonstrated an association between a polymorphism in the promoter of the HMOX1 gene and the presence of FVIII inhibitors. In parallel, I report for the first time an exacerbated oxidative status in FVIII-deficient mice as compared to control mice, and demonstrate that the control of the oxidative status in vivo reduces the anti-FVIII immune response. Conversely, the exposure of FVIII to ROS ex vivo increases the immunogenicity of the FVIII molecule.

Taken together, my results suggest that, although the pro-inflammatory molecules released upon bleeding may positively affect the anti-FVIII immune response, several strong antiinflammatory compounds are generated in vivo that dampen the potential adjuvant effect of bleedings. Ultimately, these results highlight the complex balance between the pro and antiinflammatory mediators generated upon bleeding and their effect on the anti-FVIII immune response.

TABLE OF CONTENTS

FIGURE AND TABLES LIST

ABBREVIATION LIST

. Generalities

Hemophilia A is the most frequent haemorrhagic disorder with an incidence of one newborn in ten thousands [START_REF] Lenting | The life cycle of coagulation factor VIII in view of its structure and function[END_REF]. This X-linked genetic disease follows a recessive transmission pattern and is characterized by a qualitative or quantitative defect in functional pro-coagulant factor VIII (FVIII) [START_REF] Hoyer | Hemophilia A[END_REF]. In the most severe forms of this pathology, abnormal levels of FVIII lead to spontaneous haemorrhages located mainly in joints and in soft tissues. Moreover, chronic bleedings in joints lead to the development of crippling arthropathies, decreasing the quality of life of the patients. In the most dramatic situation representing up to ten percent of the cases [START_REF] Klinge | Prevalence and outcome of intracranial haemorrhage in haemophiliacs--a survey of the paediatric group of the German Society of Thrombosis and Haemostasis (GTH)[END_REF][START_REF] Daniele | Effective management of intracranial haemorrhage with continuous infusion of highly purified von Willebrand factor/factor VIII complex concentrate in an adult with severe haemophilia A[END_REF], haemorrhages develop in the brain, engaging the vital prognosis of the patient.

I.1.1.1. Historical aspects

The first written report describing a congenital affection of blood coagulation is found in the Talmud, two centuries BC [START_REF] Rosner | Hemophilia in the Talmud and rabbinic writings[END_REF]. This text reports that circumcision should be avoided if the two previous sons passed away following their circumcision. Thirteen centuries later, the famous Arabic medical doctor Abu Al-Qasim described a haemorrhagic disease which is transmittable amongst generations. Later on, in 1803, Dr. John Conrad Otto described a haemorrhagic condition existing in some families. Comforting his hypothesis, he was able to trace back the origin of the disease affecting his patients to a woman living in Plymouth in 1720. Ten years later, John Hay described that the affected males may pass the genetic trait to their unaffected daughter (HAY, 1813). The term "Haemorrhaphilia" appears only in 1828 in the PhD manuscript by Friedrich Hopff, and is rapidly contracted to "Hemophilia" as an association of two greek words "haïma" (blood) and "philia" (affection).

The first treatments of hemophilia were reported by Patek and Taylor, two medical doctors from Harvard. They succeed to correct the coagulation defect by adding a substance derived from the plasma. They named this substance "antihemophilic globulin". However, the treatment of the affected patients with the plasma from healthy individuals was often inefficient, due to the very low concentration of the required coagulation factor. It is only in 1964 that Dr. Judith Pool discovered that the cryoprecipitation of normal plasma was generating a pellet enriched in coagulation factor [START_REF] Pool | High-Potency Antihaemophilic Factor Concentrate Prepared from Cryoglobulin Precipitate[END_REF].

I.1.1.2. Phenotypic aspects

The severity of hemophilia A is dependent on the residual activity of the endogenous circulating FVIII. Thus, hemophilia A is considered minor, when residual levels of FVIII are between 5 and 35 % of activity found in normal plasma, moderate with levels between 1 to 5% of normal and severe if FVIII levels are below 1% [START_REF] Lenting | The life cycle of coagulation factor VIII in view of its structure and function[END_REF]. The repartition of the three degree of severity is not homogenous. Indeed, half of the hemophilia A population is affected by the minor form of the disease, 10% display the moderate form and 40% are affected by the severe form of hemophilia A [START_REF] Antonarakis | Molecular etiology of factor VIII deficiency in hemophilia A[END_REF].

I.1.1.3. Genetic aspects

The FVIII encoding gene (F8 gene) was cloned between 1982 and 1984 by Gitschier and his team [START_REF] Gitschier | Characterization of the human factor VIII gene[END_REF]. The F8 gene is located at the distal part of the long arm of the X chromosome (Xq28). Its length is of 186 kbp, representing 0.1% of the total chromosome length. The coding part of the gene is spread amongst 26 exons. Once transcripted and spliced, the mature mRNA has a size of 9 kbp and represents 5% of the initial gene length. The 95% remaining sequences correspond to the intronic parts of the gene.

Despite the fact that they do not encode the FVIII protein, the intronic segments can carry some mutations responsible for FVIII deficiency. The phenotypic heterogeneity of hemophilia A reflects the wide variety of the genetic abnormalities that can affect the patients" F8 gene.

These numerous mutations are listed in the HAMSTeR (the Hemophilia A Mutation, Structures, Test and Resource website; www.factorviii-db.org). Of note, some rare hemophilia A cases are due to mutations which are not directly affecting the F8 gene, but are affecting proteins involved in FVIII production, intracellular trafficking or secretion [START_REF] Zhang | Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex[END_REF][START_REF] Oldenburg | New insight into the molecular basis of hemophilia A[END_REF]. In most hemophilia A cases (about 90% of the patients), the genetic abnormality consists in a single nucleotide mutation which is affecting the FVIII encoding gene [START_REF] Bhopale | Blood coagulation factor VIII: An overview[END_REF]. The term single nucleotide mutation engulf the missense mutations were a nucleotide substitution leads to a change in the incorporated amino acid, the non-sense mutations were a stop codon is integrated before the end of the normal gene, and the splicing mutations, when the addition or the removal of a splicing site leads to the generation of an abnormal mRNA, thus affecting the final amino-acid sequence of the FVIII protein. Small deletions or insertions and larges deletions (>50 bp) are found in 5 to 10% of hemophilia A patients. Most often, these mutations lead to a shift in the DNA reading frame, leading to a drastic modification of the protein and are generally associated with a severe form of hemophilia A [START_REF] Oldenburg | Small deletion/insertion mutations within poly-A runs of the factor VIII gene mitigate the severe haemophilia A phenotype[END_REF]. Duplication and inversions are rare, excepted for the intron 22 (Inv22) which is found inverted in 45 to 50% of patients with severe hemophilia A [START_REF] Lakich | Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A[END_REF]. Mutation Inv22 is classified as a "null" because it leads to the complete lack of the FVIII secretion. However, recent findings suggest that hemophilia A patients with Inv22 mutation produce the entire FVIII protein as two polypeptide chain [START_REF] Pandey | Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A[END_REF], although these findings remain controversial.

Moreover, in some patients with missense mutations, the FVIII molecule is secreted at a normal concentration, but is not functional. This explains why 5% of hemophilia A patients do not have any residual FVIII activity but display normal levels of circulating FVIII. These patients are referred to as "cross-reactive-material (CRM)-positive" patients [START_REF] Amano | The molecular basis for cross-reacting material-positive hemophilia A due to missense mutations within the A2-domain of factor VIII[END_REF]. Thus, the fact that hemophilia A patients have distinct genetic and phenotypic profiles makes hemophilia A a very heterogeneous disease. Moreover, the type of mutation affecting the FVIII-encoding gene lead to the production of several types of FVIII protein in different patients. From an immunological point of view, it means that during the ontogeny of the immune system, hemophilia A patients in whom FVIII is partially secreted, even though not functional, will participate in the elimination of FVIII-specific T cells. Moreover, these patients will develop regulatory T cells capable of suppressing FVIII-specific T cells in the periphery. Altogether, these mechanisms may prevent the development of the anti-FVIII immune response after infusion of therapeutic FVIII. Conversely, in patients in whom no FVIII is secreted, and particularly in CRM-negative patients, the FVIII-reactive T cells are not eliminated during T-cell ontogeny, thus increasing the risk for FVIII inhibitor development after birth/later in life.

I.1.1.4 Bleeding complications in Hemophilia A

The lack of functional pro-coagulant FVIII leads to de development of uncontrolled haemorrhages. These haemorrhages may occur at any place in the body. Thus, deep internal bleedings in the muscles lead to swelling of the affected member. Swelling may press on nerves and lead to recurring pain. Ultimately, repetition of painful episodes may result in a reluctance to use the affected member. When located in a joint, the presence of non-clotted blood, referred to as hemarthrosis, leads to the progressive destruction of the joint. It has been demonstrated that extravasation of blood within the synovial space leads to an increased concentration of erythrocyte-derived haemoglobin and heme along with the recruitment of neutrophils, monocytes, macrophages and lymphocytes. On the bleeding site, the production of pro-inflammatory cytokines and chemokines [START_REF] Lafeber | Physiopathology of haemophilic arthropathy[END_REF], combined with the deleterious pro-oxidative effect of heme-derived iron, leads to the progressive destruction of the joint. Indeed, the pro-oxidative properties of iron may directly degrade the cartilage by inducing chondrocyte apoptosis [START_REF] Roosendaal | Blood-induced joint damage: a human in vitro study[END_REF], resulting in an inhibition of the turnover of the cartilage extracellular matrix protein [START_REF] Jansen | Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: an in vitro study[END_REF]. Moreover, the proangiogenic property of heme leads to a sustained vascular neoplasia, provoking a synovial hypertrophy along with the loss of joint space [START_REF] Arnold | Hemophilic arthropathy. Current concepts of pathogenesis and management. The Journal of bone and joint surgery[END_REF]. Of note, the vicious circle of joint destruction by hemophilic arthropathy is sustain by the neovascularization of the affected joint; indeed, the increase in the number of small vessels has been proposed to increasing the risk of future bleeding events at the same location [START_REF] Lafeber | Physiopathology of haemophilic arthropathy[END_REF]. (Figure 1)

Figure 1. Physiopathology of hemophilic arthropathy. The presence of blood within the synovial space triggers several pro-inflammatory events. Thus, in the joint, the recruited immune cells release pro-inflammatory cytokines and chemokines. In combination with heme-derived iron, these pro-inflammatory mediators lead to synovial hypertrophy, chondrocyte apoptosis and progressive destruction of the joint.

I.1.2 The coagulation cascade

Any kind of alteration of the vascular endothelium integrity leads to the exposure of sub-endothelial structures/molecules such as collagen or tissue factor which, upon direct contact with blood, induce/activate primary and secondary haemostasis.

I.1.2.1 Primary haemostasis

Upon vascular damage, a localized vasoconstriction occurs as a response to a reflex neurogenic mechanism and to the secretion of endothelium-derived vasoconstrictors such as endothelin. This phenomenon reduces the blood flow upstream of the vascular breach, thus limiting the blood loss and facilitating the interaction of the mediators required for the initiation of haemostasis. Immediately following the vasoconstriction, primary haemostasis occurs. Platelets are recruited and get activated, allowing their adhesion to the exposed subendothelial matrix. Platelet activation results in dramatic changes in their shape, along with the release of the content of secretory granules. The platelet-derived mediators recruit other platelets, causing more platelet to adhere to the sub-endothelial matrix and to aggregate to each other at the vascular breach. Platelet aggregation ultimately results in the formation of a primary haemostatic plug, referred to as the "red thrombus" (Figure 2).

I.1.2.2 Secondary haemostasis

The goal of secondary haemostasis is to form a stable plug when the bleeding is severe.

During secondary haemostasis, also known as the coagulation cascade, the activated endothelium in association with activated platelets will allow the initiation of a succession of reactions leading to the generation of an insoluble fibrin clot, referred to as the "white clot".

Two pathways have been identified as being responsible for the initiation of the coagulation cascade, the extrinsic and the intrinsic pathways.

I.1.2.2.1 The extrinsic pathway

Upon exposure of the sub-endothelial matrix proteins and ensuing platelet aggregation, a membrane-bound pro-coagulant factor called "tissue factor" (TF) is expressed, which complexes and activates coagulation factor VII. The activated form of factor VII (FVIIa) in turn activates factor X (FXa), which associates with the activated factor Leiden (also known as factor V, FVa) to form a complex called the pro-thrombinase complex. The pro-thrombinase complex activates pro-thrombin (FII) into thrombin (FIIa). Eventually, the thrombin generated allows the polymerisation of fibrin monomers and activates factor XIII.

To achieve the white thrombus, the fibrin polymers are stabilised by the activated factor XIII giving rise to an insoluble fibrin clot, that act as a "net", helping the platelets to stabilize at the site of bleeding.

I.1.2.2.2 The intrinsic pathway

The intrinsic pathway, also known as the "contact factor pathway" initiates upon binding of factor Hageman (factor XII) to the negatively charged surface provided by the activated sub-endothelium. This binding results in the auto-activation of FXII to FXIIa. FXIIa induces the conversion of pre-kallikrein to kallikrein and factor XI to FXIa. FXIa activates in turn FIX to FIXa [START_REF] Scott | Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function[END_REF]. FIXa then associates with activated FVIII (FVIIIa) and with platelet-derived phospholipids (PL). In the presence of calcium, this complex, referred to as the "tenase" complex, allows the conversion of FX to FXa that sustains the downstream generation of thrombin initiated by the extrinsic pathway. in turn activates factor X (FXa), which associates with activated factor V, (FVa) to form a complex called the pro-thrombinase complex. The pro-thrombinase complex activates prothrombin (FII) into thrombin (FIIa). Eventually, the thrombin generated allows the polymerisation of fibrin monomers and activates factor XIII. Then, fibrin polymers are stabilised by the activated factor XIII giving rise to a stable fibrin clot, that act as a "net", helping the platelets to stabilize at the site of bleeding. The intrinsic pathway, initiates upon binding of factor XII to the negatively charged surface provided by the activated sub-endothelium. This binding results in the auto-activation of FXII to FXIIa. FXIIa induces the conversion of pre-kallikrein to kallikrein and factor XI to FXIa. FXIa in turn activates FIX to FIXa. FIXa then associates with activated FVIII (FVIIIa) and with platelet-derived phospholipids (PL). In the presence of calcium, this complex, called "tenase", allows the conversion of FX to FXa that sustains the downstream generation of thrombin initiated by the extrinsic pathway.

I.2.2.3 Pro-coagulant factor VIII I.2.2.3.1 Synthesis, structure and post-traductional modifications

For years, the type of cells that are producing FVIII has been under debate. Initially, the secretion of FVIII was thought to be mainly mediated by hepatocytes [START_REF] Ingerslev | Synthesis of factor VIII in human hepatocytes in culture[END_REF] and later on by liver sinusoidal endothelial cells [START_REF] Hellman | Secretion of coagulant factor VIII activity and antigen by in vitro cultivated rat liver sinusoidal endothelial cells[END_REF]. However, recent advances strongly suggest that, while sinusoidal endothelial cells from the liver presumably account for an important part of FVIII synthesis, they might not be the exclusive source for its production [START_REF] Jacquemin | FVIII production by human lung microvascular endothelial cells[END_REF][START_REF] Everett | Murine coagulation factor VIII is synthesized in endothelial cells[END_REF][START_REF] Fahs | A conditional knockout mouse model reveals endothelial cells as the predominant and possibly exclusive source of plasma factor VIII[END_REF]. The primary structure of the molecule is a multi-domain protein containing 2351 amino acids translated in the endoplasmic reticulum. After a first maturation step, a 19 amino-acid signal peptide is cleaved, thus reducing the size of the protein to 2332 amino acids. In the endoplasmic reticulum, N-glycosylations are added to the FVIII molecule before been translocated to the Golgi apparatus. In the Golgi apparatus, complex carbohydrate modifications on N-linked sites, addition of carbohydrate to serine and threonine residues and sulfation of several tyrosine residues in the acidic regions of FVIII, occur [START_REF] Kaufman | Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells[END_REF]. Eventually, a proteolysis step at residues Arg1313 and Arg1648 leads to the secretion of a protein with an apparent molecular weight of 280 kDa [START_REF] Vehar | Structure of human factor VIII[END_REF][START_REF] Lenting | The life cycle of coagulation factor VIII in view of its structure and function[END_REF]. Within the circulation, the mature form of FVIII corresponds to an heterodimer, composed of a heavy chain comprising the A1a1, A2a2 and B domains, and a light chain comprising the a3A3, C1 and C2 domains [START_REF] Wood | Expression of active human factor VIII from recombinant DNA clones[END_REF], linked by a divalent ion (Figure 4). The a3 acidic region, in association with the C2 domain, allows the binding of the FVIII protein to its chaperon molecule, the von Willebrand factor (VWF). The binding of VWF to the FVIII molecule prevents its premature degradation and allows the addressing of FVIII to the bleeding site [START_REF] Kaufman | Biosynthesis, assembly and secretion of coagulation factor VIII. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis[END_REF].

I.2.2.3.2 Activation of FVIII

FVIII is addressed to the bleeding site by its chaperon molecule VWF. Thrombin then activates the FVIII molecule through a proteolytic cleavage of the heavy chain (residues Arg372 and Arg740) and the light chain (Arg1689) (Figure 5). The cleavage of the light chain leads to the dissociation of the FVIII molecule from VWF. Once released from VWF, the FVIII molecules binds to phospholipids and a structural change in FVIII orientation occurs, allowing its interaction with FIXa and FX. Binding to FIXa involves a sequence from the A2 domain, spanning Ser558 to Gln565, and a part of the A3 domain, spanning Glu1811 to Lys1818 [START_REF] Lenting | The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX[END_REF][START_REF] Mertens | Factor VIII-factor IX interactions: molecular sites involved in enzyme-cofactor complex assembly[END_REF]. The activated form of FVIII (FVIIIa) is able to exert its co-factor activity towards FIXa within the tenase complex that is composed of FVIIIa, FIXa and FX. In association with the phospholipids exposed at the surface of the activated platelets or by the damaged endothelium, the tenase complex activates FX. The presence of FVIIIa in the tenase complex enhances FX activation by 10 5 folds [START_REF] Van Dieijen | The role of phospholipid and factor VIIIa in the activation of bovine factor X[END_REF]. Later on in the coagulation cascade, FXa participates in the formation of the prothrombinase complex, involved in the generation of insoluble fibrin.

I.2.2.3.3 Life cycle of FVIII, catabolic receptors, in vivo distribution, site of elimination

Once released in the circulation, FVIII rapidly associates with VWF [START_REF] Vlot | The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor[END_REF][START_REF] Dimitrov | Thermodynamic analysis of the interaction of factor VIII with von Willebrand factor[END_REF]. The VWF-FVIII interaction increases FVIII half-life from about few minutes, as seen in patients with von Willebrand disease who lack FVIII-binding VWF, to 10-12 hours. VWF stabilizes the heterodimeric structure of FVIII, protects FVIII from proteolytic degradation, avoids binding to phospholipids and association to FIXa and prevents the cellular uptake of FVIII [START_REF] Lenting | Clearance mechanisms of von Willebrand factor and factor VIII[END_REF]. After having played its role within the tenase complex, FVIII is inactivated through two distinct mechanisms: proteolytic degradation or spontaneous dissociation of the A2 domain. The proteolytic degradation of activated FVIII is mediated by activated FIX, activated FX and activated protein C (APC) and involves a cleavage in the acidic region a1 of the heavy chain at position 336 [START_REF] Koedam | Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor[END_REF][START_REF] Rick | Factor IXa and von Willebrand factor modify the inactivation of factor VIII by activated protein C[END_REF].

This proteolytic cleavage increases the dissociation rate of the A2 domain, thus impairing FVIII ability to bind and activate FX [START_REF] Regan | Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase[END_REF]. APC also cleaves the A2 domain at position 562 thus disrupting the binding site of FVIII for FIXa [START_REF] Fay | Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity[END_REF]. The spontaneous dissociation of FVIII is believe to be the consequence of the low affinity of the A2 domain for the metal ion-linked A1/A3-C1-C2 dimer [START_REF] Persson | Kinetics of the interaction between the human factor VIIIa subunits: effects of pH, ionic strength, Ca2+ concentration, heparin, and activated protein C-catalyzed proteolysis[END_REF].

Clearance studies have revealed that after injection to hemophilia A patients, the removal of FVIII from the circulation follows a biphasic model characterized by a fast phase and a slow phase [START_REF] Saenko | Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism[END_REF]. Several catabolic receptors have been identified that mediate FVIII clearance.

I.2.2.3.4 Catabolic receptors for FVIII

The catabolism of FVIII implicates its uptake by specific receptors expressed on immune or non-immune cells. One of the first receptor identified as a catabolic receptor for FVIII uptake was the low density lipoprotein receptor-related protein (LRP, CD91) [START_REF] Saenko | Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism[END_REF]. LRP is a major endocytic receptor which is expressed in the liver but is also present in the placenta, the brain and the lungs [START_REF] Strickland | LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism[END_REF]. In vitro and in vivo studies have confirmed that LRP binds FVIII through the terminal part of the C2 domain in the light chain [START_REF] Lenting | The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein[END_REF] and the A2 domain of the heavy chain [START_REF] Saenko | Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism[END_REF] and leads to FVIII endocytosis and degradation. These observations have been confirmed by the use of a specific LRP inhibitor, the receptor-associated protein (RAP). Thus, the treatment of cells expressing LRP with RAP inhibited the uptake and the subsequent degradation of FVIII in vitro. Subsequently, the intravenous administration of RAP into mice completely inhibited the fast phase of FVIII clearance [START_REF] Saenko | Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism[END_REF].

Few years later, the membrane-bound heparan sulphate proteoglycans (HSPG) receptor was found to participate in the LRP-mediated FVIII uptake. The uptake of FVIII by HSPG was proposed to be mediated through the heparin-binding domain of FVIII located in the A2 domain. The identification of HSPG as a catabolic receptor for FVIII led to the description of two distinct pathway for FVIII elimination: an LRP-dependent pathway and an LRPindependent pathway [START_REF] Sarafanov | Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein[END_REF]. However, the fact that blockade of both LRP and HSPG receptors does not completely prevent FVIII endocytosis in vitro suggests the implication of other receptors [START_REF] Dasgupta | Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation[END_REF]. In this respect, the mannose-ending sugars present on FVIII have been implicated in FVIII endocytosis through the "macrophage mannose receptor" or CD206 [START_REF] Dasgupta | A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes[END_REF]. Other sugar residues, particularly within the B domain of FVIII have been found to mediate FVIII uptake through the asialoglycoprotein receptor, an endocytic receptor for lectins, following exogenous removal of capping sialic acids using neuraminidase [START_REF] Bovenschen | The B domain of coagulation factor VIII interacts with the asialoglycoprotein receptor[END_REF]. Recently, the sialic acid capping moiety on N-linked carbohydrate has been found to mediate FVIII endocytosis through the sialic-acid binding immunoglobulin-like lectin 5 (Siglec-5) [START_REF] Pegon | Factor VIII and von Willebrand factor are ligands for the carbohydratereceptor Siglec-5[END_REF].

To date, the nature of the receptors involved in FVIII uptake is still under investigation. We can speculate that structural changes in FVIII may lead to the generation of non-native molecular patterns which could participate in FVIII endocytosis through other receptors. In particular, as discussed later, one of our hypothesis is that oxidation of FVIII at the site of bleeding, where reactive oxygen species are released by the activated platelets and neutrophils may alter FVIII structure and immunogenicity.

I.2.2.3.5 In vivo distribution of FVIII

Following its infusion in the patient, FVIII circulate within the bloodstream. In vivo experiment in FVIII-deficient mice have demonstrated that the infused FVIII rapidly concentrates in the spleen and in the liver [START_REF] Navarrete | Splenic marginal zone antigenpresenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A[END_REF]. In the spleen, FVIII colocalizes with the metallophilic macrophages in the marginal zone.

I.1.3 Prevention or treatment of bleeding episodes in patients with hemophilia A

I.1.3.1 Factor VIII products

Two different kinds of products are now available on the market. The first type of product that was used is called plasma-derived FVIII (pdFVIII). This product is obtained by purifying FVIII from the plasma of thousands of healthy donors. In a recent analysis, plasmaderived products were found to contain at least 124 other protein, among which some albumin, traces of fibronectin and VWF, which are co-purified during the manufacturing process [START_REF] Basilico | Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach[END_REF]. The second type of product is called "recombinant" FVIII (rFVIII). Recombinant FVIII products are synthetized in vitro in stably transfected cell lines.

Depending on the manufacturer, the rFVIII is produced in Chinese Hamster Ovary cells (CHO) or in Baby Hamster Kidney cells (BHK). Despite the fact that rFVIII is produced in vitro, the final formulation was found to contain at least 41 other proteins [START_REF] Basilico | Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach[END_REF]. More recently, another type of rFVIII was developed in which the B domain, that has no known pro-coagulant function, has been deleted; this product is referred to as B-domaindeleted FVIII (BDD-FVIII).

I.1.3.2 Infusion regiment

FVIII can be administered to the patients following two distinct regimens, either ondemand or on a prophylactic basis. For on-demand treatments, the quantity of infused FVIII varies with the severity of the bleeding. Treatment of slight bleeds, such as epistaxis or moderate hematuria, can be achieved by restoring 30 to 50% of FVIII level. In cases of more severe bleeds, such as acute joint bleeds or large hematomas, FVIII is infused in the patients until bleeding stops. In cases of life-threatening bleeds, the continuous infusion of FVIII was found to reduce the consumption of FVIII [START_REF] Batorova | Intermittent injections vs. continuous infusion of factor VIII in haemophilia patients undergoing major surgery[END_REF]. In the latter case, FVIII infusion may be prolonged for a period of up to 10 days, to prevent the recurrence of hemorrhages. In contrast with on-demand treatments, prophylactic treatments consist in the bi-or tri-weekly administration of FVIII and aim at maintaining a FVIII concentration between 3 and 5% of the normal levels. By preventing bleedings, this infusion regiment increases the patients" quality of life. Moreover, setting an early primary prophylaxis regiment has been shown to prevent the development of arthropathies, thus delaying or reducing the need for early joint replacement. To date, the development of easy-handling FVIII preparations has considerably improved the quality of life of the patients by allowing them to self-administrate FVIII.

I.1.3.3 Complications of the treatment

Until the 90"s, the most serious complication of pdFVIII infusion was the transmission of infectious agents such as HIV or hepatitis B and C [START_REF] Mauser-Bunschoten | Comorbidity in the ageing haemophilia patient: the down side of increased life expectancy[END_REF]. Since then, progress in virus inactivation procedures along with the development of recombinant FVIII molecules have reduce the incidence of viral infection. Nowadays, the major complication consecutive to FVIII administration is the development of antibodies raised against therapeutic FVIII. These antibodies are capable of inhibiting FVIII activity and are thus called "inhibitors". The development of an immune response towards FVIII abrogates the efficiency of the treatment and increases patient"s mortality. This complication occurs in 25 to 30% of patients with severe hemophilia A, and 5% of patients with moderate/minor hemophilia A [START_REF] Oldenburg | Genetic risk factors for inhibitors to factors VIII and IX[END_REF].

I.1.3.4 Treatment of patients with FVIII inhibitors

The treatment of hemophilia A patients with FVIII-inhibitors can be achieved through several approaches. Classically, if a FVIII inhibitor is discovered in a hemophilia A patient, the first approach is the initiation of an immune tolerance induction (ITI) protocol. Indeed, almost 40 years ago, Brackmann and Gormsen reported that the repetitive injections of large amounts of FVIII was eradicating the FVIII inhibitors [START_REF] Brackmann | Massive factor-VIII infusion in haemophiliac with factor-VIII inhibitor, high responder[END_REF].

Further studies have demonstrated that this protocol was efficient in up to 80% of the cases of inhibitor-positive patients [START_REF] Dimichele | Analysis of the North American Immune Tolerance Registry (NAITR) 1993-1997: current practice implications. ISTH Factor VIII/IX Subcommittee Members[END_REF][START_REF] Lenk | The German National Immune Tolerance Registry, 1997 update. Study Group of German Haemophilia Centres[END_REF]. From a mechanistic point of view, the injection of high dose FVIII (currently ranging from 50 to 250 IU/kg/day) was found to eradicate FVIII-memory B cells in mice [START_REF] Hausl | High-dose factor VIII inhibits factor VIIIspecific memory B cells in hemophilia A with factor VIII inhibitors[END_REF] as well as in patients [START_REF] Gilles | Neutralizing antiidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A[END_REF][START_REF] Sakurai | Association of anti-idiotypic antibodies with immune tolerance induction for the treatment of hemophilia A with inhibitors[END_REF][START_REF] Van Helden | Factor VIII-specific B cell responses in haemophilia A patients with inhibitors[END_REF]. However, it should be noted that all patients are not eligible for ITI. Moreover, many factors have been found to influence the success of ITI, such as the inhibitory titre at the time of ITI initiation [START_REF] Mariani | International immune tolerance registry, 1997 update[END_REF], the delay between inhibitor detection and ITI initiation [START_REF] Kreuz | Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up[END_REF], as well as the age of the patient and the inhibitor peak titre after the start of ITI [START_REF] Mariani | Immune tolerance in hemophilia-principal results from the International Registry. Report of the factor VIII and IX Subcommittee[END_REF][START_REF] Kreuz | Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up[END_REF]. When all these parameters are fulfilled, then ITI may be initiated.

However, if the ITI is not applicable to the patient or in case of ITI failure, other strategies can be used. If the patient is at high risk of bleeding, activated factor VII can be used to restore the coagulation through the extrinsic pathway. Additionally, a therapeutic preparation comprising multiple activated clotting factors can be used to bypass the FVIII inhibitors and is called FEIBA ® (Factor Eight Inhibitor Bypassing Agent).

I.2 The anti-factor VIII immune response

The development of FVIII inhibitors is consecutive to the triggering of an immune response to the exogenous FVIII. It is considered to be a classical allogeneic immune response, which is T-cell dependent [START_REF] Lacroix-Desmazes | Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A[END_REF]. The antigen-presenting cells (APCs) are the cell population that recognize and endocytose the antigen. Within the endosome of the cells, the antigen is cleaved into small peptides which associate with the class-II major histocompatibility complex (MHC-II). The peptide, in association with the MHC-II is exposed at the surface of the cell to be presented to the naïve T cells. Upon reception of an adequate activation signal, the APCs activate the FVIII-specific naïve T cells.

In turn, the activated T cells activate B cells. Ultimately, the activated B cells differentiate into memory B cells and anti-FVIII IgG producing plasmocytes [START_REF] De Groot | Immunogenicity of protein therapeutics[END_REF].

I.2.1 FVIII as seen by the immune system in patients with hemophilia A

As explained above, a wide range of mutations affecting the F8 gene leads to severe hemophilia A. However, despite the fact that all the patients with severe hemophilia A are characterized by a FVIII activity below 1%, they differ in the sense that some patient do not have circulating FVIII antigen whereas some do. From an immunological point of view, this is critical. Indeed, during the ontogeny of the immune system, tolerance mechanisms are triggered. However, triggering of these mechanisms relies on the presence of the selfantigens. These mechanisms are maintained during the entire lifespan of the individuals to prevent auto-immune reactions. It seems obvious that tolerance to FVIII cannot be triggered in CRM-negative patients, who do not have any circulating FVIII antigen. This explains why the incidence of FVIII inhibitors is higher in these patients. Yet, the fact that only up to 30-50% of CRM-positive patients develop FVIII inhibitors suggest that other factors, in addition to the absence of immune tolerance to FVIII, are required for the initiation of the anti-FVIII immune response [START_REF] Brown | The effect of naturally occurring antibodies to factor VIII on an immunoradiometric assay for factor VIII coagulant antigen. Observation on a cross-reacting material-positive (CRM+) hemophiliac with a factor VIII inhibit[END_REF]. The development of FVIII inhibitors in CRM-positive patient could be triggered because their endogenous FVIII is slightly different from the functional FVIII [START_REF] Pashov | In silico calculated affinity of FVIIIderived peptides for HLA class II alleles predicts inhibitor development in haemophilia A patients with missense mutations in the F8 gene[END_REF]. However, this hypothesis does not explain why not all CRM-negative patients develop FVIII inhibitors. Taken together, this suggests that the absence of endogenous FVIII is not sufficient to explain the development of an anti-FVIII immune response.

I.2.2 Antigen-presenting cells

An immune response starts with the capture of the target antigen by an appropriate antigen presenting cell such as dendritic cells (DCs), macrophages or B cells.

I.2.2.1 Dendritic cells

Considered as professional APCs, DCs are able to initiate primary immune responses by activating naïve T cells [START_REF] Banchereau | Immunobiology of dendritic cells[END_REF]. Originating from monocytes, the immature DC possess the capacity to endocytose the antigens. Following their migration to the secondary lymphoid organs, DCs mature and express a wide panel of markers, endowing them with the capacity to activate T cells (Figure 6).

I.2.2.2 Macrophages

Macrophages are often described as scavenger cells, owing to the fact that they express a wide variety of endocytosis receptors on their surface [START_REF] Taylor | Macrophage receptors and immune recognition[END_REF]. Within the bloodstream, they circulate as monocytic precursors and differentiate into macrophages after their extravasation in the inflamed tissue. A wide variety of sub-populations of macrophages are also present in the spleen. In the spleen, metallophilic and marginal zone macrophages have been described to mediate exogenous FVIII endocytosis in FVIII-deficient mice and to participate in the initiation of the anti-FVIII immune response [START_REF] Navarrete | Splenic marginal zone antigenpresenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A[END_REF]. Their capacity to activate T cells is lower than that of DCs and the endocytosed antigen have been proposed to mostly go towards the degradation pathway rather than the presentation pathway [START_REF] Trombetta | Cell biology of antigen processing in vitro and in vivo[END_REF].

I.2.2.3 B cells

B cells have been described to endocytose circulating antigens through their B cell receptor (BCR) which is a membrane bound immunoglobulin. Thus, it has been demonstrated that memory B cells are key players in secondary immune responses. The initiation of primary immune responses by B cells has been documented; however their role in initiating the anti-FVIII immune response in the context of hemophilia A has been poorly studied.

I.2.3 The spleen

The spleen comprises three distinct regions: the red pulp, the white pulp and the marginal zone. Blood borne antigens enter the spleen through the splenic artery, and are filtered by the macrophage-rich red pulp and the marginal zone. The marginal zone contains B cells, several macrophages sub-populations and DCs. To initiate an immune response, the antigen-loaded APC reach the white pulp where they activate T cells [START_REF] Nolte | Isolation of the intact white pulp. Quantitative and qualitative analysis of the cellular composition of the splenic compartments[END_REF]. Thus, the spleen has a tremendous role in both the capture and presentation of blood borne antigens [START_REF] Mebius | Structure and function of the spleen[END_REF]. This is in line with the facts that, in mice, the administered FVIII was found to accumulate in the spleen and that splenectomy prior to the administration of FVIII reduces the anti-FVIII immune response [START_REF] Navarrete | Splenic marginal zone antigenpresenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A[END_REF].

I.2.4 The anti-FVIII immune response in hemophilia A patients I.2.4.1 T-cell activation

After its internalization by an appropriate APC, the FVIII molecule will be proteolysed in the endosomes. The peptides that are generated associate with the MHC class II and are presented at the cell surface. Within the secondary lymphoid organs, CD4 positive T cells "sense" the presence of their specific antigen in association with the MHC class II through their T cell receptor (TCR). This constitutes the first activation signal for T cells. If no other activation signal is provided, the T cells are anergized and may acquire a regulatory phenotype [START_REF] Quaratino | Fully competent dendritic cells as inducers of T cell anergy in autoimmunity[END_REF]. Thus, co-stimulatory molecules have to be expressed on APCs to activate T cells. These co-stimulation markers are expressed only if the APCs receive a "danger signal". Once such a signal is provided, the APC matures, leading to the overexpression of surface molecules such as CD80, CD86 and CD40 (Figure 6). The expression of these molecules contributes to the activation of T cells. Thus, the CD80 and CD86 molecules expressed on APCs interact with CD28 on T cells, and CD40 interacts with the CD40L that is expressed on activated CD4+ T cells. Such a cross-talk contributes to T-cell activation but also strengthen the stimulation of APCs. It has been shown that the in vivo invalidation of CD80 or CD86, but also the blockade of CD40, abrogates the anti-FVIII immune response [START_REF] Qian | Prevention and treatment of factor VIII inhibitors in murine hemophilia A[END_REF]. To achieve the activation of T cells, a third signal is required. Indeed, activated APCs secrete different types of cytokines, depending on the danger signal that they initially received. The released cytokines polarize the differentiation of T cells into T helper 1, 2 or 17 cells (Figure 7). In inhibitor-positive patients with hemophilia A, all three subsets of T cells were found [START_REF] Ettinger | Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti-factor VIII immune responses[END_REF]. However, patients with low inhibitory titres seem to have a Th1 profile whereas patients with high inhibitory titres present with a Th2 profile, as evaluated by analysis of the dominant IgG subclasses [START_REF] Reding | Distribution of Th1-and Th2-induced anti-factor VIII IgG subclasses in congenital and acquired hemophilia patients[END_REF].

I.3 Risk factors

As presented earlier; the initiation and amplification of an immune response requires the participation of inflammatory mediators. This phenomenon is inherent to the danger theory, initially proposed by P. Matzinger. Thus, several genetic and non-genetic risk factors have been identified or proposed to participate in the initiation of the anti-FVIII immune response.

I.3.1 The danger signal theory

The concept of danger signal is derived from the danger theory proposed in 1994 by P.

Matzinger [START_REF] Matzinger | Tolerance, danger, and the extended family[END_REF]. According to this, APCs are activated by danger signals. These signals can originate from invading pathogens, which express several structures able to trigger the immune response. These structures are called "pathogen-associated molecular patterns" (PAMP). It can also originate from self-cells, which secrete or expose several endogenous molecules in response to cellular stress. In 2002, P. Matzinger merged these two kinds of molecules (i.e., endogenous and exogenous PAMPs) under the same terminology, i.e.,

"damage-associated molecular patterns" (DAMP).

I.3.1.1 Exogenous danger signals

PAMPs correspond to molecular components found only in microorganisms such as bacteria, viruses or fungi. These microorganisms possess specific structures such as carbohydrates or glycolipids that are not present in the mammalian host. These components may derive from the microorganism wall such as lipopolysaccharide (LPS) and peptidoglycan for bacteria, or carbohydrates for viruses. They can also originate from the cytosolic compartment of the microorganism, such as nucleic acids, that are exposed upon pathogen lysis or phagocytosis [START_REF] Sirisinha | Insight into the mechanisms regulating immune homeostasis in health and disease[END_REF].

I.3.1.2 Endogenous danger signals

The first identified danger signals were the CD40 ligand (CD40L), the tumor necrosis factoralpha (TNF-α) and the interleukine-1 beta (IL1-β). These molecules are able to directly induce the maturation of APC [START_REF] Koide | Interleukin 1 enhances T-dependent immune responses by amplifying the function of dendritic cells[END_REF][START_REF] Santiago-Schwarz | Mechanisms of tumor necrosis factor-granulocyte-macrophage colony-stimulating factor-induced dendritic cell development[END_REF][START_REF] Caux | Activation of human dendritic cells through CD40 cross-linking[END_REF].

Under physiological conditions, these factors are stored inside the cells and are not in contact with their specific receptors on the immune cells. In case of cellular stress or cellular damage, these mediators are released in the extracellular microenvironment. The general example is provided by necrotic cells, which release several molecules able to activate the immune system such as heat shock proteins (HSP) [START_REF] Quintana | Heat shock proteins as endogenous adjuvants in sterile and septic inflammation[END_REF], the chromatin associated high mobility group box 1 (HMGB1) [START_REF] Scaffidi | Release of chromatin protein HMGB1 by necrotic cells triggers inflammation[END_REF], and some purinemetabolism derived molecules such as ATP or uric acid [START_REF] Bours | Adenosine 5'-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation[END_REF].

I.3.2 Genetic risk factors

Several immune-related genes have been under investigation to assess their role in facilitating the anti-FVIII immune response. Amongst them are found the type of FVIII mutation, the HLA haplotype, and polymorphisms in a wide variety of immune-related genes. The type of FVIII mutation is particularly important because, as explained previously, it dictates the establishment of a regulatory response to FVIII during the ontogeny of the immune system.

The HLA haplotype is also of particular interest because it is a key component in antigen presentation. Eventually, the identification of immune-related gene polymorphisms may allow the identification of patient at higher risk of FVIII inhibitors development.

I.3.2.1 FVIII mutations

As detailed in section I.1.1.3, several types of mutations are affecting the F8 gene. Although the common feature of mutations in severe hemophilia A patients is the complete abolition of FVIII activity, the different mutations may have different effects on the production, function and structure of FVIII. Thus, the presence of a non-functional FVIII in the circulation imparts the patient with a lower risk for inhibitor formation compared to a patient in whom the FVIII molecule is not synthesized or secreted. Thus, the risk for inhibitor development has been linked to the type of mutation as presented in Table 1. Surprisingly, in patients with the Inv22 mutation, the incidence of FVIII inhibitors is lower than in the case of patients with large deletions. In Inv22 patients, since no circulating FVIII antigen is detected, one would expect the absence of thymic education towards FVIII, and the ensuing failure to eliminate FVIII-specific CD4+ T cells and to generate regulatory T cells.

However, as explained in section I.1.1.3, the work from Pandey et al. has suggested that, in patients with the Inv22 mutation, FVIII is synthesized as two polypeptides that remain inside the cells. In particular, Pandey et al. claimed to have detected FVIII antigen in the peripheral blood mononuclear cells (PBMCs) of healthy donors and patients. These data lead the authors to propose that immunological tolerance to FVIII might be triggered at a better level in Inv22 patients as compared to patients where the F8 gene mutation abrogates the production of the protein. These data are however contradicted by recent publications documenting the preponderant role of endothelial cells in FVIII synthesis and absence of detection of the FVIII mRNA in peripheral blood cells [START_REF] Everett | Murine coagulation factor VIII is synthesized in endothelial cells[END_REF].

I.3.2.2 HLA haplotype

Several studies have emphasized the associating between the HLA haplotypes of the patients with the development of FVIII inhibitors. Thus, it appears that the HLA class II alleles DRB1*15 and DQB1*0602 are associated with a higher risk for FVIII inhibitor development [START_REF] Hay | HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party[END_REF][START_REF] Pavlova | Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A[END_REF]. Conversely, the HLA class II alleles DRB1*11 and DQB1*03 have recently been associated with a reduced risk for FVIII-inhibitor development [START_REF] Pergantou | Impact of HLA alleles and cytokine polymorphisms on inhibitors development in children with severe haemophilia A[END_REF]. It is important to keep in mind however that haemophilia A is a rare disease and that it is extremely challenging to constitute large enough cohorts of patients to accurately determine the importance of respective HLA haplotypes.

I.3.2.3 Polymorphisms in immune-related genes

The analysis of single nucleotide polymorphisms (SNPs) in different immune-related genes resulted in the identifications of variants in cytokine genes that were associated with the development of FVIII inhibitors. Thus, SNPs in the promoter of the genes encoding IL-10, TNF-α and CTLA-4 were associated with a higher incidence of FVIII inhibitor in patients with hemophilia A (Astermark et al., 2006a;[START_REF] Gouw | The multifactorial etiology of inhibitor development in hemophilia: genetics and environment[END_REF][START_REF] Pavlova | Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A[END_REF]. Indeed, the presence of a 134-bp-long variant of a CA repeat microsatellite in the promoter region of the IL-10 gene, previously associated with high levels of autoantibodies in several auto-immune diseases, was also associated with the presence of FVIII inhibitors in patients with severe hemophilia A (Astermark et al., 2006b). Similarly, within the TNF-α promoter, four SNPs were identified (-308 G>A, -827 C>T -238 G>A and -670 A>G) that affect the transcription of the TNF-α encoding gene. Subsequently, patients with severe hemophilia A possessing the combination of alleles -308AA -827CC -238GG and -670AA were found to be more susceptible to develop an inhibitor (Astermark et al., 2006a).

Similarly, carriage of the G allele at position +49 of the CTLA-4 gene is associated with a decreased expression of CTLA-4, leading to an increased proliferation of activated T-cell, was associated with a higher incidence of inhibitor in patients with severe hemophilia A [START_REF] Astermark | Polymorphisms in the CTLA-4 gene and inhibitor development in patients with severe hemophilia A[END_REF]. Recently, the Fc gamma receptor IIa (FcγRIIa or CD32) R131H polymorphism, that increases the binding affinity of the FcγRIIa to IgG1 and IgG2 isotypes was associated with inhibitor development in patients with severe hemophilia A [START_REF] Eckhardt | The Fc gamma receptor IIa R131H polymorphism is associated with inhibitor development in severe hemophilia A[END_REF]. Moreover, a genome-wide association study (GWAS) was conducted using a combined cohort involving 833 patients with severe hemophilia A from three independent cohorts (HIGS, MIBS and HGDS). This GWAS analysis evaluated the association of 13,331 SNPs from 1,081 genes with the presence of FVIII inhibitor in patients with severe hemophilia A. The authors identified 53 SNPs as significant predictors of inhibitor status.

More precisely, within the 13 SNPs that were concordant in the three cohorts analyzed, 5 were associated with an increased risk for the development of FVIII-inhibitor (MAPK9, DOCK2, CD44, IQGAP2 and CSF1R) and 8 were found to be protective (PDGFRB, PCGF2, HSP90B1, F13A1, IGSF2, ALOX5AP, MAP2K4 and PTPRN2) [START_REF] Astermark | The polygenic nature of inhibitors in hemophilia A: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort[END_REF].

I.3.3 Non-genetic risk factors

The idea that the anti-FVIII immune response is not only dictated by patients" intrinsic risk factors originates from the observation that homozygous hemophilic twins do not respond equally to FVIII infusion from an immunological point of view: some might develop inhibitors while other might not [START_REF] Astermark | Basic aspects of inhibitors to factors VIII and IX and the influence of non-genetic risk factors[END_REF]. Thus, the age of the patient at the start of the treatment, the mode and intensity of FVIII administration at the first bleeding episode and the state of the immune system at the time of FVIII administration, are among the most debated non-genetic risk factors.

I.3.3.1 Age at the start of treatment

Lorenzo et al reported in 2001 a higher incidence of inhibitor formation in Spanish patients where FVIII infusion was started before the age of 6 month [START_REF] Lorenzo | Incidence of factor VIII inhibitors in severe haemophilia: the importance of patient age[END_REF]. The same conclusion was reached in another cohort from the Hemophilia Center in Utrech where a time-dependence between the age at first FVIII administration was correlated with inhibitor formation [START_REF] Van Der Bom | Age at first treatment and immune tolerance to factor VIII in severe hemophilia[END_REF]. However, this was not confirmed in the Milanese cohort where no association was found [START_REF] Santagostino | Environmental risk factors for inhibitor development in children with haemophilia A: a case-control study[END_REF]. It has since been proposed that early replacement therapy in young patients reflects a more severe clinical conditions that require more intensive treatments, thus suggesting the participation of other risk factors, such as treatment intensity.

I.3.3.2 Mode and intensity of FVIII administration

Different patients with hemophilia A do not display similar bleeding patterns. Thus, the treatment that they received differs depending on their bleeding phenotype. In line with this observation, the question of the repercussion of the treatment schedule and FVIII dosage on the development of the anti-FVIII immune response has been raised. To answer this question, the RODIN study evaluated the incidence of the mode of administration of FVIII and intensity of treatment on FVIII inhibitor formation. This was done using a large cohort of 606 patients involving 29 hemophilia treatment centres. The results from this study indicate an increased risk for inhibitor development after intensive FVIII treatment and a decreased risk when prophylaxis was used. However, the risk for inhibitor development was the same during the first 20 exposure days, irrespectively of the infusion regimen. As a conclusion, the authors suggest that patients may be divided into three groups: the first group of patients in whom inhibitors will never develop independently of the treatment regimen, patients in whom inhibitors will develop depending on the treatment, and patients who will develop inhibitors in all cases [START_REF] Gouw | Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study[END_REF].

I.3.3.3 State of the immune system at the time of FVIII infusion

Infectious disease, vaccination, severe bleeds or surgeries are events associated with the activation of the pro-inflammatory machinery, and hence of the immune system. Thus, it appears plausible to hypothesize that the concomitant infusion of FVIII along with one of these events would result in a higher risk for inhibitor development. This concept is supported by the observation that, in some inhibitor-negative hemophilia A patients, an anti-FVIII immune response was observed after a surgical procedure [START_REF] Sharathkumar | Intensive exposure to factor VIII is a risk factor for inhibitor development in mild hemophilia A[END_REF]. This was however not confirmed in the case-control study by Santagostino et al, where no association between the immune status at start of the treatment and inhibitor development was found.

Deciphering the participation of some of these factors represents the main goal of my PhD work.

I.4 Purpose of the PhD work

The global aim of my PhD work was to decipher the bleeding-associated risk factors in the development of FVIII inhibitors. This general question will be addressed in four parts. The first part focuses on the association of a polymorphism in a gene expressed in response to bleeding events with the development of FVIII inhibitors in patients with severe haemophilia A. The second part exploits a mouse model of severe haemophilia A in order to evaluate the impact of severe bleedings in the anti-FVIII immune response. The third chapter of the result section concentrates on whether modulation of the oxidative state may affect the anti-FVIII immune response in vivo. Lastly, I assess whether oxidative modifications of the FVIII molecule enhance its immunogenicity. For a better understanding of these objectives, these four objectives will be introduced separately.

II. RESULTS

II.1. Development of inhibitory antibodies to therapeutic factor VIII in severe

hemophilia A is associated with microsatellite polymorphism in the HMOX1 promoter

As described in section I.3.2.3, several SNPs in immune-related genes have been associated

with the development of FVIII inhibitors in patients with severe hemophilia A.

Previous experimental observations from the lab have demonstrated that the administration of hemin to FVIII-deficient mice leads to the systemic induction of the heme-degrading enzyme, heme oxygenase-1 (HO-1), and was drastically reducing the anti-FVIII immune response [START_REF] Dimitrov | Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII[END_REF]. In normal conditions, heme-bound hemoglobin is confined within erythrocytes, where it participates in the fixation and transport of oxygen. However, in the case of vascular damage, erythrocytes are lysed and their content is released in the microenvironment. Free heme, due to its redox properties, is potentially harmful in the sense that it can oxidize and damage any surrounding molecule. Thus, in response to heme exposure, several mechanisms are triggered, leading to the expression of HO-1. To date, three polymorphisms have been described in the HO-1 encoding gene (HMOX1), among which, two SNPs (-413 T>A and -1135 G>A) and one (GT) n repeat dinucleotide length polymorphism [START_REF] Exner | The role of heme oxygenase-1 promoter polymorphisms in human disease[END_REF]) (Figure 9). Although the nature of the SNPs does not seem to significantly modify the activity of the HMOX1 promoter, the length of the (GT) n repeats has been associated with the yield of expression of the HO-1 protein. From a mechanistic point of view, it is probable that the succession of (GT) n repeats confers a Z-conformation to the DNA, thus reducing the affinity for transcription factors. Thus, promoters with long (GT) n repeats would have less affinity for the transcription factors as compared to promoter with shorter (GT) n repeats. The association between the number of (GT) n repeats has been associated with the outcome of various inflammatory conditions found in pulmonary, cardiovascular and autoimmune diseases. Results from these studies either associates long (GT) n repeats with a unfavourable outcome or short (GT) n repeats with a favourable outcome in these diseases [START_REF] Exner | The role of heme oxygenase-1 promoter polymorphisms in human disease[END_REF].

Here, we wondered whether polymorphisms in the promoter of the HMOX1 gene could be associated with the development of FVIII inhibitors in patients with severe hemophilia A.

Using a multi-centric cohort including 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic FVIII, we demonstrated that patients with inhibitors have a higher frequency of alleles with large (GT) n repeats (L: n≥30) compared to patients without inhibitors (19.2% vs 9.3% respectively)

and that genotypes containing the L allele were more frequent in patients with an inhibitor (odd ratio 2.31; 95% CI 1.46-3.66; P<0.001).

In this work, we demonstrated for the first time an association between a polymorphism in the promoter of the stress-inducible HMOX1 gene with the risk of developing FVIII inhibitors.

Moreover, these results suggest that the capacity of the patients to cope with inflammation, associated or not with hemorrhages may be a critical parameter influencing the initiation of the anti-FVIII immune response.

As presented in section I.3.2.3, a recent GWAS analysis was conducted using a combined cohort involving 833 patients with severe hemophilia A from three independent cohorts (HIGS, MIBS and HGDS). This GWAS analysis, including 13,331 SNPs from 1,081 genes, resulted in the identification of 53 SNPs as significant predictors of inhibitor status. The 5 SNPs that were concordant in the three cohorts and are associated with a higher risk for the development of inhibitors have odd ratios as high as 4.29. However, GWAS analyses focus only on SNPs, and do not include length polymorphism, thus explaining why the number of (GT) n repeats in the promoter of the HMOX1 gene was not identified in the GWAS analysis conducted by Prof Astermark. In contrast to the GWAS strategy, which screens thousands of genes, our approach was candidate-gene driven, based on the experimental finding that induction of HO-1 in mice had a protective effect on the development of FVIII inhibitors.

While having two opposite starting points, these two strategies are complementary in the identification of the patient"s genetic profiles associated with higher risks for the development of FVIII inhibitors.

II.2. Hemarthrosis and arthropathies do not favor the development of factor VIII inhibitors in hemophilia A mice

As described in section I.1.1.4, the lack of functional endogenous FVIII in patients with severe hemophilia A leads to the development of hemarthrosis and ensuing arthropathies.

Beside the crippling effects of such complications, little is known about the participation of such events on the development of FVIII inhibitors. However, the presence of concomitant bleedings as a risk factor for the development of FVIII inhibitor has been suggested and is detailed in section I.3.3.3. Unlike humans, the mouse model for FVIII-deficiency does not have the same pathophysiological development. Indeed, FVIII-deficient mice do not present spontaneous bleedings, and do not develop arthropathies. However, the insertion of a needle below the patella of FVIII-deficient mice generates a localized hemarthrosis which is resorbed within 14 days, and leads to the development of knee arthropathy [START_REF] Hakobyan | Experimental haemophilic arthropathy in a mouse model of a massive haemarthrosis: gross, radiological and histological changes[END_REF].

Thus, using an established model of knee puncture in FVIII-deficient mice, we asked the question of the development of FVIII inhibitors in this particular situation To follow the anti-FVIII immune response in the mouse model of hemarthrosis or arthropathy, FVIII replacement therapy was initiated 1, 7 or 14 days after puncture of the knee. Anti-FVIII IgG titres and inhibitory titres were determined in the serum of mice by ELISA and Bethesda assay three days after the second, third and fourth injection of therapeutic FVIII. The results obtained from these experiments demonstrate that the presence of hemarthrosis or arthropathies do not represent a risk factor for the anti-FVIII immune response, as the anti-FVIII IgG titres were not increased in mice with hemarthrosis and arthropathies compared to un-injured mice. Conversely, we observed a significant reduction of the anti-FVIII IgG titres in mice where the FVIII replacement therapy was initiated 7 days after knee injury. Because of the presence of blood at the site of injury, and after having ruled out an increased consumption of FVIII, we assessed the induction of HO-1 in these mice by western-blot.

Indeed, systemic induction of HO-1 in mice drastically reduces the anti-FVIII immune response [START_REF] Dimitrov | Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII[END_REF]. We found that HO-1 was induced 7 days after puncture of the knee, and was concomitant with the reduction in the anti-FVIII immune response.

Moreover, we analysed the expression of maturation markers in the splenic antigen presenting cells by flow cytometry. We observed that 7 days after puncture of the knee of FVIII-deficient mice, the level of expression of MHC class II and CD86 were significantly reduced in dendritic cells and macrophages. Taken together, our results demonstrate that bleedings in FVIII-deficient mice do not represent a danger signal that adjuvant the anti-FVIII immune response.

Our observations are important in view of the fact that bleeding, surgery and vaccination have often been proposed as risk factors for the development of FVIII inhibitors. Importantly, no scientific demonstration has been provided in favour of any of these factors. Potential limitations of the present work reside in the use of the mouse model of severe hemophilia A.

Indeed, in contrast to patients in whom the severe hemophilia A phenotype may be due to different types of mutation, with all the possible consequences on the ontogeny of the immune system as described in section I.1.1.3, the mouse model of hemophilia A is completely naïve for the human FVIII molecule. The identification of the risk factors for the development of FVIII inhibitors is complicated by the fact that administration of FVIII in FVIII-deficient mice systematically triggers an anti-FVIII immune response. Accordingly, due to the congenic nature of the FVIII-deficient mice strain, their ability to turn on the antiinflammatory machinery upon bleeding is probably similar in every individual, and do not reflect the heterogeneity that is the hallmark of the human situation, as highlighted in section II.1.

II.3. Restoration of the oxidative balance in factor VIII-deficient mice reduces the immunogenicity of therapeutic factor VIII

As described in section I.3.3.3, a pro-inflammatory state of the immune system at the time of FVIII administration is considered as a risk factor facilitating the development of FVIII inhibitors. Thus, bleedings are associated with the triggering of the respiratory burst in innate immune cells that are recruited at the site of injury. The respiratory burst leads to the generation of reactive oxygen species (ROS) the primary function of which is to destroy potentially invading pathogens. However, ROS are not specific to pathogens and will act nonspecifically on any molecule present in the surrounding environment. Thus, excessive accumulation of ROS has a deleterious effect and may lead to damages to self-proteins. To counteract these undesirable effects of excessive ROS accumulation, several enzymatic and non-enzymatic antioxidant systems have evolved.

In this work, we hypothesized that the lack of functional endogenous FVIII in FVIII-deficient mice is associated with an increase ROS accumulation. Because the quantification of ROS is complicated by their short half-life, we aimed at determining the status of the endogenous antioxidant systems, as an indirect proof of chronic ROS generation. Thus, we compared the oxidant radical absorbance capacity (ORAC) of plasma from FVIII-deficient mice to that of wild-type C57Bl/6 mice. We found that plasma from FVIII-deficient mice has lower ORAC value compared to that of C57Bl/6 mice (2511±32 vs 2666±44 Trolox equivalent, P<0.05).

This observation was confirmed using the ferric reducing ability of plasma (FRAP) assay.

We then hypothesized that the supplementation of mice with the antioxidant N-acetyl-Lcysteine (NAC) would restore the redox balance and will affect the anti-FVIII immune response. Thus, NAC (0.142g/L) was added to the drinking water of mice throughout the FVIII-immunization protocol. We validated that the supplementation with NAC was restoring the ORAC value of the plasma of FVIII-deficient mice. Moreover, we found that mice treated with NAC had lower anti-FVIII IgG titres compared to that of untreated mice (675.0±129.3 vs 358.5±74.3, P < 0.05). Importantly, the treatment of mice with NAC was not impairing the ability of their immune system to mount an immune response, as the total IgG titres and the immune response to tetanus toxin were equal in both NAC-treated and control group.

Taken together, these results demonstrate that the lack of FVIII in FVIII-deficient mice is associated with a reduced capacity of the plasma to scavenge ROS. This might be due to a chronic generation of ROS that might originate from chronic bleedings, despite the absence of overt hemorrhages. These identify the redox state as a risk factor for the development of the anti-FVIII immune response. However, these data were obtained using the FVIII-deficient mouse model. It would be interesting to determine whether these results are reproducible in patients with hemophilia A. If so, the determination of the redox state in patient might be seen as a predictive tool for the development of inhibitors. Moreover, the use of antioxidant compounds might lower the risk of patients with hemophilia A to develop FVIII inhibitors.

II.4. Oxidation of therapeutic factor VIII aggravates its immunogenicity in factor-VIII deficient mice

Manuscript in preparation (discussion pending, but results are discussed in chapter III)

The occurrence of cellular damage triggers the inflammatory machinery. A major arm of the inflammatory machinery resides in the recruitment and activation of phagocytic cells at the damaged site. The role of activated phagocytes at the site of injury is to limit the invasion of pathogens. To achieve this goal, phagocytes dispose of a wide arsenal to directly destroy pathogens, or to facilitate their neutralization by the other components of the immune system.

Indeed, in 1973, Babior et al. reported that phagocytes utilize a membrane-bound NADPH oxidase to generate large amounts of reactive oxygen species (ROS) [START_REF] Babior | Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent[END_REF]. The process of ROS generation and secretion is described as the "respiratory burst" or "oxidative burst". This system comprises a wide variety of mediators acting on several types of molecules.

From a chemical point of view, the membrane-bound NADPH oxidase generates a superoxide anion (O2 •-), which is then dismuted to H 2 O 2 and oxygen by the superoxide dismutase (SOD).

Later on, H 2 O 2 is converted into hypochlorous acid (HOCl). Although the generated ROS may directly destroy the target antigens, they also exert multiple functions on the immune response, which can be direct, by affecting the signalling of immune cells, or indirect, through alterations of the antigens that are addressed to the immune system.

ROS react rapidly with the surrounding proteins, DNA or lipids. In the case of HOCl however, it seems that proteins are likely to be the main target. As a selective oxidant, HOCl reacts with particular amino acids. Thus, the order of reactivity for protein side chain is Met > Cys >> Cystine ~ His ~ α-amino > Trp > Lys >> Tyr > Arg > Gln ~ Asn [START_REF] Pattison | Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds[END_REF]. Once the reactive side-chain residues have been consumed, and if the concentration of HOCl increases, the backbone amides of the protein are targeted by HOCl. The attack of the backbone amides by HOCl results in the fragmentation of the target protein. Thus, oxidative damages on a singular protein depend on the composition and the accessibility of the amino acids of the protein.

From an immunological point of view, the oxidative alteration of antigens has been repeatedly associated with a higher immunogenicity. In this work, we hypothesized that the oxidative modification of FVIII, which may occur at the site of bleeding, modulates its immunogenicity. As a model for ROS, we used hypochloric acid (HOCl), which is the most abundant ROS released by neutrophils. Our results demonstrate that exposure of FVIII to HOCl result in a dose-dependent loss of FVIII pro-coagulant activity. To characterize the impact of the exposure of FVIII to HOCl, we studied the association of native or HOClexposed FVIII to its natural binding partners. We found no major changes in the ability of HOCl-exposed FVIII to bind VWF, phosphatidyl serine, and four monoclonal antibodies (BO2C11, BO2BII, ESH8 and 77IP52H7). We demonstrate that the exposure of FVIII to a 25-fold molar excess of HOCl results in the disappearance of free amines without the generation of carbonyls groups. This suggests that the exposure of FVIII to a 25-fold molar excess of HOCl results in a mild oxidation of the FVIII molecule. In vivo, we demonstrate that, as hypothesized, exposure of FVIII to HOCl modulates FVIII immunogenicity. Indeed, FVIII-deficient mice treated with FVIII that was exposed to a 25-fold molar excess of HOCl, mounted a higher anti-FVIII immune response compared to mice treated with native FVIII. In an attempt to understand the underlying mechanism, we demonstrate that the exposure of FVIII to a 25-fold molar excess of HOCl does not inducing the maturation of human DCs in vitro. However, the incubation of splenocytes from FVIII-primed mice with HOCl-exposed FVIII resulted in a higher proliferation compared to that of splenocytes incubated with untreated FVIII.

Further work is required to complete our observations. Thus, the binding of FVIII or HOClexposed FVIII to FIXa has not been explored yet. Similarly, the endocytosis of HOClexposed FVIII has to be investigated, although a first set of experiment suggested no change in FVIII uptake following exposure to HOCl. Several studies suggest that the oxidative modification of antigens is associated with a modified susceptibility of the antigen to be processed in the endosome of antigen-presenting cells. Thus, we will compare the susceptibility of FVIII and HOCl-exposed FVIII to be cleaved by endosomal proteases (e.g. cathepsins).

III. DISCUSSION

In patients with severe hemophilia A, the lack of functional FVIII leads to prolonged bleeding time. Thus, the time-lapse for the resolution of hemorrhages is higher as compared to healthy individuals. To correct hemorrhages, exogenous FVIII is administered to the patients. Damage to the endothelium triggers an inflammatory response, which is required for postinjury regeneration and tissue repair. Inflammation following endothelium damage is a dynamic process, which is driven by numerous inflammatory mediators. The innate and the adaptive immune system can be activated by endogenous signals that originate from stressed or injured cells, that signify "danger" to the host [START_REF] Matzinger | Tolerance, danger, and the extended family[END_REF]. Endogenous danger signals released from stressed cells which trigger the inflammatory response after injury have been termed alarmins or danger-associated molecular patterns (DAMPs) [START_REF] Harris | Alarmin(g) news about danger: workshop on innate danger signals and HMGB1[END_REF][START_REF] Bianchi | DAMPs, PAMPs and alarmins: all we need to know about danger[END_REF]. Additionally, at the site of injury, the lysis of erythrocytes leads to the release of hemoglobin and heme, the latter being known to promote inflammation and to activate immune cells [START_REF] Dutra | Heme on innate immunity and inflammation[END_REF]. Damage of the endothelium also triggers the hemostatic system. Exposure of the sub-endothelium induces the arrest and activation of platelets. At the same time, expression of tissue factor (TF) triggers the initiation of the coagulation cascade. These two independent but complementary systems are limiting the loss of blood and represent the first step of wound healing. As the vessel wall is repaired, inflammation is resolved, and homeostasis is restored. However, in patients with severe hemophilia A, the lack of functional FVIII, by increasing the time for bleeding arrest might also increase the quantity of inflammatory mediators released at the bleeding site. This was the central hypothesis of my PhD work. Here, I will discuss the effect of some mediators that are released on the bleeding site on the immune system.

Focus on the alarmins resulting from the damage of the endothelium

Following trauma, many endogenous molecules such as DNA, ATP, uric acid, DNA-binding proteins and reactive oxygen species (ROS) are released in the extracellular environment.

These molecules are referred to as alarmins or DAMPs and signal tissue and cell damage to the immune system. The binding of alarmins to their specific receptors induces different effects on the cells of the immune system, such as cell differentiation, cell death or the secretion of pro-or anti-inflammatory mediators. Examples of alarmins, receptors and associated effects are summarized in table 2.

As a prototypic alarmin, the high-mobility group box protein 1 (HMGB1) has been shown to play an important role in the response to sterile injury, such as ischemia/reperfusion and hemorrhagic shock [START_REF] Lotze | High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal[END_REF]. This ubiquitous protein is passively released upon disruption of cell membrane or actively secreted by macrophages and monocytes exposed to inflammatory cytokines or to the complement activation product C5a [START_REF] Rittirsch | Functional roles for C5a receptors in sepsis[END_REF]. The activity of HMGB1 is mediated by its interaction with the toll-like receptor (TLR) 2 and 4. It also interacts with the receptor for advanced glycation endproducts (RAGE), the latter being involved in the rapid initiation of the inflammatory response.

Interestingly, HMGB1 also drives coagulation towards a pro-coagulant state by inducing TF expression and inhibiting tissue plasminogen activator on endothelial cells. Furthermore, HMGB1 was found to promote ROS production by neutrophils [START_REF] Tang | High-mobility group box 1, oxidative stress, and disease[END_REF]. In view of the fact that we observed an increased oxidative state in FVIII-deficient mice, probably due to an increased or chronic production of ROS (article 3), it might be of interest to determine whether levels of HMGB1 in plasma are higher in FVIII-deficient mice, and/or whether they correlate with the inhibitor status of patients with severe hemophilia A. Saïd-Sadier et al., 2012 at the bleeding site. Free hemoglobin rapidly converts into methemoglobin and releases the heme molecule. Free heme has multiple effects on the immune effectors that are recruited to the site of bleeding and on any surrounding molecules. In vivo, the injection of heme was found to increase vascular permeability, leukocyte recruitment and increases the expression of acute phase proteins [START_REF] Lyoumi | Heme and acute inflammation role in vivo of heme in the hepatic expression of positive acute-phase reactants in rats[END_REF][START_REF] Wagener | Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase[END_REF]. The presence of heme leads to the activation of various types of cells. Thus, heme activates endothelial cells and induces the expression of intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, P-selectin, von Willebrand factor (VWF) [START_REF] Wagener | Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells[END_REF][START_REF] Belcher | Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease[END_REF] and the secretion of IL-8 [START_REF] Natarajan | Hypoxia inducible factor-1 modulates hemin-induced IL-8 secretion in microvascular endothelium[END_REF]. Heme also recruits and activates neutrophils, in a protein kinase C (PKC)-dependent manner. Activated neutrophils produces large amount of ROS and releases their DNA content, leading to the formation of neutrophil extracellular traps (NETs). Moreover, heme was found to increase the lifespan of neutrophils by inhibiting apoptosis [START_REF] Arruda | Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB[END_REF]. This phenomenon possibly amplifies the accumulation of pro-inflammatory molecules at the site of injury.

Apart from its direct activating effect on immune cells, heme also generates large amounts of ROS through different mechanisms. First, it was demonstrated that heme triggers ROS production upon positively stimulation/induction of the NADPDH oxidase enzyme pathway [START_REF] Barcellos-De-Souza | Heme modulates intestinal epithelial cell activation: involvement of NADPHox-derived ROS signaling[END_REF]. Second, heme and heme-derived iron generate free radicals through the Fenton reaction. Third, heme, due to its lipophilic nature, binds lipids (i.e., lipids from the membrane of cells) and, in the presence of hydrogen peroxide, leads to the generation of highly reactive alkoxyl (RO • ) and peroxyl (ROO • ) radicals [START_REF] Tappel | The mechanism of the oxidation of unsaturated fatty acids catalyzed by hematin compounds[END_REF]. The effects of ROS on the immune system and their implications in modulating the immunogenicity of antigens will be discussed later on.

To prevent damages associated with the presence of free heme, several systems have evolved.

Thus, the hemoglobin released from erythrocytes is taken up by haptoglobin, which prevents the dissociation of the heme group. The haptoglobin-hemoglobin complex is then addressed to the reticulo-endothelial system, mostly in the spleen. Heme, if dissociated from hemoglobin is cleared by hemopexin and albumin. Heme-bound hemopexin is removed from the circulation mostly in the liver, through the interaction of the heme-hemopexin complex with the lowdensity lipoprotein receptor-related protein (LRP or CD91) [START_REF] Hvidberg | Identification of the receptor scavenging hemopexin-heme complexes[END_REF]. Results from our laboratory have demonstrated that heme is also capable of interacting with FVIII.

This interaction results in a decreased pro-coagulant activity of FVIII due to the inhibition of the interaction of FVIII with activated FIX (FIXa) (annexe 1, [START_REF] Repesse | Heme binds to factor VIII and inhibits its interaction with activated factor IX[END_REF]).

However, the presence of heme on the FVIII molecule did not alter the immunogenicity of FVIII. Thus, beside the fact that the heme-hemopexin complex is taken up by LRP, the endocytosis by human dendritic cells of heme-treated FVIII was similar to that of non-treated FVIII (personal observation). As mentioned above, heme exerts a pro-inflammatory effect on the effector cells of the immune system. In this sense, it sounds logical to hypothesize that heme, upon binding to FVIII, increases its immunogenicity. However, administration of heme-treated FVIII to FVIII-deficient mice elicited a similar immune response as that induced by non-treated FVIII (personal observation).

Another way to detoxify free heme relies on the induction of the heme-degrading enzyme heme oxygenase-1 (HO-1). Heme-oxygenase-1 mediates the degradation of heme into carbon monoxide (CO) and biliverdine, that both exert anti-inflammatory and cyto-protective effects [START_REF] Gozzelino | Mechanisms of cell protection by heme oxygenase-1[END_REF]. Indeed, CO inhibits hemoglobin oxidation, thus preventing additional release of heme [START_REF] Ferreira | Sickle hemoglobin confers tolerance to Plasmodium infection[END_REF]. Additionally, the expression of HO-1 in leukocytes was found to decrease the expression of adhesion molecules and dampen their migration capacity [START_REF] Wagener | The heme-heme oxygenase system: a molecular switch in wound healing[END_REF]. Although heme is a pro-inflammatory molecule, the feedback activity of HO-1 controls inflammation, contributes to wound-healing thus participating in the restoration of homeostasis.

Results from our laboratory have demonstrated that the systemic induction of HO-1, as well as the injection of biliverdine or CORM-3, a CO-releasing molecule, in FVIII-deficient mice reduces the immune response to FVIII [START_REF] Dimitrov | Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII[END_REF]. Likewise, in the mouse model of hemarthrosis, we observed a reduced anti-FVIII immune response in mice bearing 7 dayold knee punctures. This reduced immune response was concomitant with the local induction of HO-1 (article 2). The increased expression of HO-1 at the level of inflammatory sites has been previously reported in patients with rheumatoid arthritis or with osteoarthritis [START_REF] Fernandez | Expression of heme oxygenase-1 and regulation by cytokines in human osteoarthritic chondrocytes[END_REF][START_REF] Kobayashi | Regulatory role of heme oxygenase 1 in inflammation of rheumatoid arthritis[END_REF], as well as in the joint lesions of a rat model of adjuvant-induced arthritis [START_REF] Devesa | Potential role of heme oxygenase-1 in the progression of rat adjuvant arthritis[END_REF]. Heme-mediated induction of HO-1 thus protects cartilage destruction in vitro and in vivo by inhibiting osteoclast formation [START_REF] Zwerina | Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption[END_REF]. Likewise, the administration of CORM-3 to mice protects the animals from collagen-induced arthritis through the inhibition of the production of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 [START_REF] Ferrandiz | Treatment with a COreleasing molecule (CORM-3) reduces joint inflammation and erosion in murine collagen-induced arthritis[END_REF]. All these examples point towards a anti-inflammatory role of HO-1. However, in the human population, different individuals are not equal in terms of the capacity to induce HO-1. Indeed, the expression levels of HO-1 are regulated at the transcriptional level by several polymorphisms. Amongst these, the (GT) n repeat polymorphism, which is present within the promoter region of the HO-1 encoding gene (HMOX1), was associated with the outcome of several conditions such as pulmonary, cardiovascular, neurological and autoimmune diseases [START_REF] Exner | The role of heme oxygenase-1 promoter polymorphisms in human disease[END_REF]. Our observation that long (GT) n repeat polymorphisms are associated with the occurrence of FVIII inhibitors in patients with severe hemophilia A suggests that the capacity of the patients to cope with bleeding-induced inflammation affects the development of the anti-FVIII immune response.

Whether the modulation of HO-1 levels dictates the development of FVIII inhibitors would require additional studies. protein immunogenicity. Advanced glycation endproducts (AGE) are generated upon the exposure of sugar residues to oxidants. Advanced-glycated proteins then acquire the capacity to bind the receptor for AGEs (RAGE) and display enhanced proinflammatory signaling activity as shown in the case of albumin [START_REF] Cohen | Glycated albumin increases oxidative stress, activates NF-kappa B and extracellular signal-regulated kinase (ERK), and stimulates ERK-dependent transforming growth factor-beta 1 production in macrophage RAW cells[END_REF]. The engagement of RAGE by AGE-modified proteins induces a hyper responsive state in monocytes and macrophages, resulting in an increased secretion of proinflammatory cytokines such as IL-12 and TNF-α, and stimulates the generation of reactive oxygen intermediates (ROIs) through the stimulation of the respiratory burst enzyme NAPDH oxidase [START_REF] Schmidt | RAGE: a novel cellular receptor for advanced glycation end products[END_REF]. Whether such adducts, originating from lipids or carbohydrates, are present on the FVIII molecule and whether they participate in triggering the anti-FVIII immune response remain unclear. The study of such events and their in vivo relevance is however complicated by the low amount of FVIII that is found in circulation, and the ensuing difficulties to detect oxidative alterations on FVIII in vivo. However, this hypothesis should not be ruled out immediately, owning to the fact that FVIII was found to associates with oxidized but not native LDL [START_REF] Kumagai | Inhibition of plasma coagulation through interaction between oxidized low-density lipoprotein and blood coagulation factor VIII[END_REF]. Because oxidized LDL have been described to trigger an anti-LDL immune response, it is tempting to speculate that oxidized LDL, in association with FVIII, may be considered as an immunogenic carrier for FVIII.

Focus on hypochlorous acid

Hypochlorous acid (HOCl) is generated by myeloperoxidase (MPO), a major constituent of the primary granules of neutrophils. HOCl reacts rapidly with proteins, DNA and lipids.

However, proteins are likely to be the major target for HOCl modification. Neutrophil recruitment and activation correspond to early events during inflammation. Upon activation, neutrophils release large amounts of MPO thus increasing the local concentration of ROS.

Recent finding on neutrophils have also uncovered several functions linking innate and adaptive immunity. Amongst them, it was demonstrated that following antigen endocytosis, neutrophils can be internalized by DCs, resulting in the presentation of the "neutrophilengulfed" antigen to T cells [START_REF] Alfaro | Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes[END_REF]. Thus, the antigens presented by DCs derive either from the inflammatory environment or from the endosomes of the neutrophils. These considerations suggest that, in both cases, the antigen has encountered a pro-oxidative environment either due to MPO released at the inflammatory site or during endosomal trafficking within the neutrophils. From an immunological point of view, the modification of proteins by HOCl was shown to affect their immunogenic properties. Thus, advanced oxidation protein products (AOPP), generated upon exposure of a model antigen, such as albumin, to HOCl, was found to activate neutrophils and monocytes [START_REF] Witko-Sarsat | AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: a potential target for N-acetylcysteine treatment in dialysis patients[END_REF], and to activate dendritic cells [START_REF] Alderman | The role of advanced oxidation protein products in regulation of dendritic cell function[END_REF]. In our study, the treatment of FVIII with HOCl did not induce the maturation of dendritic cells (Article 4). The low amount of FVIII incubated with the DCs, as compared to the situation with albumin, may explain our result. Alternatively, it is tempting to speculate that the presence of AOPP consecutive to chronic inflammation in the plasma of hemophilia A patients may pre-activate dendritic cells, thus increasing the risk for initiating an immune response to therapeutic FVIII. Apart from the direct activation of innate immune cells, the oxidative modification of antigens was found to increase their cellular uptake, processing and presentation. HOCl-modified ovalbumin was found to be endocytosed through the lectin-like oxidized low density lipoprotein receptor 1 (LOX-1), a multiligand receptor that also acts as a danger sensing molecule. Importantly, LOX-1 was initially described to bind oxidized but not native LDL. As told earlier, FVIII was found to associate with oxidized LDL. Thus, LOX-1 could mediate the "immunogenic" uptake of FVIII either through the direct oxidation of FVIII by HOCl or through the interaction of FVIII with oxidized LDL. In our experiment, we did not observed an increased uptake of HOCl-exposed FVIII by MO-DCs compared to untreated FVIII (data not shown).

Another consequence of oxidation of proteins resides in the susceptibility for proteolytic cleavage by endosomal enzymes such as cathepsins. Indeed, HOCl was described to enhance the intracellular processing and presentation of antigens. The presentation of peptides by antigen-presenting cells requires the cleavage of the endocytosed protein within the endosome. Within the endosome, the slow degradation of the protein allows the interaction of the peptides with MHC class-II molecules. Thus, macrophages have a higher proteolysis rate of antigens as compared to dendritic cells or B cells. The consequence of the fast proteolysis of an antigen is the limited presentation of T-cell epitopes, thus explaining why macrophages do not present antigens as efficiently as dendritic cells [START_REF] Delamarre | Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate[END_REF]. In line with these observations, oxidation of tetanus toxin was found to reduce its susceptibility to proteolysis, resulting in a higher presentation to specific T cells [START_REF] Pernollet | OH. treatment of tetanus toxin reduces its susceptibility to limited proteolysis with more efficient presentation to specific T cells[END_REF].

However, the impact of oxidative modifications of antigens in view of their immunogenicity is not clear. Thus, the type as well as the degree of oxidation modulates the fate of the antigen. Indeed, Weiskopf et al. reported that oxidation of antigenic peptides decreases the affinity of the TCR for its specific peptide, resulting in a decreased T-cell response [START_REF] Weiskopf | Oxidative stress can alter the antigenicity of immunodominant peptides[END_REF]. However, the latter work evaluated the oxidative modification of a peptide and not of a full protein. Our experimental data show that splenocytes incubated with HOCl-exposed FVIII have a higher proliferation index compared to that of splenocytes incubated with untreated FVIII (article 4). Although no increase in the uptake or maturation of DC was observed, these results suggest that upon processing of HOCl-exposed FVIII, the peptides that are generated are either presented in higher amounts or are individually more immunogenic owing to the presence of oxidative adducts.

  I. INTRODUCTION ............................................................................................................ I.1. Hemophilia A ................................................................................................................. I.1.1. Generalities .............................................................................................................. I.1.1.1. Historical aspects .............................................................................................. I.1.1.2. Phenotypic aspects ............................................................................................ I.1.1.3. Genetic aspects .................................................................................................. I.1.1.4 Bleeding complications in Hemophilia A .......................................................... I.1.2 The coagulation cascade ........................................................................................... I.1.2.1 Primary haemostasis .......................................................................................... I.1.2.2 Secondary haemostasis ...................................................................................... I.1.2.2.1 The extrinsic pathway ................................................................................. I.1.2.2.2 The intrinsic pathway .................................................................................. I.2.2.3 Pro-coagulant factor VIII ................................................................................... I.2.2.3.1 Synthesis, structure and post-traductional modifications ............................ I.2.2.3.2 Activation of FVIII ...................................................................................... I.2.2.3.3 Life cycle of FVIII, catabolic receptors, in vivo distribution, site of elimination ................................................................................................................. I.2.2.3.4 Catabolic receptors for FVIII ...................................................................... I.2.2.3.5 In vivo distribution of FVIII ........................................................................ I.1.3 Prevention or treatment of bleeding episodes in patients with hemophilia A .......... I.1.3.1 Factor VIII products ........................................................................................... I.1.3.2 Infusion regiment ............................................................................................... I.1.3.3 Complications of the treatment .......................................................................... I.1.3.4 Treatment of patients with FVIII inhibitors ....................................................... I.2 The anti-factor VIII immune response ............................................................................ I.2.1 FVIII as seen by the immune system in patients with hemophilia A ................... I.2.2 Antigen-presenting cells ....................................................................................... I.2.2.1 Dendritic cells ................................................................................................ I.2.2.2 Macrophages .................................................................................................. I.2.2.3 B cells ............................................................................................................. I.2.3 The spleen ............................................................................................................. I.2.4 The anti-FVIII immune response in hemophilia A patients ................................. I.2.4.1 T-cell activation .............................................................................................. I.2.4.2 B-cell activation ............................................................................................. I.3 Risk factors ...................................................................................................................... I.3.1 The danger signal theory .......................................................................................... I.3.1.1 Exogenous danger signals .................................................................................. I.3.1.2 Endogenous danger signals ................................................................................ I.3.2 Genetic risk factors ................................................................................................... I.3.2.1 FVIII mutations .................................................................................................. I.3.2.2 HLA haplotype ................................................................................................... I.3.2.3 Polymorphisms in immune-related genes .......................................................... I.3.3 Non-genetic risk factors ............................................................................................ I.3.3.1 Age at the start of treatment ............................................................................... I.3.3.2 Mode and intensity of FVIII administration ...................................................... I.3.3.3 State of the immune system at the time of FVIII infusion ................................. I.4 Purpose of the PhD work ................................................................................................ II. RESULTS ......................................................................................................................... II.1. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphism in the HMOX1 promoter ........................... II.2. Hemarthrosis and arthropathies do not favor the development of factor VIII inhibitors in hemophilia A mice ........................................................................................................... II.3. Restoration of the oxidative balance in factor VIII-deficient mice reduces the immunogenicity of therapeutic factor VIII ........................................................................... II.4. Oxidation of therapeutic factor VIII aggravates its immunogenicity in factor-VIII deficient mice ....................................................................................................................... III. DISCUSSION ............................................................................................................. IV. REFERENCES ............................................................................................................ V. ANNEXES ......................................................................................................................

FigureFigure 5 .

 5 Figure 1. Physiopathology of hemophilic arthropathy

Figure 6 .

 6 Figure 6. FVIII endocytosis and presentation on major histocompatibility

Figure 7 .

 7 Figure 7. CD4 + T-cell activation by APC.

FigureFigure 9 .

 9 Figure 8. B-cell activation.

Figure 2 .

 2 Figure 2. Primary hemostasis. Upon binding to the exposed endothelium, platelets are activated, leading to morphological changes and release of platelet chemoattractant mediators. At the site of endothelium injury, the aggregation of platelets results in the formation of the red thrombus which is consolidate by the fibrin generated during the secondary hemostasis.

Figure 3 .

 3 Figure3. Secondary hemostasis. Upon exposure of the sub-endothelial matrix proteins and ensuing platelet aggregation, extrinsic pathway starts with the expression of the pro-coagulant tissue factor (TF), which complexes and activates coagulation factor VII. The activated form of factor VII (FVIIa) in turn activates factor X (FXa), which associates with activated factor V, (FVa) to form a complex called the pro-thrombinase complex. The pro-thrombinase complex activates prothrombin (FII) into thrombin (FIIa). Eventually, the thrombin generated allows the polymerisation of fibrin monomers and activates factor XIII. Then, fibrin polymers are stabilised by the activated factor XIII giving rise to a stable fibrin clot, that act as a "net", helping the platelets to stabilize at the site of bleeding. The intrinsic pathway, initiates upon binding of factor XII to the negatively charged surface provided by the activated sub-endothelium. This binding results in the auto-activation of FXII to FXIIa. FXIIa induces the conversion of pre-kallikrein to kallikrein and factor XI to FXIa. FXIa in turn activates FIX to FIXa. FIXa then associates with activated FVIII (FVIIIa) and with platelet-derived phospholipids (PL). In the presence of calcium, this complex, called "tenase", allows the conversion of FX to FXa that sustains the downstream generation of thrombin initiated by the extrinsic pathway.

Figure 4 .

 4 Figure 4. FVIII structure. Following the translation of FVIII mRNA, the immature 2352 amino-acid form of FVIII is translocated to the endoplasmic reticulum. There, the proteolytic cleavage of the signal peptide reduces its size to 2332 amino-acids. After its translocation to the Golgi apparatus, a proteolytic cleavage at Arg1313 and Arg1648, lead to the formation of an heterodimer. The heavy chain comprises the domains A1-a1-A2-a2-B and the light chain the domains a3-A3-C1-C2. The two chains are linked through a divalent ion. Within the circulation, FVIII circulates in association with VWF.

Figure 5 .

 5 Figure 5. Activation of FVIII. Thrombin activates the FVIII molecule through a proteolytic cleavage of the heavy chain (residues Arg372 and Arg740) and the light chain (Arg1689). Activated FVIII adopts a heterotrimeric conformation and its affinity for VWF is reduced.

Figure 6 .

 6 Figure 6. FVIII endocytosis and presentation on major histocompatibility class II (MHC II) molecules.The FVIII molecule is endocytosed through receptor(s) present at the surface of the APC. After FVIII internalization in the endosomes, the FVIII-derived peptides associate with MHC II molecules and are addressed at the surface of the APCs.

Figure 7 .

 7 Figure 7. CD4 + T-cell activation by APC. Initially, the TCR recognizes the antigenic peptide in association with the MHC class II. The presence of a danger signal (danger associated molecular pattern, DAMP) leads to the expression of co-stimulation markers on the APC (CD80:86 and CD40). These markers engage the CD28 and CD40L expressed on T-cells. This second signal leads to the secretion of cytokines by the APC which finalizes the activation of the T cell.I.2.4.2 B-cell activation

Figure 8 .

 8 Figure 8. B-cell activation. After the internalization of a soluble antigen through their BCR, the B cells process and present the antigenic peptide on MHC II molecules. The recognition of the complex peptide/MHC II by an activated antigen-specific T cell leads to the maturation and differentiation of the B cell into memory B cells and plasmocytes. Plasmocytes are producing antigen-specific immunoglobulins.

Figure 9 .

 9 Figure9. HMOX1 promoter organization. The promoter region of the HO-1-encoding gene contains several transcription factor consensus binding sites, such as heat shock element (HSE), nuclear factor-κB (NFκB) and the activator protein-2 (AP-2). In addition to the (GT) n repeat dinucleotide length polymorphism (depicted in red), these additional transcription factor binding site possibly regulates the activity of the HMOX1 promoter.

  However, in up to 30% of patients with severe hemophilia A, an anti-FVIII immune response develops. The anti-FVIII immune response is characterised by the synthesis of inhibitory anti-FVIII antibodies. The anti-FVIII immune response follows a classical development. Thus, the development of the anti-FVIII immune response requires the endocytosis of FVIII by professional antigen-presenting cells (APC) and the presentation of FVIII-derived peptides in association with the class-II major histocompatibility complex (MHC class-II) to naïve FVIIIspecific T cells. However, the complete activation of T cells requires the presence of danger signals. Although several risk factors have been proposed to favour the development of the anti-FVIII immune response, the origin of the danger signal remains mysterious. Bleeding severity, surgery or vaccination are some of the risk factors that have been proposed to increase the risk for FVIII inhibitor development. However, no scientific evidence has convincingly demonstrated their participation in the initiation of the anti-FVIII immune response.

Table 1 . F8 gene mutations and risk for inhibitor development.

 1

Table 2 . Examples of alarmins.

 2

Table 1 .

 1 F8 gene mutations and risk for inhibitor development

	F8 gene mutation	Risk for inhibitor development (%)
	Large deletions	68
	Nonsense mutations on FVIII light chain	50
	Intron 22 inversion	35
	Small insertions or deletions	26
	Nonsense mutation outside of FVIII light chain	14
	Large deletions involving only one exon	12
	False-sense mutation on FVIII light chain	12
	False-sense mutation outside of FVIII light chain	4
	Splicing anomalies	2
		Table adapted from the 2006 AFSSAPS report

Table 2 . Examples of alarmins

 2

	Alarmin	Receptor	Effect upon receptor ligation
	Nucleotides (ATP, ADP) PI, P2X and P2Y receptors	Dendritic cell (DC) maturation,
			chemotaxis, secretion of
			cytokines (IL-1ß, IL-18)
	Heat shock proteins	CD14, CD91, scavenger	DC maturation, cytokine
		receptors, TLR4, TLR2, CD40	induction, DC migration to
			lymph nodes
	HMGB1	RAGE, TLR2, TLR4	Chemotaxis, cytokine
			induction, DC activation,
			neutrophil recruitment,
			activation of immune cells
	Oxidative stress	Intracellular redox-sensitive	Cell death, release of
		proteins	endogenous DAMPs
	Laminin	Integrins	Neutrophil recruitment,
			chemotaxis
	S100 proteins	RAGE	Neutrophil recruitment,
			chemotaxis, cytokine secretion,
			apoptosis
	Hyaluronan	TLR2, TLR4, CD44	DC maturation, cytokine
			production, adjuvant activity
		Adapted from

Apart from HMGB1, many other mediators have been described to affect the development of the immune response and are listed in table 2. In an attempt to evaluate the participation of bleedings in the initiation of the anti-FVIII immune response, we implemented a model of knee hemarthrosis that is induced by puncture of the knee joint capsule. In contrast to our initial hypothesis, our data demonstrate that acute joint bleeding in hemophilic mice is not associated with an increased immune response to therapeutic FVIII (article 2). However, the mouse model of FVIII-deficiency might not be suitable for the assessment of the adjuvant effect of bleedings on the immune response to FVIII. Indeed, upon administration of FVIII, FVIII-deficient mice systematically mount an anti-FVIII immune response, which is not the case in the human where 70% of patients with severe hemophilia A do not develop FVIII inhibitors. It would be interesting to evaluate the impact of bleedings in the model of FVIIIdeficient mice that are tolerant against human FVIII [START_REF] Van Helden | Maintenance and break of immune tolerance against human factor VIII in a new transgenic hemophilic mouse model[END_REF]. Using this mouse model would permit to evaluate the triggering effect of hemorrhages on the initiation of the anti-FVIII immune response. In other words, it would clarify whether bleedings are sufficient enough to overcome/break a pre-existing tolerance to FVIII.

Focus on the effects of heme

Heme is used in many proteins as a prosthetic group. It participates in redox reactions or is used for electron and gas transport. Heme also participates in cell signalling and regulates the expression of several genes. However, owing to its redox properties, the heme molecule is potentially harmful. Thus, under physiological conditions, heme is confined inside the cells where it is in complex with several hemoproteins, such as hemoglobin or cytochromes. In the case of cellular damage, heme may be released in the circulation. Within erythrocytes, heme is found in large quantities bound to hemoglobin where it ensures the transport of oxygen.

However, in the case of bleeding, erythrocytes are lysed and release their hemoglobin content Different animal models should be developed in order to better understand the interaction between the strength of HO-1 induction and the development of the anti-FVIII immune response. For instance, the use of conditional knock-out models for the HO-1 gene, using the Cre-LoxP system, on a FVIII-deficient background, would allow the inactivation of HO-1 without putting the survival of the mice at stake [START_REF] Poss | Heme oxygenase 1 is required for mammalian iron reutilization[END_REF]. Alternatively, transgenic FVIII-deficient mouse models having different susceptibilities to the induction of HO-1 upon heme exposure would allow a direct comparison of the immunogenicity of FVIII in the presence of strong or weak ant-inflammatory signals.

Focus on the effect of ROS.

As told earlier, the presence of vascular damages is associated with pro-inflammatory events that trigger the generation of ROS. Apart from their direct effect on immune effector cells, ROS are capable of oxidizing proteins. Protein oxidation was described to alter protein stability, enzymatic activity and modify protein immunogenicity. The oxidative modification of surface moieties leads to the generation of neo-epitopes, either through the modification of preexisting residues (carbohydrates or amino-acid side chains), or through the addition of oxidation by-products. Thus, the fragmentation of lipid hydroperoxides leads to the generation of reactive aldehydes, such as malondialedehyde (MDA) or 4-hydroxynonenal (HNE). These two products were shown to form covalent adducts on different amino acid side chains. Such adducts on proteins are targets for the B-cell immune response, leading to the generation of antibodies against the modified antigen. Moreover, introduction of such adducts may disturb the established tolerance to self-protein and potentially induce autoimmune disease. In the case of FVIII, it is possible that the addition of immunogenic adducts to the therapeutic protein participates in the development of the anti-FVIII immune response. Oxidative modification of sugar residues has also been described to generate new particles that modify

Concluding remarks

Despite the higher immunogenicity that we observed in mice upon exposure of FVIII to HOCl in vitro (article 4), the underlying mechanism still remains unknown. Further work will be conducted to decipher the role of antigen-processing enzymes (such as cathepsins) and their effect on the generation of immunogenic peptides. Moreover, the in vivo determination of FVIII modifications by oxidants is still lacking and is complicated by the low amount of FVIII found in the circulation. Answering this question will require the development of new tools. My work highlights the complexity of the physio-pathological response to bleedings in the case of FVIII deficiency, and its consequences on the anti-FVIII immune response.

Although several pro-inflammatory mediators are released upon bleedings, and potentially adjuvant the anti-FVIII immune response, the restoration of homeostasis triggers several systems, such as HO-1, that down modulate immune responses. Deciphering the participation of each of these systems and their variability in humans may represent a novel strategy to explain the development of the anti-FVIII immune response that is found in 30% of patients with severe hemophilia A.