Inflammation in the central nervous system leads to demyelination and neurodegeneration in patients with multiple sclerosis (MS). While the etiology of the disease is not known, genetic studies have highlighted 110 genetic variants responsible for MS susceptibility.

The processes responsible for the spectrum of disease severity are not well understood.

However, the severity of MS is closely correlated with the efficacy of myelin repair, or remyelination, a process whose effectiveness is very heterogeneous among patients. Why remyelination fails or succeeds in patients remains largely unknown, mainly because this process has never been addressed in a humanized pathological context.

To tackle this question, we developed a new model combining focal demyelination in nude mice and graft of MS or healthy donor (HD) lymphocytes (LT). We were able to reproduce at least partially remyelination heterogeneity, with some patients LT inducing a defect in remyelination and some others inducing a myelin repair comparable to HD. Comparing the secretory profile of patient LT according to their repair capacities, we highlighted an heterogeneous cytokine response to the same stimulus, leading to a differential microglial activation and ultimately an efficient or inefficient differentiation of precursor cells during remyelination.

To decipher why LT from patients have differential abilities to respond to a stimulus, we hypothesized that the genetic variants known for MS susceptibility could also drive disease severity by influencing LT functions and therefore remyelination. We found that the interaction of variants associated with genes responsible for T Folicular Helper and naïve Th0 cells functions induce the secretion by patient LT of a cytokine profile leading to a proinflammatory activation in microglia, leading to remyelination failure.

Overall, we discovered a new role for LT in remyelination: they orchestrate MIG activation and are therefore at least partially responsible for the success or failure of the myelin repair process. Capitalizing on patients with high repair capacities to understand the cellular and molecular actors leading to successful remyelination in pathological conditions seem to be a key approach to develop therapeutical targets.  APC: Antigen presenting cell  Arg-1: Arginase 1  BBB: Blood brain barrier  CC1: Adenomatous polyposis coli protein clone antibody CC1  CCL: C-C motif chemokine ligand  CCR: C-C motif chemokine receptor  CD: Cluster of Differentiation  CNPase: 2',3'-Cyclic-nucleotide 3'phosphodiesterase

Résumé

Les processus inflammatoires médiés par le système immunitaire induisent une demyélinisation et une mort neuronale dans le système nerveux central des patients atteints de sclérose en plaques (SEP). L'étiologie exacte de la maladie est inconnue, même si la connaissance de sa composante génétique a été enrichie par la découverte de 110 variants responsable de sa prédisposition.

Les évènements responsables de l'hétérogénéité de sévérité de la SEP chez les patients sont encore mal compris. Néanmoins, la capacité de remyélinisation (un procédé endogène durant lequel la myéline détruite est reformée), elle aussi très hétérogène chez les patients, est intimement corrélée à la sévérité des symptômes. Les raisons des différences d'efficacités de remyélinisation chez les patients ne sont pas connues, principalement car cette hétérogénéité n'a jamais été étudié dans un contexte expérimental approprié.

Pour mieux comprendre ce phénomène, nous avons développé un nouveau modèle murin humanisé en combinant une démyélinisation focale chez des souris nude à une greffe de lymphocytes (LT) provenant de donneurs sains (DS) ou de patients SEP. Nos résultats ont démontré que cette greffe permet de reproduire au moins partiellement l'hétérogénéité de remyélinisation présente chez les patients : Certains LT de patients induisaient un défaut de remyélinisation, pendant que d'autres induisaient une réparation de la myéline comparable à celle induite par les LT de DS. En comparant le profil de sécrétion des patients selon leur capacité à induire un contexte favorable à la remyélinisation, nous avons mis en évidence des différences dans la sécrétion de certaines cytokines, ce qui induisaient selon le cas une activation bénéfique ou délétère des cellules microgliales ayant pour conséquence une différentiation efficace ou entravée des cellules précurseurs d'oligodendrocytes pendant la remyélinisation.

Pour comprendre pourquoi les LT de patients répondaient de façon différente à une même stimulation, nous avons émis l'hypothèque qu'une interaction entre les variants génétiques de prédisposition à la SEP pouvait également modifier la fonctionnalité des LT pendant la remyélinisation. Nous avons mis en évidence que les LT de patients ayant des variants associés à des gènes impliqués dans la fonction des lymphocytes T CD4+ folliculaires (TFH) et des lymphocytes T naïfs (Th0) induisaient un défaut de remyélinisation lorsque greffés dans une lésion démyélinisée, et ce en dirigeant l'activation microgliale vers un phénotype délétère.

En résumé, nous avons mis en évidence un nouveau rôle pour les lymphocytes lors de la remyélinisation : ils influencent l'activation microgliale et sont donc au moins partiellement responsables de l'échec ou de la réussite de la réparation de myéline chez les patients. La

I.

Multiple sclerosis: Etiology and treatments

Foreword

Multiple sclerosis (MS) is an auto-immune disease leading to demyelination and neurodegeneration in the central nervous system (CNS). In this pathology, an abnormal immune response initiated by lymphocytes (LT) [START_REF] Compston | Multiple sclerosis[END_REF][START_REF] Dendrou | Immunopathology of multiple sclerosis[END_REF] is leading to a chain of events resulting in an invasion of the CNS by both innate and adaptive immune cells, causing neuroinflammation. The inflammatory attacks induce myelin destruction and oligodendrocyte (OL) death. Then, demyelinated axons degenerate causing a heterogeneous spectrum of symptoms in MS patients. The cause of disease onset is still not understood, but MS appears in patients with a genetic predisposition and who are exposed to environmental factors contributing to the triggering of the abnormal immune response.

Myelin

A. Structure and physiological myelination

Myelin structure and molecular composition

Myelin is a lipid-rich substance wrapping the axons of neurons. Myelin's structure results from the wrapping of successive layers of plasma membrane of myelinating cells (Figure 1). Myelin is composed by 70% of lipids and 30 % of proteins. In the CNS, myelin is formed by OL. In the peripheral nervous system (PNS), myelin is formed by Schwann Cells (SC). The PNS and the CNS myelin are fairly similar, up to a few exceptions in their protein composition [START_REF] Aggarwal | Central nervous system myelin: Structure, synthesis and assembly[END_REF][START_REF] Kursula | Structural properties of proteins specific to the myelin sheath[END_REF]. Therefore, specific markers exist to discriminate between the two kind of myelin: the Myelin Oligodendrocyte Glycoprotein (MOG) only expressed in the CNS myelin, whereas the Protein 0 (P0) is exclusively expressed in the PNS. Structurally, one SC only wraps one axon, whereas one OL can form myelin on up to 60 segments of axons. Myelin along the axon is not a continuous structure: it forms segments, called internodes, which have an average length of 1µm, separated by a structure called node of Ranvier where the axon is not myelinated. In the node of Ranvier, in the neuron plasma membrane, a high number of sodic and calcic voltage dependent channels are concentrated (Figure 3).

Chapter I : Introduction

Oligodendrocyte and Schwann cells differentiation during myelination

During the myelination process occurring during development, myelinating OL are derived from the differentiation of oligodendrocytes precursor cells (OPCs). Several waves of OPC migration from ventral and dorsal domain occur during embryonic life. After migration, OPCs differentiate into mature myelin forming OL [START_REF] Bercury | Dynamics and mechanisms of CNS myelination[END_REF]. During the differentiation process, cells of the oligodendroglial lineage go through different stages that can be characterized by specific markers. For instance, OPCs express A2B5 and platelet derived receptor-α (PDGFRα), Pre-OL express O4, mature OL express galactocerebroside (GalC) and adenomatous polyposis coli clone CC1 (CC1) and myelin producing OL express myelin basic protein (MBP), 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) and myelin Modified from [START_REF] Poliak | The local differentiation of myelinated axons at nodes of Ranvier[END_REF] Chapter I : Introduction oligodendrocyte glycoprotein (MOG). The immunostaining against those proteins allow the characterization of the progress of the differentiation process (Figure 2A).

SC arise from neural crest, a multipotent cell population formed in the dorsal part of the neural tube. SC precursors are formed after specification of a subpopulation of neural crest cells when they encounter and contact axons [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF]. Then, SC become immature and wrap (without forming myelin) several axons. They can then form myelinating or non-myelinating SC, in part depending on the diameter of the axon they contact (Figure 2B).

).

Figure 2: Representation of the OL and SC lineage progression during differentiation. The associated specific markers for each stages are indicated between brackets. Modified from [START_REF] Jessen | The origin and development of glial cells in peripheral nerves[END_REF][START_REF] Nishiyama | Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity[END_REF].

Chapter I : Introduction

B. Function

Fast axonal conduction

During evolution, organisms grew in size. As a result, axonal conduction speed from the CNS to the extremities of the body was one of the requirements for a fast circulation of the information in the body [START_REF] Zalc | The origin of the myelination program in vertebrates[END_REF]. Myelin allowed an acceleration of the speed of conduction up to 100 times faster by two mechanisms: first, its fat-enriched composition acts like a natural insulator, reducing the capacitance of the axon membrane and therefore accelerating axonal conduction (Figure 3A). Second, the myelin is clustering the voltagedependant channels at the Node of Ranvier leading to so called saltatory conduction: the axonal influx is going to "jump" from one Node of Ranvier to another, inducing the propagation of the depolarization only in this structure (Figure 3B) [START_REF] Hartline | Rapid Conduction and the Evolution of Giant Axons and Myelinated Fibers[END_REF]. This way of transmitting depolarization is considerably faster than if the electric current had to pass from one channel to another all along the axon.

Metabolic support and protection of axons

The myelin sheath wrapping the axon also has other, more recently discovered, roles [START_REF] Fünfschilling | Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity[END_REF][START_REF] Simons | Oligodendrocytes: Myelination and axonal support[END_REF]. OL provide a metabolic support to neurons by transforming glucose into lactate and pyruvate. These metabolites can be transferred from the OL to the neuron cytoplasm, and used as a source of energy by the neuron. In CNPase 1 knock-out model (in which the gene is inactivated), the myelination occurs but its structure is abnormal [START_REF] Rasband | CNP is required for maintenance of axon-glia interactions at nodes of ranvier in the CNS[END_REF]. This hinders the metabolite exchange between OL and neurons, and leads to axonal transport defects in neurons resulting in an early death of animal due to neuroinflammation and neurodegeneration. In addition, it has been demonstrated that OL can secrete neuronal pro survival factors such as insulin growth factor-1 (IGF-1) and neurotrophins [START_REF] Byravan | Murine oligodendroglial cells express nerve growth factor[END_REF][START_REF] Dai | Neuronal signals regulate neurotrophin expression in oligodendrocytes of the basal forebrain[END_REF][START_REF] Dai | The trophic role of oligodendrocytes in the basal forebrain[END_REF][START_REF] Wilkins | A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons[END_REF] . Finally, myelin represents a physical barrier between the axon and the extracellular domain, protecting it from inflammatory stimuli occurring during neuroinflammation.

In summary, myelin is a multi-functional and an indispensable element of the healthy CNS. Chapter I : Introduction

Etiology

A. MS: a complex disease

Diseases can be caused by environmental factors such as viruses, microbes, parasites or toxins but can also be purely genetic, in which the disease is the consequence of a DNA mutation and in which the environment has no effect on disease triggering. The simplest form of genetic diseases is monogenic pathologies: they result from a mutation in a single gene. This mutation triggers an impairment of function in the protein coded by the gene leading to the disease.

These mutations are rare, and the disease is hereditary according to Mendel's law. MS is a complex disease, in which both genetic and environmental factors are involved. It occurs in patients carrying predisposition variants and exposed to environmental factors increasing the odds of disease onset. Each of the variants is frequent in the general population and is neither sufficient nor necessary to trigger the disease.

B. Genetic predisposition

Evidence of a genetic component in MS

The simplest and definitive evidence that MS has a genetic component comes from studies of families in which there is an MS patient. MS has a familial recurrence rate of about 20% (20 [START_REF] Compston | Multiple sclerosis[END_REF] Chapter I : Introduction % of MS patients have at least one affected relative). The risk for a monozygote twin to develop the disease when its twin is affected is around 30%, as compared to 5% when the twins are dizygote (Figure 4) [START_REF] Compston | Multiple sclerosis[END_REF][START_REF] Hansen | Risk for multiple sclerosis in dizygotic and monozygotic twins[END_REF], demonstrating a genetic involvement in the probability to develop MS. However, as stated above, the genetic causes of MS explain only a part of the susceptibility.

HLA genes and MS

Human leukocyte antigen (HLA) genes are involved in the Human Major Histocompatibility

Complex (MHC) located on chromosome 6. This zone of the genome is highly polymorphic, and HLA genes can be divided into two majors groups: class I HLA and class II HLA. They both encode for cell-surface glycoproteins [START_REF] Strominger | Biology of the Human Histocompatibility Leukocyte Antigen (HLA) System and a Hypothesis Regarding the Generation of Autoimmune Diseases[END_REF]. Class I HLA molecules are expressed by almost all cell types and they will by default present a subset of peptides that have been degraded in the cytoplasm. If a cell presents an exogenous peptide on its class I HLA (for instance, in case of intracellular infection), it will be recognized and the cell will be killed by CD8+ T cells. Class II HLA molecules are only expressed by antigen presenting cells (APCs), which phagocytose and present peptide debris on the surface of the glycoproteins. This can be recognized by CD4+ T cells, triggering the adaptive immune response if the peptide is exogenous. HLA molecules are in this way implicated in immune surveillance and tolerance.

The presence of the allele HLA DRB1*1501 (of the HLA class II) has been known to be a risk for developing MS since the 1970s. The risk to develop MS in individuals homozygous for HLA DRB1*1501 is around 3 times higher compared to someone not carrying the risk allele. This is the genetic factor with the largest impact on the risk to develop MS. In nearly all studies of genetic predisposition, the frequency of this allele was higher in the MS population compared to the healthy controls. Other variants of HLA molecules are known to be either a risk factor (HLA DRB1*03, DRB1*08:01) or a protective factor (HLA DRB1*14:01) [START_REF] Hollenbach | The immunogenetics of multiple sclerosis: A comprehensive review[END_REF].

GWAS and Immunochip: a revolution in the genetics of MS

Until a few years ago, little progress had been made in the understanding of MS genetics. Only a few variants were discovered in addition to the HLA related ones, related to IL7R and IL2RA

genes [START_REF] Gregory | Interleukin 7 receptor α chain (IL7R) shows allelic and functional association with multiple sclerosis[END_REF][START_REF] Munoz-Culla | The genetics of multiple sclerosis: review of current and emerging Bibliography candidates[END_REF]. The real revolution happened when genome wide association studies (GWAS) were realized. GWAS is a method screening the genome for single nucleotide polymorphisms (SNP) and evaluating their association with 5) . The vast majority of the SNPs are closely associated with genes having a role in immune pathways [START_REF] Sawcer | Multiple sclerosis genetics[END_REF].

Linking genotypes to MS susceptibility and severity

After the discovery of several susceptibility variants for MS, attempts were made to link the genotype of patients with their phenotype to predict disease course and severity. For instance, patients carrying the (HLA) DRB1*1501 allele show cognitive impairments due to more important neuronal degeneration [START_REF] Okuda | Genotype-phenotype correlations in multiple sclerosis: HLA genes influence disease severity inferred by 1HMR spectroscopy and MRI measures[END_REF]. A recently discovered polymorphism in the oligoadenylate synthetase 1 gene is linked to increased disease activity and relapse frequency in patients carrying the risk allele [START_REF] O'brien | OAS1: A multiple sclerosis susceptibility gene that influences disease severity[END_REF].

Several other polymorphisms have well established consequences on LT functions: A loss of function on regulatory anti-inflammatory processes is involved in MS susceptibility. For instance, Regulatory T cells (Treg) of patients carrying the risk allele of the CD226 gene showed reduced immunosuppressive capacity and therefore could contribute to a decrease of the peripheral tolerance leading to the survival and the proliferation of autoreactive T cells [START_REF] Piédavent-Salomon | Multiple sclerosis associated genetic variants of CD226 impair regulatory T cell function[END_REF]. Mice carrying the risk allele also had a loss of function of Treg cells leading to an exacerbated disability score when Experimental autoimmune encephalomyelinitis (EAE), an animal model of MS, was induced. A gain of function of proinflammatory processes is also responsible for MS onset: One of the variants associated to the SLC9A9 gene led to a reduced expression of its mRNA in MS patient carrying this risk allele and this reduction induced an increased expression of IFN-γ by T-cells [START_REF] Esposito | A pharmacogenetic study implicates SLC9a9 in multiple sclerosis disease activity[END_REF].

Mechanistically, a reduced expression of SLC9A9 favors differentiation into T helper 1 (Th1) Interferon-γ (IFN-γ) secreting cells among CD4 + cells. Th1 cells are one of the pathogenic cell type in MS, and it is therefore likely that this variant can play a role in disease onset.

Interestingly, MS predisposition polymorphisms can also drive disease severity by influencing the myelin repair process: remyelination. In a murine model of demyelination/remyelination, a polymorphism in the epidermal growth factor gene can severely impede the myelin repair process [START_REF] Bieber | Allelic variation in the Tyk2 and EGF genes as potential genetic determinants of CNS repair[END_REF]. Combined, these data indicate that the SNPs carried by patients not only predispose them to MS, but can also drive the disease evolution by worsening inflammatory attacks or preventing myelin repair. However, the genotype of patients is not yet routinely used in the clinic, and further investigation is needed to predict even partially disease evolution and severity using genomic data.

Epigenetic component of MS

Several elements argue in favor of an epigenetic component in MS: the established interaction between genes and environmental factors (smoking with HLA-DRB1*15:01 for instance [START_REF] Olsson | Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis[END_REF]) and the fact that the loci discovered for MS susceptibility only explain half of the genetic predisposition risk for MS [START_REF] Zheleznyakova | Epigenetic research in Multiple Sclerosis: progress, challenges and opportunities[END_REF]. But perhaps the most convincing evidence is the study of monozygotic twins. Despite identical genetic background, the risk of the twin of an MS patient developing MS themselves is only 30%.

Therefore, another mechanism of gene expression regulation are likely to be involved and epigenetic is the most likely hypothesis [START_REF] Xiang | The epigenetic mechanism for discordance of autoimmunity in monozygotic twins[END_REF].

To highlight an epigenetic effect on MS susceptibility, the epigenome of twins discordant for MS have been studied. However, no differences were found in the methylation of CpG islands (the most studied epigenetic trait) of 18000 genes [START_REF] Baranzini | Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis[END_REF]. However, this negative result does not exclude the epigenetic hypothesis, as several other epigenetic mechanisms exist (e.g. acetylation of histones and non-coding RNA) and these have not been studied in detail in MS. Furthermore, recent evidence argues in favor of a critical role of methylation in MHC related genes in patients with the relapsing form of MS [START_REF] Maltby | Genomewide DNA methylation profiling of CD8+ T cells shows a distinct epigenetic signature to CD4+ T cells in multiple sclerosis patients[END_REF][START_REF] Maltby | Differential methylation at MHC in CD4 + T cells is associated with multiple sclerosis independently of HLA-DRB1[END_REF].

Multiple other putative epigenetic mechanisms have been proposed to be responsible for MS susceptibility and severity [START_REF] Küçükali | Epigenetics of multiple sclerosis: an updated review[END_REF], e.g. miRNA inducing a defect in phagocytosis, methylation of anti-inflammatory genes (i.e FoxP3) or acetylation of genes in the Th17 pathway, but further studies are needed to validate these hypothesis.

C. Environmental triggers

As previously stated, MS is a complex disease: the disease is triggered in individuals with a genetic predisposition who are exposed to environmental risk factors. Several of these risk factors are known.

North-south gradient of MS prevalence and vitamin D

There is a North-South gradient of the prevalence of MS in the world (Figure 6). Likewise, there is a strong inverse correlation between ultra violet radiation (UV) exposure and risk for MS. In other words, it is likely sun exposure decreases the risk of developing MS.

Vitamin D and its active derivative cholecalciferol and ergocalciferol, has a well described role in calcium metabolism and notably in skeleton remodeling. The main source of Vitamin D in humans is the skin which synthesizes it after exposure to UV. Therefore, there is also a North-South gradient of blood levels of Vitamin D in the world. These phenomena are only correlative and not demonstrated to be causative, but there is an accumulation of clues in the direction of low vitamin D levels as a susceptibility factor for MS [START_REF] Ascherio | Vitamin D and multiple sclerosis[END_REF][START_REF] Lucas | Ultraviolet radiation, vitamin D and multiple sclerosis[END_REF]: Retrospective studies show that in average MS patients had lower vitamin D level in the blood before the disease onset than the general population and people that follow a vitamin D treatment have lower risk of developing MS [START_REF] Duan | Vitamin D status and the risk of multiple sclerosis: A systematic review and meta-analysis[END_REF][START_REF] Martinelli | Vitamin D levels and risk of multiple sclerosis in patients with clinically isolated syndromes[END_REF]. Finally, migration studies show that individuals who have moved from their country of origin to a more southern country have a lower risk of MS [START_REF] Gale | Migrant studies in multiple sclerosis[END_REF].

Vitamin D has potent immunomodulatory effects that could explain its protective role for MS [START_REF] Ascherio | Vitamin D and multiple sclerosis[END_REF][START_REF] Koch | Environmental factors and their regulation of immunity in multiple sclerosis[END_REF][START_REF] Prietl | Vitamin D and immune function[END_REF]. Notably, vitamin D has been shown

to increase suppressive properties of Tregs, induce tolerogenic antigen presenting cells, reduce the invasion of macrophages in the CNS during EAE, and foster Th cell differentiation towards the Th2 phenotype which has immunomodulatory properties. In addition, vitamin D levels are lower in MS patients, and there is a correlation between low level of Vitamin D and severity of the disease. However, while vitamin D treatment ameliorated the wellbeing of patients, it did not show any promising effect on disease severity or frequency of relapses.

Other factors could contribute to the north-south gradient of MS prevalence, such as viral infections and alimentary habits. These putative causes are detailed below.

Viral infection

Infection with Herpes virus and Epstein-Barr virus (EBV) increase the risk to develop MS. The link between EBV infection and MS is well established from epidemiological studies, with an approximate 2-fold increase of the risk of developing MS in patients that developed mononucleosis during their lifetime.

The mechanism by which being infected by EBV increases the risk to develop MS is not known.

The most intuitive hypothesis is molecular mimicry: if one of the antigens of the virus is very close in its molecular structure from a protein of the myelin, it could trigger the generation of Myelin-reactive T cells [START_REF] Koch | Environmental factors and their regulation of immunity in multiple sclerosis[END_REF]. T cells of MS patients present a cross reactivity between the myelin protein MBP and some of the EBV antigens. Once the virus is eliminated from the system, the inflammatory attack could still occur against myelin that is recognized as Modified from oysterhc.com similar to the pathogen. Other hypotheses could also explain the increased risk of MS induced by EBV infection, such as that myelin specific T cells could be activated by a generic inflammatory context induced by the virus, or that the B cells in the meninges, which are often affected by EBV, would secrete antibodies against EBV antigens that cross react with myelin protein, inducing myelin destruction.

Other environmental factors

Smoking has more recently been recognized as a factor leading to an increased risk of developing MS and is associated with a greater disease activity and higher disability [START_REF] Healy | Smoking and disease progression in multiple sclerosis[END_REF][START_REF] Manouchehrinia | Tobacco smoking and disability progression in multiple sclerosis: United Kingdom cohort study[END_REF] . There is a dose effect of the number of cigarette smoked daily on the risk for MS [START_REF] Hernán | Cigarette smoking and incidence of multiple sclerosis[END_REF] but the mechanism behind this risk increase remains unclear even if some putative mechanism have been proposed. In the lung of smokers MS patients, there is an increase of T cell proliferation and an increased number of macrophages. This global immune response could be partially responsible for triggering MS [START_REF] Ockinger | T-cell activation and HLA-regulated response to smoking in the deep airways of patients with multiple sclerosis[END_REF]. A salt-enriched diet is also a risk factor for MS, as high dose of sodium chloride leads to a more severe phenotype in EAE. Sodium chloride induces a pathogenic Th17 polarization in CD4 + cells and inhibit Treg functions [START_REF] Hernandez | Sodium chloride inhibits the suppressive function of FOXP3+ regulatory T cells[END_REF][START_REF] Kleinewietfeld | Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells[END_REF].

Other factors could be implicated, such as a fat-enriched diet, alcohol and coffee consumption or night work. However, the experimental proofs are insufficient (due to low numbers of cases or unreliability of the measures) and no causative proofs are established.

Microbiota

The gut-associated lymphoïd tissue (GALT) represents around 80% of the total body's immune system. Every day, because of food consumption, foreign components are constantly passing through the gut and therefore a strong immune tolerance must be put in place to avoid constant immune activation against alimentary products. Bacteria of the gut, or gut microbiota, takes advantage of this immune tolerance to colonize the intestinal tract. Because of the strong interaction between the immune system and the microbiota, a dysfunction of the immune tolerance induced by the microbiota is likely to promote the triggering of autoimmune diseases [START_REF] Colpitts | Influence of the Gut Microbiome on Autoimmunity in the Central Nervous System[END_REF].

The immune tolerance in the gut is a perpetually moving equilibrium between the induction of pro-inflammatory Th17 cells, essential for our defense against bacterial and fungal pathogens, and Tregs, critical for peripheral tolerance and inflammation resolution. Both of these cell types are numerous in the gut, and bacterial exposure is critical for their maturation [START_REF] Kamada | Role of the Gut Microbiota in the Development and Function of Lymphoid Cells[END_REF]. As a result, germ-free mice (mice bred in sterile conditions) that do not have a microbiota are protected against EAE because of the drastic reduction of pathogenic Th17 cells [START_REF] Lee | Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis[END_REF]. The equilibrium in the gut can be broken by a pathogenic population of bacteria, or a non-pathogenic bacteria inducing a strong immune reaction. If this reaction appears in genetically predisposed patients, it could lead to the appearance of MS. In the last few years, a lot of studies have been performed to evaluate if the microbiota composition is different in MS patients. It was found that the diversity of the microbiota is reduced in a subpopulation of MS patients, and some bacteria are found enriched in these patients compared to healthy controls [START_REF] Chen | Multiple sclerosis patients have a distinct gut microbiota compared to healthy controls[END_REF][START_REF] Lee | Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis[END_REF].

Further studies are necessary to understand how the microbiota influences the disease triggering and severity.

Clinical description & existing treatments

A. Epidemiology and social cost MS affects more than 2.4 million people worldwide. The average incidence is about 100/100 000 per year (0.1%) in North America and Europe, and of 2/100 000 (0.002%) in Eastern Asia and Africa. This disease is therefore not classified as a rare disease in the western world. The disease affects more often women than men (sex ratio of 2.7 women per 1 man in 2014 in

France [START_REF] Foulon | Prevalence and mortality of patients with multiple sclerosis in France in 2012 : a study based on French health insurance data[END_REF]), the underlying cause remaining purely speculative.

It is the second cause of disability in young adults in Europe [START_REF] Compston | Multiple sclerosis[END_REF]. Even if worldwide data on the mean age of disease onset are difficult to get (due to difference of diagnosis criteria, lack of public available data and other causes) the estimated average age at disease onset is around 30. Because of its occurrence in early adult life and its disabling symptoms, unemployment rate is high in patients. In addition to that, frequent hospitalization and frequent life-long treatment make the societal cost high in MS: 50 k€ per year and per patient in average in Europe [START_REF] Kobelt | Costs and quality of life in multiple sclerosis in Europe: method of assessment and analysis[END_REF].

B. Symptoms and diagnosis

MS lesions can occur anywhere in the CNS and the symptoms of patients will depend on the function of the neurons affected by demyelination and neurodegeneration. Therefore MS patients can experience a large spectrum of symptoms (Figure 7), from loss of vision to cognitive impairment. The diagnosis of MS is certain when two lesions in the white matter, separated in time (>1month) and in space appears in the CNS. Usually, the lesions are visualized by magnetic resonance imaging (MRI) [START_REF] Compston | Multiple sclerosis[END_REF]. Other diagnosis tools are available to the clinicians such as the high concentrations of IgG antibodies and/or oligoclonal bands in the cerebrospinal fluid (CSF), or measurement of evoked potential (visual, brainstem, sensory) which can be slowed down in case of demyelination [START_REF] Compston | Multiple sclerosis[END_REF]. In the clinical routine, disease evolution and severity is calculated only taking the walking difficulty into account (Expanded Disability Status Scale or EDSS), or the EDSS weighted by disease duration (Multiple Sclerosis Severity Score or MSSS).

C. MS clinical forms

The pathology of MS includes three distinct clinical forms [START_REF] Loma | Multiple Sclerosis: Pathogenesis and Treatment[END_REF]. The most common one, affecting 70-85% of patients is the Relapsing-Remitting form (RRMS), characterized by acute relapses that can last days or weeks, during which patients experience a sudden worsening of symptoms, interspersed by remission phases that can last for several months (Figure 8A). During remission, RRMS patients partially or totally recover the neurological functions that were affected during relapses. Twenty years after diagnosis, 80-90% of RRMS patients have developed the Secondary Progressive form (SPMS) [START_REF] Scalfari | The natural history of multiple sclerosis, a geographically based study 10: Relapses and long-term disability[END_REF][START_REF] Trojano | The transition from relapsing-remitting MS to irreversible disability: Clinical evaluation[END_REF], in which, after several round of relapses and remission, deterioration of neurological function is progressive and steady (Figure 8B). The third common form of MS is the Primary Progressive form (PPMS), usually the most severe form, which starts from disease onset and continues throughout the patient's life with a progressive neurological deterioration in patients, without or with very little recovery and without relapses (Figure 8C).

In the progressive phase of MS, the worsening of disabilities and symptoms is the consequence of axonal loss leading to cerebral atrophy (0.6-1% a year compared to 0.1-0.3% a year in healthy controls) [START_REF] Miller | Measurement of atrophy in multiple sclerosis: pathological basis, methodological aspects and clinical relevance[END_REF]. A fourth form of MS exists, the Progressive-Relapsing form, but the fact that it is often misdiagnosed as PPMS in early stages and that it affects less than 5% of the patients, make studies to characterize this disease form extremely challenging.

The clinical evolution is largely unpredictable in patients. An accumulating number of proofs show that the clinical or radiological characteristics gathered during two years of the disease course do not have predictive value [START_REF] Cree | Long-term evolution of multiple sclerosis disability in the treatment era[END_REF]

D. FDA-approved treatments

Nowadays, there is no cure for MS. However, huge improvements have been made in the treatment of patients, especially patients affected with RRMS, in the last two decades.

Treatments for MS can be divided in two major categories: first line medications, with moderate efficacy but limited side effect and second line medications, prescribed to patients with severe disease disease form and/or in which first line therapy has failed (Table 1). Second line medications have usually a better efficacy but can induce severe side effects. All disease modifying treatments on the market target the inflammatory component of MS, but proremyelinating and neuroprotective treatment are under pre-clinical and clinical trial and could revolutionize the therapeutic strategy of MS.

Treatments for RRMS

The first FDA-approved treatment was Interferon-β (IFN-β) in the early 1990s. Currently, there are several variants of IFN-β on the market, with limited severe side effects but a modest (around 1/3) reduction of relapse rate (Table 1). The mechanisms of action of this drug are not totally understood, but administration of IFN-β reduces the production of IFN-γ and reduces antigen presentation by macrophages leading to decreased activation of myelin specific T cells [START_REF] Yong | Interferon beta in the treatment of multiple sclerosis: mechanisms of action[END_REF]. Like IFN-β, Glatimer acetate (GA) is an injectable drug, with a comparable efficacy on relapse rate and even rarer serious side effects. GA is a polymer of four amino acids found in the myelin protein MBP. Here again, the mechanisms of action are not totally understood but the injection leads to shift in CD4+ cells, from a Th1 pro-inflammatory to a Th2 anti-inflammatory population. However, GA did not show any effect on disability progression in randomized trials, contrary to IFN-β (Loma and Heyman, 2011) (Table 1). Another immunomodulatory drug, dimethyl fumarate, is available. It seems that this treatment acts on several deleterious events occurring during MS: it has a pro-apoptotic effect on mature T cells while promoting Th2 differentiation in naïve CD4+ cells, and reduces antigen presentation in macrophages and dendritic cells [START_REF] De Jong | Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate[END_REF][START_REF] Linker | Dimethyl fumarate in multiple sclerosis: latest developments, evidence and place in therapy[END_REF][START_REF] Treumer | Dimethylfumarate is a potent inducer of apoptosis in human T cells[END_REF].

In the 21th century, antibody-based therapy emerged in the treatment of MS. Natalizumab seemed to be a great improvement in the treatment of RRMS: the reduction of the frequency of relapse was of 68% against placebo, twice more efficient than existing treatments and a reduced disability progression of 42 % ( [START_REF] Cross | Established and novel disease-modifying treatments in multiple sclerosis[END_REF][START_REF] Winkelmann | Multiple Sclerosis treatment and infectious issues: Update 2013[END_REF]. Another antibody-based treatment, Alemtuzumab is available on the market. It targets CD52, an antigen carried by T and B cells, and leads to their depletion. But here again, due to severe side effects (infections, secondary autoimmune diseases) it is only used to treat patients with extremely severe cases of MS [START_REF] Havrdova | Alemtuzumab in the treatment of multiple sclerosis: key clinical trial results and considerations for use[END_REF] (Table 1).

As listed above, several treatments targeting the immune system are available for RRMS.

However, they either display limited efficacy or strong side-effects. This is one of the characteristics of treatment against auto-immune diseases: it is extremely challenging to target specifically the deleterious effects of the immune system while conserving the indispensable functions of immunity against pathogens.

Treatment for progressive MS

The progressive form of MS, corresponding to slow but steady neurodegeneration, is not as well understood as the inflammatory part occurring in RRMS (see II. Immunopathology of MS).

More than 50 molecules have been tested in phase II or III clinical trials since the 1980s [START_REF] Abdelhak | Primary Progressive Multiple Sclerosis: Putting Together the Puzzle[END_REF] with moderate side-effects [START_REF] Montalban | Ocrelizumab versus Placebo in Primary Progressive Multiple Sclerosis[END_REF]. This treatment has very limited efficacy, but was nevertheless FDA-approved because it is the first molecule demonstrated to be more efficient than placebo in PPMS.

The therapeutic situation in patients for PPMS is much worse than for RRMS patients, with very few available treatments. However, the recently discovered role of B cells in the pathology gives hope for the treatment of progressive MS, and tens of molecule are under clinical trial currently [START_REF] Shirani | Therapeutic Advances and Future Prospects in Progressive Forms of Multiple Sclerosis[END_REF].

Future treatments: remyelination therapy

In the last decade, MS research has been notably focusing on preventing the transition between the relapsing and the progressive phase of MS, characterized by a massive axonal loss. To preserve axons from degeneration, the most promising strategy is to enhance remyelination of demyelinated axons. Remyelination can fail in patients because of OPC recruitment, survival or differentiation, and some pro-remyelinating compounds are ongoing clinical trial [START_REF] Plemel | Remyelination therapies :a new direction and challenge in multiple sclerosis[END_REF].

Future treatments: cell therapy

Cell therapy was deeply explored in the last 20 years as a potential treatment for MS. Three major approaches were or are still tried: replacing the destroyed myelin by transplanting myelinating cells, enhancing repair and neuronal survival by grafting cells with pro-repair and neurotrophic properties and calming the inflammatory attacks using cells with immunomodulatory properties [START_REF] Ben-Hur | Cell Therapy for Multiple Sclerosis[END_REF]). We will focus on the latest strategy, as it is the most advanced one in terms of clinical trials.

Mesenchymal stem cell (MSC) injection is one of the most promising cell therapy in trial for MS. MSC are multipotent cells that can give rise to a wide variety of cell types (e.g. neural cells, fibroblasts, myocytes). The routine source of adult MSC in the body is the bone marrow.

MSC can easily be amplified in vitro. Therefore, technically these cells are extremely practical as they can be obtained by a moderately invasive procedure, can be amplified easily and autologous graft can be performed. Their potential beneficial role in MS is important. They can differentiate into neurons and they have strong immunomodulatory properties. MSC can, among all the processes studied, induce the induction of Treg, inhibit Th17 response and antigen presentation [START_REF] Caprnda | Immunomodulatory effects of stem cells: Therapeutic option for neurodegenerative disorders[END_REF][START_REF] Cobo | Mesenchymal stem cells expressing vasoactive intestinal peptide ameliorate symptoms in a model of chronic multiple sclerosis[END_REF][START_REF] Payne | Human adipose-derived mesenchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity[END_REF]. It is interesting to note that the immunomodulatory effect of MSC is systemic: it does not only act in the CNS [START_REF] Salinas Tejedor | Mesenchymal stem cells do not exert direct beneficial effects on CNS remyelination in the absence of the peripheral immune system[END_REF]. They can inhibit apoptosis of neurons, induce angiogenesis, and promote neuroprotection and remyelination [START_REF] Caprnda | Immunomodulatory effects of stem cells: Therapeutic option for neurodegenerative disorders[END_REF][START_REF] Teixeira | Mesenchymal stem cells secretome: A new paradigm for central nervous system regeneration?[END_REF]. In EAE, MSC treatment gave very interesting results with an important decrease of disease severity due to an impairment of inflammatory processes leading to demyelination and neuronal death [START_REF] Fisher-Shoval | Transplantation of placenta-derived mesenchymal stem cells in the EAE mouse model of MS[END_REF][START_REF] Zappia | Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy[END_REF]. MSC injected intrathecally in patients are well tolerated [START_REF] Connick | The mesenchymal stem cells in multiple sclerosis (MSCIMS) trial protocol and baseline cohort characteristics: an open-label pre-test: post-test study with blinded outcome assessments[END_REF] and the preliminary results in phase I and II show globally a deceleration of disease progression in SP and PPMS patients [START_REF] Meamar | The role of stem cell therapy in multiple sclerosis: An overview of the current status of the clinical studies[END_REF].

Another serious candidate for cell therapy is hematopoietic stem cells (HSC). HSC are multipotent cells giving rise to hematic and immune cells (macrophages, dendritic cells, LT).

HSC do not have a sensible impact on neuroprotection nor remyelination. However, transplanting HSC allow to reboot the immune system, with the hope that newly formed cells will not induce an autoimmune response. They are tested in very aggressive forms of MS after ablation of all immune cells by chemotherapy or total body irradiation. HSC seem to be more efficient than MSC to slow disease progression in the preliminary results of phase I and II studies [START_REF] Burt | Autologous non-myeloablative haemopoietic stem cell transplantation in relapsing-remitting multiple sclerosis: a phase I/II study[END_REF]. However, the medical procedures involve the total suppression of the patient's immune cells, inducing a strong risk of secondary infection.

Finally, neural stem cells are extremely interesting for MS therapy as they have both systemic immunomodulatory and myelinating properties and are undergoing clinical trial for some leukodystrophies (a group of pathology affecting the myelin) and other neurodegenerative diseases [START_REF] Goldman | Stem and Progenitor Cell-Based Therapy of the Central Nervous System: Hopes, Hype, and Wishful Thinking[END_REF],.

II. Pathophysiology and Immunopathology of MS 1. Inflammation, demyelination and neurodegeneration

The hallmark of MS is the presence of several demyelinated plaques disseminated within the CNS. MS lesions were first characterized in the white matter but lesions can also be found in the gray matter of patients. These demyelinating plaques are the consequences of an autoimmune attack against myelin mediated by LT and invading macrophages crossing the BBB and by resident microglia (MIG) (Figure 9). The autoimmune attack occurs because of a failure of suppression of autoreactive T cells and of a dysregulation of the global inflammatory response [START_REF] Compston | Multiple sclerosis[END_REF][START_REF] Dendrou | Immunopathology of multiple sclerosis[END_REF].

A. Relapsing phase of MS

MS can be considered as a disease with two phases: The RR phase characterized by a strong inflammation and the progressive phase (including SP and PP) in which neurodegeneration occurs with a decreased presence of inflammation in the CNS.

In RRMS, relapses are triggered by a massive inflammatory attack leading to demyelination.

What is causing the immune system to trigger the inflammatory storm during a relapse is not known. Chronic inflammation will induce OL death, leading to chronic demyelination. In this stage of the disease, several gadolinium positive active plaques are found, the vast majority in the white matter. Once the inflammatory storm is over, myelin repair can occur. When this process is efficient, axons do not degenerate and a normal axonal function is restored [START_REF] Duncan | Extensive remyelination of the CNS leads to functional recovery[END_REF]. This process is believed to allow a total or a partial remission in patients .

However, axonal death is already present in lesion of RRMS patients in early disease course [START_REF] Hauser | The Neurobiology of Multiple Sclerosis: Genes, Inflammation, and Neurodegeneration[END_REF], that could explain why the remission is sometimes not total in patients.

B. Progressive phase of MS

In the progressive phase of MS, there is a chronic and steady axonal loss correlating with disability progression in the patient. The progressive phase is characterized by neuronal death leading to brain atrophy affecting white and grey matter [START_REF] Fisher | Gray matter atrophy in multiple sclerosis: A longitudinal study[END_REF][START_REF] Jacobsen | Brain atrophy and disability progression in multiple sclerosis patients: a 10-year follow-up study[END_REF][START_REF] Lanz | Brain atrophy and cognitive impairment in multiple sclerosis: A review[END_REF][START_REF] Losseff | Progressive cerebral atrophy in multiple sclerosis. A serial MRI study[END_REF]. Inflammation is less prominent in plaques.

The mechanisms leading from the inflammatory component to the neurodegenerative component are not fully understood and several hypotheses have been proposed: 1) MS is a primary neurodegenerative disorder and the inflammatory processes are not causing axonal death.

2) The inflammation and neurodegeneration are interlinked, and neurodegeneration is the result of chronic demyelination due to inflammatory processes.

The first hypothesis is supported by the fact that MRI studies show subtle changes in the white-matter of patients before the appearance of a lesion and breakdown of the BBB [START_REF] Filippi | Magnetization transfer changes in the normal appering white matter precede the appearance of enhancing lesions in patients with multiple sclerosis[END_REF]. However, experimental and clinical data are arguing for the second hypothesis in which neurodegeneration is driven by inflammation. In EAE, neurodegeneration can be triggered by priming T cell against myelin antigen (Kornek et al., 2000a). In patients, active cortical plaques are always associated with immune cell infiltration (T and B cells) in the meninges and there is a correlation between B cell infiltration and disability progression [START_REF] Kutzelnigg | Cortical demyelination and diffuse white matter injury in multiple sclerosis[END_REF]. Diffuse white matter injury associated with perivascular and parenchymal infiltration of T cells and MIG activation is also found in patients suffering from progressive MS [START_REF] Prineas | Immunopathology of secondary-progressive multiple sclerosis[END_REF].

Globally, the view on neurodegenerative processes in progressive MS as being inflammationindependent is unlikely. There is a large amount of diffuse, perivascular and meningeal inflammation mediated by T cell, B cell and substantial MIG activation [START_REF] Lassmann | What drives disease in multiple sclerosis: Inflammation or neurodegeneration?[END_REF].

These inflammatory processes are strongly correlated with neuronal death [START_REF] Frischer | The relation between inflammation and neurodegeneration in multiple sclerosis brains[END_REF].

Role of T cells in MS and animal models

MS is a disease of the immune system. The vast majority of predisposition polymorphism are associated with genes having functions in immune pathway and among the immune genes

A. Th1 and Th17 cells in EAE and MS

The vast majority of cellular and molecular mechanisms believed to cause myelin destruction and degeneration in MS were discovered in the murine model EAE. In EAE, the immune system is primed against myelin by injection of myelin peptides. With the addition of adjuvants to stimulate the innate immune system, this causes a violent inflammatory response against myelin leading to demyelination and neurodegeneration. EAE is the most accurate model of MS as it reproduces some of the clinical and immunological aspects of MS. CD4+ cells are the main effectors of EAE as the injection of myelin-specific CD4+ cells is enough to trigger the disease [START_REF] Pettinelli | Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2-T lymphocytes[END_REF]. More precisely, Th1 and Th17 are the pathogenic cell types in EAE as they can both induce the disease (Figure 10). In EAE, these two cell types are able to cross the BBB and induce a strong neuroinflammation leading to demyelination and/or neurodegeneration. In MS, the role of Th1 and Th17 cells is more obscure even if some hints argue for an important role in the disease. IFN-γ production (a Th1 produced cytokine) is partially associated with relapses in RRMS patients and microarrays in MS lesions showed an upregulation of the production of IL-17 (a Th17 produced cytokine) [START_REF] Lock | Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis[END_REF]. Finally, treatment by GA, inducing a switch from Th1 to a Th2 immune response, reduces relapse rate in patients [START_REF] Cross | Established and novel disease-modifying treatments in multiple sclerosis[END_REF].

B. Th2 and Treg cells in EAE and MS

The role of Th2 cells in MS and even in EAE is not fully understood. However, this cell type seems to be protective [START_REF] Nagelkerken | Role of Th1 and Th2 cells in autoimmune demyelinating disease[END_REF]. For instance, there is an upregulation of Th2 secreted cytokines in the blood of patients in remission phase [START_REF] Oreja-Guevara | TH1/TH2 Cytokine profile in relapsing-remitting multiple sclerosis patients treated with Glatiramer acetate or Natalizumab[END_REF] and there is an upregulation of Th2 cells produced in the remission phase of EAE induced in SJL mice. Treg cells role, on the contrary, is better understood both in EAE and MS pathology.

Transferring Treg cells after priming of the immune system against myelin is sufficient to prevent the appearance of EAE in mice, indicating a crucial role of these cells in peripheral tolerance and in preventing autoimmune reaction [START_REF] Sakaguchi | Regulatory T Cells and Immune Tolerance[END_REF]. The number of Treg cells does not seem to be decreased in MS patients [START_REF] Feger | Increased frequency of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple sclerosis patients[END_REF] but some studies indicate that their suppressive capacities could be affected: In some patients, Tregs are no longer able to suppress the proliferation of myelin-specific T cells , and are also not able to proliferate efficiently to calm the inflammatory storm [START_REF] Haas | Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis[END_REF][START_REF] Kumar | CD4+CD25+FoxP3+ T lymphocytes fail to suppress myelin basic protein-induced proliferation in patients with multiple sclerosis[END_REF];

C. CD8+ cells in EAE and MS

EAE is proven to be mainly a CD4+ cells dependent disease. However in MS, CD8+ cells seem to be pathogenic as a great number are found in MS plaques. On average, autopsies of MS patients show that there are more invading CD8+ than CD4+ cells [START_REF] Hauser | Immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions[END_REF] . CD8+

cells are located at close distance from damaged axons and are going through oligoclonal expansion in the CSF, blood and brain of MS patients [START_REF] Babbe | Clonal Expansions of Cd8 + T Cells Dominate the T Cell Infiltrate in Active Multiple Sclerosis Lesions as Shown by Micromanipulation and Single Cell Polymerase Chain Reaction[END_REF][START_REF] Jacobsen | Oligoclonal expansion of memory CD8+ T cells in cerebrospinal fluid from multiple sclerosis patients[END_REF][START_REF] Junker | Multiple sclerosis: T-cell receptor expression in distinct brain regions[END_REF][START_REF] Skulina | Multiple sclerosis: brain-infiltrating CD8+ T cells persist as clonal expansions in the cerebrospinal fluid and blood[END_REF]. Moreover, CD8+ cells are likely to be found in cortical plaques, a location strongly associated with clinical progression in MS [START_REF] Lucchinetti | Inflammatory Cortical Demyelination in Early Multiple Sclerosis[END_REF].

In animal models, MOG reactive CD8+ cells can induce severe EAE [START_REF] Sun | Myelin Antigen-Specific CD8+ T Cells Are Encephalitogenic and Produce Severe Disease in C57BL/6 Mice[END_REF] with more neuronal death than if MOG reactive CD4+ cells are injected. In patients, axonal damage is correlated with the number of CD8+ but not CD4+ cells in the lesion [START_REF] Bitsch | Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation[END_REF]. All these data suggest than CD8+ cells could be the main actor responsible for axonal damage in MS. [START_REF] Kowarik | CXCL13 is the major determinant for B cell recruitment to the CSF during neuroinflammation[END_REF] and CCL19 [START_REF] Krumbholz | CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions[END_REF], among others [START_REF] Blauth | The ins and outs of B cells in multiple sclerosis[END_REF].

Role of B cells in MS and animal models

B cells from MS patients can induce demyelination by secreting IgG antibodies that target myelin in vivo and in vitro [START_REF] Elliott | Functional identification of pathogenic autoantibody responses in patients with multiple sclerosis[END_REF] confirming the long debated hypothesis of myelin specific antibodies. Several myelin specific antibodies are now known to target MBP, MOG, SPAG16 and other myelin components [START_REF] Claes | B cells are multifunctional players in multiple sclerosis pathogenesis: Insights from therapeutic interventions[END_REF]. One of the major discoveries of the last few years was that B cells aggregate in the CNS in areas called ectopic lymphoid follicles, located in the brain meninges, from which they induce inflammatory response (Figure 11). From these locations, B cells can induce myelin specific T cell expansion, and induce inflammation efficiently by secreting a vast panel of cytokines that leads to neurodegeneration and disability progression in the progressive phase of MS [START_REF] Magliozzi | Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology[END_REF].

Role of Macrophages and Microglia in MS and animal models

Like T and B cells, invading macrophages and MIG are found in MS plaques suggesting a role in the physiopathology of MS. In EAE, the disease progression is linked to the infiltration of macrophages in the CNS [START_REF] Ajami | Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool[END_REF] and inhibition or depletion of macrophages generally attenuate the paralysis progression occurring in EAE [START_REF] Agrawal | Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis[END_REF][START_REF] Bhasin | Modulation of microglial/macrophage activation by macrophage inhibitory factor (TKP) or tuftsin (TKPR) attenuates the disease course of experimental autoimmune encephalomyelitis[END_REF][START_REF] Martiney | Prevention and Treatment of Experimental Autoimmune Encephalomyelitis by CNI-1493, a Macrophage-Deactivating Agent 1[END_REF] .

A. Direct role on demyelination and neurodegeneration

Macrophages and MIG participate directly to the demyelination and neurodegeneration in MS: a huge number of macrophage and/or MIG secreted cytotoxic compounds or cytokines are found in active MS plaques and the surrounding tissue [START_REF] Lassmann | Mechanisms of white matter damage in multiple sclerosis[END_REF]. One of the well described mechanisms of demyelination is the action of the tumor necrosis factor α (TNF-α):

It is toxic at high doses for OL and induce their death [START_REF] Selmaj | Tumor Necrosis Factor Mediates Myelin and Oligodendrocyte Damage In Vitro[END_REF][START_REF] Zajicek | Interactions between oligodendrocytes and microglia[END_REF]) and this mechanism is mediated notably through cell contact mechanisms. Nitric oxide (NO), normally used for intracellular signaling can also be secreted by MIG leading to OL death in vitro [START_REF] Merrill | Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide[END_REF]. Similarly, a lot of other cytokines are toxic to human OL in vitro, such as IFN-γ and Interleukin-6.

The innate immune system also plays a role in axonal loss and neurodegeneration occurring after demyelination. The CSF and the conditioned media of MS patient macrophages in late stage disease are able to induce neurotoxicity in human neurons in vitro [START_REF] Alcazar | Axonal damage induced by cerebrospinal fluid from patients with relapsing-remitting multiple sclerosis[END_REF][START_REF] Piani | Involvement of the cystine transport system xc-in the macrophage-induced glutamatedependent cytotoxicity to neurons[END_REF]. The cytotoxic elements released by innate immune cells during the demyelination process can also induce neuronal death. Of note, reactive oxygen species, released in great quantity during neuroinflammation, can cause DNA damage inducing neurodegeneration in patients [START_REF] Vladimirova | The activation of protein kinase C induces higher production of reactive oxygen species by mononuclear cells in patients with multiple sclerosis than in controls[END_REF] and antioxidant treatments efficiently suppress the appearance of EAE [START_REF] Marracci | Alpha lipoic acid inhibits T cell migration into the spinal cord and suppresses and treats experimental autoimmune encephalomyelitis[END_REF]. NO, produced by the iNOS (Nitric oxide synthetase) enzyme in macrophages and MIG, is also cytotoxic to neurons, inducing their apoptosis [START_REF] Emerson | Heme oxygenase-1 and NADPH cytochrome P450 reductase expression in experimental allergic encephalomyelitis: An expanded view of the stress response[END_REF]. Glutamate clearance by OL and astrocytes is crucial for neuronal survival as a high extracellular concentration in neurons can induce excitotoxicity in neurons leading to their apoptosis. In EAE like in MS the oligodendroglial death prevents glutamate clearance, and activated macrophages secrete more of this neurotransmitter in inflammatory conditions than in a physiological context [START_REF] Fine | Tumor Necrosis Factor Inhibits Glutamate Uptake by Primary Human Astrocytes[END_REF][START_REF] Hendriks | Macrophages and neurodegeneration[END_REF]. In addition, inflammatory cytokines prevent glutamate uptake by astrocytes. Therefore, macrophages induce directly and indirectly neuronal death by glutamate excitotoxicity by secreting large amount of glutamate and preventing its processing by OL and astrocytes. Other mechanisms have been discovered more recently involving the toxicity of macrophages secreted metalloproteases [START_REF] Lu | Involvement of tissue plasminogen activator in onset and effector phases of experimental allergic encephalomyelitis[END_REF].

B. Role in lymphocytes recruitment, antigen presentation and lymphocyte activation

To invade the CNS, T cells, B cells and macrophages require appropriate attracting signal, a role usually attributed to C-C chemokine ligand 2 (CCL2). CCL2, produced by MIG is one of the most understood signals triggering CNS invasion by T cells. In CCL2 knock-out mice, there is a large decrease of the invasion of CNS by leukocytes during EAE leading to less severe symptoms [START_REF] Kim | Astrocyte CCL2 sustains immune cell infiltration in chronic experimental autoimmune encephalomyelitis[END_REF] and high levels of CCL2 are found in MS plaques [START_REF] Mcmanus | MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: An immunohistochemical and in situ hybridization study[END_REF]. After being activated in the periphery, invading T and B cells can also be reactivated against myelin in the CNS. To do so, they need MHC class II antigen presentation in addition to co-stimulation. During EAE, MIG increase the expression of MHC class II molecules and co stimulatory molecules such as CD86 [START_REF] Olson | Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs[END_REF]. These data indicate that after invading the CNS, T and B cells are reactivated against myelin and proliferate, worsening the extent of the inflammatory attack. Other mechanisms implicating the innate immune system are suspected to be part of the pathophysiological process of MS: The B7-H1/PD-L1 is a molecular pathway implicating a cross-talk between macrophages and CD4+ cells. This pathway is normally an inhibitor of the maturation of CD4+ cells. In MS, there is a dysregulation of this pathway leading to an increase of the IL-17 secretion inducing a preferential maturation of myelin-reactive Th17 cells [START_REF] Chastain | The role of antigen presenting cells in multiple sclerosis[END_REF].

MS Lesions

The histopathological landmark of MS is demyelinated lesions in the CNS called plaques. They can appear anywhere in the brain and the spinal cord and are not only located in the white matter. They can be visualized by MRI, but to determine at what stage the lesion is, postmortem analysis or biopsy are indispensable. MS lesions can be classified according to the extent of myelin loss, inflammation, neurodegeneration, and remyelination.

A. Acute active plaques

In early stages of RRMS, acute active plaques are found frequently: they are characterized by the presence of huge inflammatory infiltrates. The infiltrates are composed in vast majority of macrophages distributed equally all over the lesion. Activated MIG are also present throughout the lesion. In addition to macrophages, the infiltrate is composed of T cells (with a large part of CD8+ cells) and B cells, usually in close proximity to blood vessels. The amount of demyelination in acute active plaques is variable and can be measured directly by oil-red O staining or indirectly by evaluating myelin phagocytosis by macrophages [START_REF] Popescu | Pathology of Multiple Sclerosis : Where Do We Stand ? Continuum[END_REF]. The heterogeneity of demyelination extent and other features led to a more complex classification of acute plaques, in which four different patterns can be found [START_REF] Lucchinetti | Heterogeneity of multiple sclerosis lesions : implications for the pathogenesis of demyelination[END_REF]. Patterns I and II are very close histopathologically: myelin destruction is the consequence of a predominant T cell-and macrophage-induced inflammation. In pattern II plaques (the most frequent type of lesions), there is a strong presence of myelin specific antibody and complement activation indicating a very important role of B cells in the demyelination process. Most of the time, the infiltrates are centered on veins and venules. In pattern III, the demyelination and OL apoptosis are usually very strong and are preferentially affecting periaxonal myelin components, but only in the plaque borders (in contrary to pattern I and II). Demyelination is even affecting the white matter around the lesion. In pattern IV, a type of lesion found very rarely in patients, the OL death is not apoptotic, suggesting a mechanism of OL death independent of inflammatory attacks. The common features of acute plaques include demyelination but axonal damage can also occur.

B. Chronic plaques

Chronic plaques are most often seen in the progressive phase of MS. They can be defined as active, when demyelination is still ongoing (in this case, foamy macrophages containing myelin fragments can be seen) or inactive when demyelination is complete. Inflammation is less prominent in chronic plaques than in acute plaques and its pattern is different. In chronic plaques, T and B cells are localized in vast follicular structures from which they induce neurodegeneration [START_REF] Popescu | Pathology of Multiple Sclerosis : Where Do We Stand ? Continuum[END_REF][START_REF] Wu | The immuno-pathophysiology of multiple sclerosis[END_REF]. It is in chronic plaques that the axonal damage and neuronal death is mainly occurring, by several mechanisms: axonal degeneration due to lack of metabolic support by OL, oxidative stress, cytotoxicity inflammation-induced, and glutamate toxicity, among others.

While this classification is complete, it is complex and difficult to apply in a clinical routine. A simpler classification, aiming to unify all the data analyzed for comparability has been proposed recently [START_REF] Kuhlmann | An updated histological classification system for multiple sclerosis lesions[END_REF]. This new classification incorporates the majority of the histopathological elements used in previous classification. By analyzing the presence and the nature of infiltrates and of demyelination, lesions are defined as active, mixed active/inactive and inactive with our without demyelination.

III. Remyelination 1. Forewords

Remyelination is one of the few regenerative processes of the nervous system. In remyelination, OPCs proliferate, migrate toward the lesion and differentiate into new myelinforming OL to wrap demyelinated axons. It is very efficient in the healthy CNS, it restores fast axonal conduction and allows functional recovery. The several remyelination steps are under the control of the inflammatory environment, and reinforcing its efficacy in MS patients is one of the most promising therapies to treat MS.

Histological description and clinical relevance

A. Evidence of remyelination in the human brain

Existence of remyelination was demonstrated in the PNS prior to the CNS. Two major arguments (reviewed in [START_REF] Hommes | Remyelination in Human CNS Lesions[END_REF]) led scientists in the 1980s to conclude that remyelination could also occur in the spinal cord and the brain: the appearance of myelin on fibers that were demyelinated and the fact that this myelin was short and thin (and therefore different from developmental myelin). Remyelination can be observed post-mortem in the CNS of MS patients: Using luxol fast blue, a dye staining myelin, the areas where the myelin is destroyed or has been repaired (shadow plaques) are observable (Figure 12A, [START_REF] Prineas | Continual Breakdown and Regeneration Sclerosis Plaques[END_REF]). The post-mortem analysis in patients allowed measurement of the efficacy of the phenomenon, by measuring the number of lesions that undergo remyelination at a certain point. However, with this analysis, one cannot be sure that the efficacy of the process observed at the moment of death is representative of the efficacy of remyelination in the disease. In other words, in vivo longitudinal studies for evaluating the extent of the repair process would be more accurate to assess remyelination in patients.

Numerous imagery techniques are used to evaluate the brain compartment of MS patients [START_REF] Filippi | Association between pathological and MRI findings in multiple sclerosis[END_REF] Myelin was stained using luxol fast blue. From [START_REF] Franklin | Why does remyelination fail in multiple sclerosis?[END_REF]

B. Remyelination in animal models

To decipher the mechanisms of MS and of remyelination, animal models are extremely useful.

Remyelination in EAE

The most accurate model of MS is EAE. In this model, an auto-immune attack against myelin is triggered. EAE is a great model of CNS inflammation and neurodegeneration. However, it is not very well suited for remyelination for several reasons [START_REF] Ransohoff | Animal models of multiple sclerosis: the good, the bad and the bottom line[END_REF][START_REF] Tanaka | Mechanisms of remyelination: Recent insight from experimental models[END_REF]: lesions are disseminated stochastically in time and in space, it is therefore hard to know where and when to look to observe the process. In addition, demyelination and remyelination occur at the same time, making it difficult to determine which process is ongoing when observed. Finally, axonal damage and neurodegeneration occur rapidly in EAE, making the remyelination process rather limited.

Toxic models of demyelination and remyelination

Two majors models of toxic demyelination are extensively used nowadays: Cuprizone feeding and lysolecithin/lysophosphatidylcholine (LPC) or ethidium bromide induced focal demyelination (Figure 12C). Cuprizone is a copper chelator which, when added to the food or water of mice, is toxic for OL: they die within 4-6 weeks of treatment and a severe demyelination is observed in the corpus callosum and in the hippocampus [START_REF] Ransohoff | Animal models of multiple sclerosis: the good, the bad and the bottom line[END_REF].

Cuprizone lesions induce glial activation, like in MS, and the lesions observed in this model resemble type II MS lesions. The principal setback of the cuprizone model is that demyelination and remyelination occurs at the same time, making difficult to study the kinetics of myelin repair [START_REF] Kipp | The cuprizone animal model: New insights into an old story[END_REF].

To study remyelination, the micro-injection of a detergent can also be performed. This is the case for LPC or ethidium bromide focal demyelination in which a small volume of the compound is injected in the spinal cord or the corpus callosum of mice, rats or cats. This causes a demyelination at the site of injection by solubilization of the myelin by the detergent. This model has been used with great success to determine the cellular and molecular of remyelination. In this model, remyelination is spontaneous and extremely efficient [START_REF] Jeffery | Remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin[END_REF]. The kinetics of the repair process are well defined. After two days, the demyelination is complete. After 7-8 days, OPCs have proliferated and migrated to the lesion.

The lesion is infiltrated by a large number of macrophages from the periphery, as well as T and B cells. After 21 days, OPC have differentiated and remyelination is complete (Figure 13C).

C. Remyelination, neuroprotection and disease severity

The first evidence that remyelination is a beneficial mechanism for recovery was the discovery that this process restores a fast saltatory conduction in remyelinated neurons [START_REF] Smith | Central remyelination restores secure conduction[END_REF]. This restauration of a proper neural influx is the consequence of sodium channel clustering at regular intervals along the axons occurring even before the formation of the new myelin sheath [START_REF] Coman | Nodal, paranodal and juxtaparanodal axonal proteins during demyelination and remyelination in multiple sclerosis[END_REF][START_REF] Freeman | Acceleration of conduction velocity linked to clustering of nodal components precedes myelination[END_REF]. In addition, locomotor symptoms induced by focal demyelination in cat spinal cord are rescued by remyelination [START_REF] Jeffery | Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination[END_REF] and this data was further confirmed in a more severe model of demyelination [START_REF] Duncan | Extensive remyelination of the CNS leads to functional recovery[END_REF].

Remyelination is beneficial in animal models but also in MS patients: in shadow plaques of post mortem tissue, damaged axons are rare (comparable to normal appearing white matter) compared to chronic lesions (Kornek et al., 2000b) indicating that remyelination protects from neurodegeneration. The mechanisms by which remyelination in patients prevents neurodegeneration are not fully understood, however the trophic support to neurons provided by OL and the fact that myelin physically protects against inflammation are believed to be the two major mechanisms of promoting axonal survive. An additional proof that remyelination allows functional recovery for at least locomotor functions is that the remyelination capacity in patients is inversely correlated to the MS severity score (evaluating walking capacities of patients) [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF].

All these data indicate that reinforcing remyelination in patients is a promising strategy to treat MS.

Mechanisms of remyelination

A. Role of oligodendrocyte precursor cells

Oligodendrocyte precursor cells: an adult precursor population

Adult OPCs are an abundant cell population in the brain, representing around 5 % of the total CNS cell population. A large amount of OPCs generated during development do not differentiate and give rise to the stock of adult OPCs. OPCs are distributed roughly equally throughout the CNS, they can therefore respond to an injury happening everywhere in the CNS. In vivo, adult OPCs have a complex morphology, with a multi arborized cell membrane that does not fit the usual representation of the embryonic bipolar cell when observed in vitro.

Adult OPCs are different from developmental OPCs from a transcriptomic point of view and in physiological conditions, and their role in physiological conditions is starting to be unraveled, with studies highlighting their role in synaptic growth , synaptic plasticity and motor skill learning [START_REF] Nishiyama | Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity[END_REF][START_REF] Polito | NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system[END_REF][START_REF] Xiao | Rapid production of new oligodendrocytes is required in the earliest stages of motor-skill learning[END_REF].

Recruitment of OPCs in response to demyelination

In physiological conditions, OPCs are a quiescent and rarely dividing cell population. After a demyelinating injury, OPCs are activated and exit their resting state in order to execute the different steps leading to forming new myelin. After sensing inflammatory stimuli, OPCs will go through morphological change [START_REF] Levine | Activation and proliferation of endogenous oligodendrocyte precursor cells during ethidium bromide-induced demyelination[END_REF]) and a deep change in their gene expression profile: They start to express genes that are well known in OL development such as OLIG2, NKX2.2, MYT1 among others (Fancy et al., 2004a;Franklin and ffrench-Constant, 2008;[START_REF] Vana | Myelin Transcription Factor 1 (Myt1) Expression in Demyelinated Lesions of Rodent and Human CNS[END_REF]. Due to mitogenic and chemoattractant factors, OPCs will go through the recruitment phase: They commence proliferation and migrate towards the demyelinated area in response notably to PDGF and FGF-2 [START_REF] Zhao | Mechanisms of CNS remyelination-the key to therapeutic advances[END_REF]. Only OPCs located at a small distance from the injury will be able to be recruited to the lesion [START_REF] Franklin | Local recruitment of remyelinating cells in the repair of demyelination in the central nervous system[END_REF].

In animal models of focal demyelination, OPC recruitment happens in the first week post remyelination, as the maximum number of precursor cells is reached at day 7 post injury [START_REF] Remington | Microglial recruitment, activation, and proliferation in response to primary demyelination[END_REF]. These first steps are crucial for the success of remyelination as OPCs need to be in close proximity to axons to reform myelin and in sufficient number to ensure a full repair of destroyed myelin in the area.

Chapter I : Introduction

40

OPC recruitment is dependent on the inflammatory micro-environment. It is therefore extremely important for a successful repair process that the inflammatory response is well controlled in time and intensity.

OPC differentiation in remyelination

Once OPCs reach the lesion, the final step of remyelination is reforming myelin around naked axons. To do so, it is believed (even if not fully demonstrated) that OPCs go through the same stage of differentiation as for developmental myelination: when they receive prodifferentiation clues, OPCs extend their processes to contact axons and wrap them to form new myelin. In animal models, this process takes up to three weeks to be completed [START_REF] Jeffery | Remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin[END_REF].

The newly formed myelin is not exactly identical to the myelin formed during development.

The internodes are on average shorter and the myelin itself is thinner. The mechanisms underlying this difference in the structure of myelin are not known, but the differences argue for a different mechanism in axon myelination and remyelination. Even though the myelin is thinner, the newly formed myelin is still sufficient to reestablish normal axonal conduction, to allow functional recovery and slow/prevent neurodegeneration [START_REF] Duncan | Extensive remyelination of the CNS leads to functional recovery[END_REF][START_REF] Liebetanz | Effects of commissural de-and remyelination on motor skill behaviour in the cuprizone mouse model of multiple sclerosis[END_REF][START_REF] Mei | Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery[END_REF][START_REF] Schultz | Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination[END_REF].

Are OPCs the only source of remyelinating cells in the CNS?

Several studies have already suggested that mature oligodendrocytes targeted in MS do not participate in remyelination and this was finally well demonstrated by the use of inducible myelin-CreER mouse lines in which only mature OL are labelled: Labelled mature OL did not show any production of new myelin, nor did they proliferate or migrate to the demyelinated area [START_REF] Crawford | Pre-existing mature oligodendrocytes do not contribute to remyelination following toxin-induced spinal cord demyelination[END_REF]. On the other hand, OPCs are present in the demyelinating lesion before the appearance of new myelin and they give rise to OL forming new myelin as demonstrated by tracking experiments [START_REF] Gensert | Endogenous progenitors remyelinate demyelinated axons in the adult CNS[END_REF].

At least two other cell types can participate in reforming myelin: Neural precursor cells (NPCs)

and SC. NPCs of the subventricular zone can also give rise to remyelinating OL in animal models and in MS [START_REF] Nait-Oumesmar | The role of SVZ-derived neural precursors in demyelinating diseases: from animal models to multiple sclerosis[END_REF], however their global contribution compared to adult OPC seems modest and limited to the corpus callosum.

In numerous MS animal models, SC can enter the CNS and form new myelin. It is particularly the case in the spinal cord, where the PNS is in close contact with the CNS. However, some other areas like the cerebellum, the optic nerve and the brain stem can also be remyelinated by SC [START_REF] Duncan | Schwann cell invasion of the central nervous system of the myelin mutants[END_REF][START_REF] Itoyama | Schwann cell remyelination of demyelinated axons in spinal cord multiple sclerosis lesions[END_REF][START_REF] Zujovic | Multiple Sclerosis and Demyelinating Disease Animal Models[END_REF]. In a subset of MS patients, SC remyelination can be extensive in the spinal cord, and was even more important than OPC remyelination in a cohort of Japanese patients [START_REF] Itoyama | Schwann cell remyelination of demyelinated axons in spinal cord multiple sclerosis lesions[END_REF]. In the spinal cord, SC remyelinate the center of the lesion, whereas OPCs remyelinate the border. While the equilibrium between SC and OPC remyelination seems to be astrocyte driven [START_REF] De Castro | Astrocyte Activation via Stat3 Signaling Determines the Balance of Oligodendrocyte versus Schwann Cell Remyelination[END_REF], the mechanism implicated in SC remyelination is not yet fully described. While some studies by lineage tracking suggested that remyelinating SC are derived from CNS glial precursors [START_REF] Zawadzka | CNS-Resident Glial Progenitor/Stem Cells Produce Schwann Cells as well as Oligodendrocytes during Repair of CNS Demyelination[END_REF], other studies have shown that a large amount of SC participating in remyelination are derived from the periphery [START_REF] Oudega | Schwann Cell Transplantation for Repair of the Adult Spinal Cord[END_REF]. In addition, it is still unknown if SC derived from the periphery are dedifferentiating to migrate and form myelin or if they are derived from a pool of PNS stem cells.

B. Role of inflammation

Inflammation is a two-edged sword in MS -it induces myelin destruction, but without inflammatory stimuli remyelination would fail. Inflammation is notably induced by cells of the innate immune system: resident MIG and invading macrophages from the periphery.

Macrophages and MIG coordinate remyelination after being activated: they phagocytose myelin debris and induce OPC recruitment and differentiation.

Macrophages and MIG activation

After an injury in the CNS, resident MIG and invading macrophages will acquire a functional phenotype to respond to the injury in a process called "activation" or "polarization". The activation profile was first believed to be dichotomous: in response to pro-inflammatory stimuli like IFN-γ or LPS, MIG and macrophages will be "classically activated" or "M1" and display pro inflammatory properties, while in response to anti-inflammatory stimuli like IL-4, MIG and macrophages will be "alternatively activated" or "M2" and display anti-inflammatory properties [START_REF] Martinez | Macrophage activation and polarization[END_REF]. This very simple dual vision of innate immune system was challenged with the discovery of novel activation phenotypes: The M2b phenotype (in response to IL-1β), which has immunoregulatory properties, and M2c (in response to IL-10), which has immunoregulatory and pro-regenerative capacities. The M2 phenotype in response to IL-4 is now called M2a [START_REF] Martinez | The M1 and M2 paradigm of macrophage activation: time for reassessment[END_REF]Murray et al., 2014a). Moreover, a large transcriptomic study showed that human macrophages have at least nine different phenotypes of activation in response to various stimuli (Xue et al., 2014a).

The M1/M2 is still used for convenience and because they are the only phenotypes of activation with a thoroughly described functional description. In remyelination, MIG and macrophages acquire an M1 pro-inflammatory phenotype that participates to the phagocytosis of myelin debris and induces recruitment of OPCs towards the lesion, before a switch toward the M2 phenotype that will allow OPC differentiation.

Phagocytosis of myelin debris

Myelin is formed by several layers of plasma membrane wrapping around axons. After a demyelinating injury, a lot of myelin debris is produced. Clearance of myelin debris is the first and a crucial step to induce myelin repair because myelin debris itself interferes with the maturation process of OPCs. In vitro, exposing OPCs to CNS myelin leads to a blockage of their differentiation [START_REF] Robinson | Contact with central nervous system myelin inhibits oligodendrocyte progenitor maturation[END_REF], and injection of exogenous myelin debris in a focal demyelination model leads to impediment of myelin repair due here again to a blockage of OPC differentiation and not recruitment [START_REF] Kotter | Myelin Impairs CNS Remyelination by Inhibiting Oligodendrocyte Precursor Cell Differentiation[END_REF]. The exact molecular mechanism of the effect of myelin is not totally unraveled. Clearing myelin debris in the CNS is one of the roles of the innate immune system : in mice with macrophages lacking CCR2, a receptor indispensable for phagocytosis function, remyelination was impeded by over-accumulation of myelin debris [START_REF] Ruckh | Rejuvenation of regeneration in the aging central nervous system[END_REF]. In the same way, in mice with phagocytosis-deficient MIG, remyelination is impaired [START_REF] Lampron | Inefficient clearance of myelin debris by microglia impairs remyelinating processes[END_REF]. These two simple experiments showed that the two components (resident and invading) of the innate immune are required for phagocytosis of myelin debris and therefore giving a proper start to myelin repair (Figure 13B).

In MS patients post-mortem tissue, remyelinated shadow plaques are most of the time surrounded by large number of macrophages positive for myelin staining indicating that efficient phagocytosis is correlated with efficient remyelination [START_REF] Prineas | Multiple sclerosis: Remyelination of nascent lesions: Remyelination of nascent lesions[END_REF], even if this correlation has not been proven causative in humans.

Recruitment of OPCs

MIG are extremely vigilant cells which actively sense their environment. When a demyelinating injury occurs, MIG will recruit monocytes from the circulation to perform phagocytosis of myelin debris. Both of these cell types will then induce OPC recruitment (proliferation and migration towards the lesion) (Figure 13C). The factors inducing these two steps are surprisingly not very well know. Among the few factors discovered, PDGF and FGF-2 (secreted by astrocytes and MIG) were demonstrated to induce OPC proliferation. In TNF receptor 1 knock-out mice, there is a huge decrease in OPC proliferation after demyelination, demonstrating a critical role for this MIG/macrophage-secreted cytokine in remyelination [START_REF] Arnett | TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination[END_REF]. The conditioned media of M1 macrophages induces proliferation and migration of OPCs in vitro, and by specific depletion of M1 macrophages in vivo, proliferation of OPCs was impeded [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF]. These data indicate a crucial role of MIG/macrophages for OPC recruitment but further investigation is needed to highlight the panel of secreted factors acting on OPCs.

OPC differentiation

There has been an intense debate about the need of external factors for OPCs to differentiate and reform myelin. In physiological conditions, OPCs myelinate axons by two mechanisms: one termed "by default" is an intrinsic property of OPCs to wrap small caliber axons, and another which is dependent of electrical activity of axons [START_REF] Sherman | Mechanisms of axon ensheathment and myelin growth[END_REF]. In remyelination however, several elements make axon wrapping more challenging: the axon can be injured, detrimental inflammation can still be present, and myelin debris and other extracellular elements detrimental for the myelin repair process can still be present.

Inflammation is the element that will trigger and accelerate OPC differentiation to make remyelination as efficient as possible.

After a specific time, there is a switch in the phenotype of activation in innate immune cells, when they become M2 and release immunomodulatory and pro-regenerative factors (Figure 13D). Several factors secreted by anti-inflammatory M2 MIG or macrophages are known to induce OPC differentiation [START_REF] Patel | Mediators of oligodendrocyte differentiation during remyelination[END_REF][START_REF] Yong | Taking Advantage of the Systemic Immune System to Cure Brain Diseases[END_REF][START_REF] Zhang | Targeting oligodendrocyte protection and remyelination in multiple sclerosis[END_REF].

Of note, IGF-1 increases OPC differentiation and myelin production in vitro and administration of IGF-1 decreases area of demyelination and increases the number of remyelinated axons in EAE [START_REF] Yao | myelin-related proteins[END_REF]. The Leukemia Inhibitory Factor (LIF) is both protective against OL death and also increases remyelination by promoting OPC differentiation [START_REF] Levy | Dysregulated production of leukemia inhibitory factor in immune cells of relapsing remitting multiple sclerosis patients[END_REF]. Activin-A, another MIG/macrophage-secreted factor enhances OPC differentiation in vitro and remyelination in vivo [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF].

Monocyte derived macrophages and MIG: different name, same function?

Macrophages derived from circulating monocytes and MIG are often both denominated as macrophages and were for a long time considered to have the same origin and functions in physiological and pathological conditions. This confusion persisted because the two cell population express the same markers (Iba-1, CD68, F4/80). It was therefore virtually impossible to study independently one cell population from another. However, it is well demonstrated now that these two cells types have different embryonic origins and that their function might be different during myelin repair: Experiments of fate mapping using transgenic lines demonstrated clearly that MIG arise from progenitors in the yolk sac (an annex embryonic structure) [START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF] whereas monocytes derived macrophages are generated by hematopoiesis in the bone-marrow.

The first argument that led to speculation that monocyte derived macrophages and MIG have different functions was the transcriptomic analysis of these cells that showed that, in physiological conditions, even if they express a large number of identical genes, there are genes expressed specifically in each cell type [START_REF] Hickman | The microglial sensome revealed by direct RNA sequencing[END_REF]. In EAE mice, the transcriptional profile also differs between MIG and macrophages: macrophages are more activated and proliferate more in the early acute phase of the disease whereas MIG have a more resting phenotype. These data could indicate that macrophages are the major pathogenic actor in the early step of EAE [START_REF] Vainchtein | In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed[END_REF]. MIG, however, upregulate MHC II genes and the co-stimulating molecules CD80, CD86 and CD40, arguing for a role in the reactivation of the invading adaptive immune cells [START_REF] Lewis | RNA sequencing of microglia and monocytederived macrophages from mice with experimental autoimmune encephalomyelitis illustrates a changing phenotype with disease course[END_REF]. In EAE, it seems that MIG have better phagocytic capacities than macrophages (even if the two cell types are both essential) as demonstrated by the upregulation of the expression of phagocytosis genes and functional studies [START_REF] Lewis | RNA sequencing of microglia and monocytederived macrophages from mice with experimental autoimmune encephalomyelitis illustrates a changing phenotype with disease course[END_REF][START_REF] Yamasaki | Differential roles of microglia and monocytes in the inflamed central nervous system[END_REF] and the secretion of growth activation, whereas an IL-4 stimulation elicited by Th2 cells would induce the secretion of trophic polyamine in macrophages, defining the M2 activation [START_REF] Mills | M-1/M-2 Macrophages and the Th1/Th2 Paradigm[END_REF]. The term was next exported to MIG, believed to have the same activation profile in response to the same stimuli. From there, markers expressed by M1 cells and M2 cells were used to define macrophages activation in pathological conditions.

This nomenclature raises several problems. The first one is that the M1 state if defined now as being the result of exposition to IFN-γ, Lipopolysaccharide or both, even though the transcriptomic responses induced in macrophages are different [START_REF] Martinez | Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression[END_REF]. This differences in the transcriptional response could indicate distinct role in physiological and pathological conditions. The same shortcut exists for the M2a phenotype, defined as IL-4 or IL-13 activated macrophages even though the resulting signature is close, but not identical [START_REF] Scotton | Transcriptional Profiling Reveals Complex Regulation of the Monocyte IL-1 System by IL-13[END_REF]. Overall, macrophages with a different response to distinct stimuli are classified as a same activation profile.

Secondly, when studied in vitro, macrophage activation is not limited to M1 and M2 activation.

A large study on human macrophage showed the existence of at least 9 unique transcriptomic signature in response to 29 different stimuli arguing for a spectrum model of activation in macrophages [START_REF] Xue | Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation[END_REF].

Another issue with this classification is the use of the term M1 and M2 in vivo. They simply not exist in situ as the stimulation received by macrophages are never just one cytokine.

Macrophage activation in vivo is a complex process, involving cytokine signaling, cell adhesion, cell-cell interactions … In pathological study, the term M1 and M2 are used when the innate immune cells analyzed express express in vivo the same marker than an M1 macrophage.

However, a simple common marker does not imply the same functional role.

The M1/M2 paradigm, despite all its weaknesses, is useful to get an overview of an extremely complex process. In the case of remyelination, we chose to keep it as it seems that there is a well characterized functional role of macrophages expressing M1 and M2 markers in the process [START_REF] Miron | Macrophages and CNS remyelination[END_REF].

The established M1/M2 framework and the uses of associated markers allowed major discoveries in the study of macrophages in neurodegenerative processes: In MS, several molecules with high potential therapeutical applications have been discovered by studying the secretion panel of macrophages positive for M2 markers. For instance, Activin-A can induce OPC differentiation during remyelination [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF].

Overall, macrophages in vivo do not have a binary choice during activation, they acquire a mixed subset of phenotypes that if probably continuous. A new nomenclature was proposed by a group of macrophages specialist and propose to define macrophages subset according to the stimulation used to polarize them (i.e. M(IL-4) for IL-4 stimulated macrophages) (Murray et al., 2014b). This solves the problem of the large number of stimulation defining the M1 or the M2 stage but does not clarify how we should define macrophages positive for M1 or M2 markers in vivo.

Role of the adaptive immune system.

The role of the adaptive immune system in the myelin repair process remains less understood compared to the innate immune system. However, some clues indicate a critical action of LT in remyelination. More than a decade ago, it was discovered than T cells play a role in remyelination. In mice deficient for T and B cells, or mice depleted specifically of CD4+ or CD8+, the myelin repair process was less efficient in a model of focal demyelination, indicating that these cells are necessary for remyelination [START_REF] Bieber | Efficient central nervous system remyelination requires T cells[END_REF]. One of the putative mechanisms is that T cells are able to induce OPC proliferation, as it was demonstrated in a mouse model (Hvilsted Nielsen et al., 2011a).

Remyelination heterogeneity: causes of remyelination failure

A. In animal models

In animal models of demyelination/remyelination and of MS, the major known factor leading to remyelination failure is aging. After a focal demyelination was induced in rats, both OPC recruitment (proliferation and migration to the lesion) and differentiation were impeded in aged animals compared to younger controls [START_REF] Sim | The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation[END_REF] leading to a decrease in remyelination efficacy [START_REF] Shields | Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination[END_REF].

The cause for OPC recruitment failure is still under debate: experimentally, repeated demyelination episodes induced by cuprizone treatment, mimicking a long disease course, led to a decrease of the number of OPCs at the lesion and therefore a diminished remyelination efficacy. The underlying mechanism is however not known and several hypotheses can be formulated: exhaustion of OPC stock, or diminished capacity of OPCs to proliferate and/or migrate. However, several studies do not argue for this OPC recruitment failure hypothesis, as repeated rounds of demyelination do not disturb remyelination in a focal demyelination model [START_REF] Levine | Activation and proliferation of endogenous oligodendrocyte precursor cells during ethidium bromide-induced demyelination[END_REF][START_REF] Penderis | Impaired remyelination and depletion of oligodendrocyte progenitors does not occur following repeated episodes of focal demyelination in the rat central nervous system[END_REF].

The effect of aging on OPC differentiation has been more extensively described and replicated.

In aged animals, the induction of critical signaling pathways and transcription factors for OL differentiation is delayed (Fancy et al., 2004b) and therefore the maturation of OPC becomes less efficient. Here again, it seems to depend on inflammation. In aged animals, macrophage function is altered. First, phagocytosis of myelin debris is less efficient in aged animals, leading to OPC differentiation failure. This poor remyelination is rescued in aged animals when they share blood circulation, and therefore innate immune cells, with young animals [START_REF] Ruckh | Rejuvenation of regeneration in the aging central nervous system[END_REF]. In the same experiment of parabiosis, OPC proliferation is increased suggesting that the rescue of remyelination is not only due to the rescue of phagocytic capacities but also in other macrophage functions. In fact, the cytokine secretion panel of macrophage changes over time [START_REF] Zhao | Differences in the early inflammatory responses to toxin-induced demyelination are associated with the age-related decline in CNS remyelination[END_REF]. Because cytokines secreted by macrophages and MIG drive OPC into an M2 phenotype in the final stage of remyelination leading to a proper OPC maturation [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF].

All these data demonstrate that in animal models, aging is affecting the efficacy of remyelination due to a less efficient inflammatory response.

B. In MS patients

Is aging the principal cause of remyelination failure in patients?

As several repair processes and more generally biological processes show decreasing efficacy with age, and because the efficacy of remyelination decreases in animal models, it would be expected to find the same results in patients: patients with long lasting disease should have less efficient remyelination. The classical view in the field is that remyelination is efficient in early disease stages, where the inflammatory component is strong and that, with time, the neurodegenerative stage of the disease takes over, leading to chronic plaques in which remyelination is absent. However, the reality seems to be more complex than that.

The first clues that can contradict this theory come from the analysis of post mortem tissues of MS patients. In a study of two patients with long (21 and 22 years) disease course, analysis of remyelination revealed that 73% of lesions were at least partially remyelinated [START_REF] Patani | Remyelination can be extensive in multiple sclerosis despite a long disease course[END_REF]. Even if the number of patients analyzed in this study is very low, it demonstrates that the idea that myelin repair does not occur after a long disease course is at least partially wrong. Furthermore, a wider study on 51 patients showed a positive correlation between age of patients and remyelination [START_REF] Patrikios | Remyelination is extensive in a subset of multiple sclerosis patients[END_REF]. This result seriously challenges the idea that aging is the cause of remyelination failure in MS patients.

One might argue that analysis of post mortem tissue is a 'snapshot' of a specific time and does not reflect the global capacity of a patient to induce myelin repair over time. Recently, new techniques combining PET-scan and MRI allowed for the first time to visualized remyelination in patients by imagery [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF]. Using a specific marker of myelin ([ 11 C]PiB), two scans were realized at 1-4 month of interval in 20 patients. The new [ 11 C]PiB positive voxels appearing on the latest scan images reflected the newly formed myelin, and a remyelination index was calculated. Interestingly, no correlation between the age or disease duration and the remyelination index was found.

All these data suggest that neither age nor disease is the principal factor of remyelination failure in patients.

What is causing remyelination failure in some patients?

The three studies mentioned above [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF][START_REF] Patani | Remyelination can be extensive in multiple sclerosis despite a long disease course[END_REF][START_REF] Patrikios | Remyelination is extensive in a subset of multiple sclerosis patients[END_REF] and some others [START_REF] Bruck | Remyelination in Multiple Sclerosis[END_REF][START_REF] Strijbis | Cortical Remyelination Is Heterogeneous in Multiple Sclerosis[END_REF] led to a consensus in the field:

Remyelination is heterogeneous in MS patients. There is an individual capacity to induce myelin repair. Some patients have this process working efficiently during all their lifetime, and this is correlated to a lower severity score [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF], while for some other patients, the repair process fails (Figure 14). Multiple histological studies have been performed to decipher the cellular mechanisms of remyelination failure in some patients. One of the causes investigated was, like in animal models, a failure of OPC recruitment and/or a depletion of the OPC stock after repeated rounds of demyelination/remyelination. This has been notably investigated in post mortem tissues of patients and, globally, OPCs are always present in demyelinated lesions, even if the number of OPCs can change with the clinical features of patients. OPCs are present in a considerable number in chronically demyelinated lesions [START_REF] Chang | Premyelinating oligodenrocytes in chronic lesions of multiple sclerosis[END_REF]Wolswijk, 1998a) and even more so in early active lesions [START_REF] Chang | NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions[END_REF][START_REF] Kuhlmann | Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis[END_REF]. All these data concur to disprove the recruitment and/or depletion theory: in MS patients, the OPC stock is never depleted and these cells can be recruited to the lesion.

While OPCs are present in the lesion, multiple pieces of evidence demonstrate a failure of OPC differentiation. In chronic and active lesions, there is a strong inter-individual capacity in the number of differentiating OPCs [START_REF] Kuhlmann | Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis[END_REF]Wolswijk, 1998b). Several markers of maturation were used in these studies, and it seems that the differentiation block is not homogeneous between patients. In some, cells are blocked in early steps of maturation whereas in another subgroup of patients, cells express markers of mature OLs but do not form myelin [START_REF] Chang | Premyelinating oligodenrocytes in chronic lesions of multiple sclerosis[END_REF].

Because it has been demonstrated recently that the innate immune system is driving remyelination is MS, a few studies on this have been conducted on biopsies and/or post mortem tissues. M2 MIG seems to be more present in early active lesions than in chronic lesions [START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF] and the activation status of innate immune cells seems to depend on the lesion type [START_REF] Peferoen | Activation Status of Human Microglia Is Dependent on Lesion Formation Stage and Remyelination in Multiple Sclerosis[END_REF].

Chapter I : Introduction 51 However, a recent study challenged the results of former groups, as most of the markers used to discriminate between M1 and M2 macrophages/MIG were co-expressed by these cells indicating that either the status of activation of these cells is more complex than the dual M1/M2 view or that the markers used previously were not representative of each activation status [START_REF] Vogel | Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status[END_REF]. Therefore, more advanced techniques (transcriptomic and proteomic) are needed to define more precisely the activation status of the innate immune system in MS lesions and explain why OPC differentiation can fail in some patients.

Globally, remyelination fails in some patients due to a deleterious micro-environment that will prevent OPC to reform new myelin. Chapter I : Introduction

IV. Aims of the project

Even if some substantial progresses have been made in the treatment of MS, there is still no cure nor both very efficient and safe disease modifying treatment. This is partly explained by the view of inflammation as only a detrimental actor in the pathophysiological process. As the demyelination was cause by inflammatory attacks, it was logical to focus research effort on neutralizing the immune system. In the last decade however, inflammation was demonstrated to be beneficial and indispensable for remyelination. There is therefore a beneficial inflammatory process that should not be targeted, but on the contrary, reinforced to provide remyelination and neuroprotection.

Understanding the cellular and molecular events leading to a successful remyelination is critical to develop innovative treatment to foster this mechanisms. The huge majority of studies studying remyelination have been performed using only murine models and were not able to reproduce nor explain the spectrum of remyelination efficacy found in patients. In MS, even if the use of those models allowed tremendous progress on understanding the pathophysiological mechanisms at stake, the use of patient cells in humanized model could bring some more precise insight on the cellular and molecular events leading to a successful remyelination.

LT invade the CNS and induce myelin destruction. To do so, they induce a pro-inflammatory environment that will cause a chain reaction and induce a large immune response. By being major actor of the induction of an inflammatory micro-environment in the CNS, LT could play a role a remyelination. Therefore, using a humanized model, we tackled first the following questions:

1) Do human LT from patients or HD influence the remyelination process ? If that is the case, what are the molecular and cellular events, influenced by LT, leading to a succesful remyelination ? Those questions will be tackled in chapter 2.

In chapter 2, we discovered that the secretory profile of LT upon stimulation can vary considerably from an individual to another leading to a chain of event inducing remyelination failure or success. In the second part of the project, we tried in a preliminary study to decipher what is causing this inter-individual variation by studying the effect of susceptibility SNP concentrated in a specific LT pathway on remyelination.

2) In patients, do SNPs concentrated in one Th cell pathway can influence remyelination outcome? Can one SNP or a combination of SNP be predictive of the success of remyelination ? Those questions will be tackled in chapter 3.

I. Introduction

After destruction of the myelin in MS, remyelination is triggered. From experimental data, this system is mainly mediated by the innate immune system and its state of activation to realize the different steps of the process.

As it was shown in vivo and in vitro [START_REF] Butovsky | Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis[END_REF][START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF]Zhang et al., 2014), LT secreted cytokines can influence the state of activation of macrophages and MIG.

However, this data use only mouse cells that make difficult to evaluate the consequences of a cross talk between LT and innate immune cells during remyelination. Moreover, remyelination has not being extensively studied using models including pathological MS cells from patients.

To tackle those questions, we created a novel model in vivo model by grafting MS or HD LT into a focally demyelinated lesion in the spinal cord of nude mice. We also developed new in vitro protocols to evaluate the influence of LT on innate immune cells. Finally, we analyzed the heterogeneity of remyelination in patients by defining the molecular landscape necessary for a successful myelin repair.

To eliminate the hypothesis of another cell type having an indirect role on MIG activation or OPC differentiation (see figure 2 and 3 of the publication), the purity of the primary culture of those two cell types was evaluated (supplementary Figure 7). The protocol that we used to isolate mouse or rat MIG and OPCs from mixed glial cells culture in commonly used, with minor variations, among researchers in the field [START_REF] Butovsky | Butovsky-Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of MS .pdf[END_REF][START_REF] Foote | Inflammation stimulates remyelination in areas of chronic demyelination[END_REF][START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF] and several protocol paper detail very precisely the different steps to isolate MIG or OPCs from rodent brains [START_REF] O'meara | Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues[END_REF][START_REF] Tamashiro | Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue[END_REF]. Using this technique, we obtained a proportion pure cells of 88,1 +/-5,5% for MIG and 69,7 +/-9,2 % for

OPCs. These values are of the same order of magnitude of what is obtained by other teams using this protocol.

Supplementary figure 7: To validate purity of the MIG and of the OPC culture, cells were stained respectively with Iba-1, a general marker of MIG, or with Olig-2 a marker of the OL lineage. Cells were also counterstained with Hoechst to reveal the nucleus of all the cells present in the cultures.

The % of Iba-1 + or of Olig-2 + cells were calculated. n= 6 independent experiments.

INVENTORS (contribution)

Zujovic Violetta (20%) Sanson Charles (20%) Fontaine Bertrand (20%) El Behi Mohamed (20%) Reibex Isabelle (20%)

SUMMARY

The present invention relates to an in vitro method for the prognostic of a subject affected by a demyelinating disease comprising detecting and quantifying the level of at least one of CCL19, IL-15, ENA78 and/or TRAIL in a biological fluid from the subject.

In one embodiment, the in vitro method further comprises detecting and quantifying the level of at least one of LIF and SDF-1.

In one embodiment, the level of the at least one of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL allows classification of the subject in a group having a low remyelination profile or in a group having a high remyelination profile.

In one embodiment, the measured levels of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL is/are compared to reference values. Preferably, said reference values correspond respectively to the median levels of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL in a group of patients with a demyelinating disease.

The present invention also relates to a composition for use in the treatment of a demyelinating disease comprising at least one modulator of CCL19, IL-15, ENA78 and/or TRAIL.

In one embodiment, the composition for use in the treatment of a demyelinating disease further comprises at least one modulator of LIF and/or SDF-1.

In one embodiment, the at least one modulator of CCL19, IL-15, ENA78, TRAIL, LIF and/or SDF-1 is an antibody or fragment or mimetic thereof, an aptamer, a small molecule, a peptide mimetic, a siRNA, an asRNA, an antagonist, an agonist or an inverse agonist.

In one embodiment, the composition for use in the treatment of a demyelinating disease comprises at least one inhibitor of CCL19, ENA78 and/or SDF-1 and/or at least one 25 activator of IL-15, LIF and/or TRAIL.

In another embodiment, the composition for use in the treatment of a demyelinating disease comprises at least one antagonist or at least one inverse agonist of CCR7, CXCR2 and/or CXCR4;

and/or at least one agonist of IL-15R, LIFR, TRAIL-RI and/or TRAILRII.

In one embodiment, the composition is for use in the treatment of a demyelinating disease selected from the group comprising inflammatory demyelinating disorders, multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, acute transverse myelitis, Guillain-Barre syndrome, brainstem encephalitis, optic neuritis, neuromyelitis optica, leukodystrophy, adrenoleukodystrophy, adrenomyeloma neuropathy, idiopathic inflammatory demyelinating disease, central pontine myelinolysis, optic neuritis, aquaporin 4 antibody-negative neuromyelitis optica, progressive multifocal leukoencephalopathy, periventricular leukomalacia, Vitamin B12 deficiency, Wernicke's encephalopathy, osmotic demyelination syndrome, Leigh's disease.

In one embodiment, the modulator is to be administered in a subject having a low remyelination profile.

CLAIMS

1.

An in vitro method for the prognostic of a subject affected by a demyelinating disease comprising detecting and quantifying the level of at least one of CCL19, IL-15, ENA78 and/or TRAIL in a biological fluid from the subject.

2.

The in vitro method according to claim 1, further comprising detecting and quantifying the level of at least one of LIF and SDF-1.

3.

The in vitro method according to claim 1 or 2, wherein the level of at least one of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL allows classification of the subject in a group having a low remyelination profile or in a group having a high remyelination profile.

4.

The in vitro method according to any one of claims 1 to 3, wherein the measured levels of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL is/are compared to reference values, preferably said reference values correspond respectively to the median levels of CCL19, IL-15, ENA78, LIF, SDF-1, and/or TRAIL in a group of patients with a demyelinating disease.

5.

A composition for use in the treatment of a demyelinating disease comprising at least one modulator of CCL19, IL-15, ENA78 and/or TRAIL.

6.

The composition for use according to claim 5, further comprising at least one modulator of LIF and/or SDF-1.

7.

The composition for use according to any one of claims 5 to 6, wherein said modulator is an antibody or fragment or mimetic thereof, an aptamer, a small molecule, a peptide mimetic, a siRNA, an asRNA, an antagonist, an agonist or an inverse agonist.

The composition for use according to any one of claims 5 to 7, comprising at least one inhibitor of CCL19, ENA78 and/or SDF-1 and/or at least one activator of IL15, LIF and/or TRAIL Chapter 3: Linking MS susceptibility variants to remyelination capacity

Article 2 and contribution

In this preliminary study, the consequences of genetic variants in MS patient LT on remyelination was evaluated.

Chapter 4: Discussion and conclusion I.

Role of lymphocytes in remyelination 1. Experimental evidence

LT were not originally thought to participate in myelin repair, whereas several investigations demonstrated a major role of the innate immune system. However, being present during remyelination, especially in acute plaques [START_REF] Popescu | Pathology of Multiple Sclerosis : Where Do We Stand ? Continuum[END_REF], LT could potentially influence the outcome of the process.

The involvement of LT in remyelination did not attract much attention until the early 2000's.

Using knock-out mice or antibody depletion, it was demonstrated that T cells are necessary an efficient remyelination [START_REF] Bieber | Efficient central nervous system remyelination requires T cells[END_REF]: in B6-Rag1 tm1Mom mice, lacking B and T cells, the density of remyelinated axons 35 days after focal demyelination of the spinal cord was reduced three-fold. In mice lacking CD4+ (CD4 tm1Mak knock-out model or depletion induced by anti-CD4 antibody) or CD8+ cells (CD8 tm1Mak or depletion induced by an anti-CD8 antibody), remyelination was also strongly impeded. This pioneer study used a focal demyelination model, in which the mice LT are not myelin-primed and therefore non pathological.

Mechanistically, these results could be partially explained by recent publications showing a direct regenerative role of Tregs in remyelination: First, there is a Tregs induction at the initiation of remyelination [START_REF] Plaisted | Remyelination is correlated with regulatory T cell induction following human embryoid body-derived neural precursor cell transplantation in a viral model of multiple sclerosis[END_REF]. In addition, in a knock-out mouse for Foxp3, in which Tregs are absent, remyelination was impeded and rescued by the graft of exogenous wild type Tregs [START_REF] Dombrowski | Regulatory T cells directly promote myelin regeneration in the Central Nervous System[END_REF]. Tregs were shown to have a direct prodifferentiation effect on OPCs in vitro mediated by the secretion of CCN3. Even if the wellknown immunomodulatory role of Tregs cannot be excluded to have played a role in the rescue of remyeliation, Tregs seem to have a direct regenerative role on promoting OPC maturation. Interestingly, it was also shown that myelin-specific T cells can induce a proliferation of OPC after axonal damage through an unknown mechanism (Hvilsted Nielsen et al., 2011b).

Other studies have pointed out a deleterious role of LT in remyelination: using only human cells, it was shown that the supernatant of Th1 and Th17 cells, pathogenic in MS, have direct cytotoxic effects and indirect differentiation blocking effects on OPCs in vitro [START_REF] Moore | Direct and indirect effects of immune and central nervous system-resident cells on human oligodendrocyte progenitor cell differentiation[END_REF]. The molecular actors of this cytotoxic effect were not revealed in the study, but the fact that the supernatant alone has an effect indicates a cytokine-mediated mechanism. In vivo, the graft of myelin-reactive Th17 cells in the cuprizone model delayed remyelination by inducing a pro-inflammatory phenotype in macrophages [START_REF] Baxi | Transfer of Myelin-Reactive Th17 Cells Impairs Endogenous Remyelination in the Central Nervous System of Cuprizone-Fed Mice[END_REF].

Overall, pathogenic T cells (Th1 and Th17), are, by inducing a pro-inflammatory cytotoxic environment are directly and indirectly inhibiting remyelination whereas T cells with immunoregulatory phenotypes are fostering remyelination by calming the inflammation storm and promoting OPC differentiation.

Cellular mechanisms involved

The accumulation of experimental evidence seems to point toward a prominent indirect action of LT on OPCs. In the study of Bieber et al., they already suggested that LT could act on MIG and macrophages to influence remyelination.

The cross-talk between adaptive and innate immune cells is well established in the immune response: antigen-presenting cells, mostly MIG and macrophages, can trigger lymphocyte activation and proliferation at the initiation of the adaptive immune response. Moreover, MIG are able to reactivate LT once they cross the BBB in multiple sclerosis, inducing chronic demyelination.

In remyelination, it is likely that this cross-talk goes both ways and that LT could influence MIG and macrophage activation: First, it was demonstrated two diametric opposite phenotypes of macrophage activation, driven by LT secreted cytokines IFN-γ and IL-4, have distinct roles in oligodendrogenesis and neurogenesis [START_REF] Butovsky | Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis[END_REF]. In vitro, it was shown that the supernatant of Th1 cells could drive an M1 response in MIG [START_REF] Prajeeth | Effector molecules released by Th1 but not Th17 cells drive an M1 response in microglia[END_REF] Moreover, in our studies, we clearly demonstrated by immunohistochemistry and qPCR that MS LT are able to influence MIG activation toward a pro-inflammatory M1 phenotype (El-Behi et al., 2011, Sanson et al. in preparation). Finally, a predominant M1 phenotype was found in macrophages after Th17 grafts in a cuprizone model compared to the non-grafted animal arguing for a cross talk between LT and innate immune cells as the cause for remyelination failure [START_REF] Baxi | Transfer of Myelin-Reactive Th17 Cells Impairs Endogenous Remyelination in the Central Nervous System of Cuprizone-Fed Mice[END_REF]. An induction of an M1 phenotype in MIG and macrophages could also explain why myelin reactive T cells induce OPC proliferation in vivo (Hvilsted Nielsen et al., 2011b), as the M1 phenotype is known to induce this effect on OPCs [START_REF] Butovsky | Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis[END_REF][START_REF] Miron | M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination[END_REF].

Direct mechanisms of LT on OPCs are also likely to influence remyelination even if less experimental proof exists: as stated above, Tregs can induce OPC differentiation and Th1 cells are cytotoxic for OPCs. However, these results must be taken with cautions as the direct effect was demonstrated in vitro. In our first study, we did not find any direct effect of MS LT on OPC differentiation [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF]. However, we also demonstrated a strong inter individual heterogeneity of the effect of MS LT on remyelination, making any conclusions difficult since the effect on remyelination is the results of pro and anti-differentiation signals from LT.

The results of the study arguing for a beneficial role versus the ones arguing for a detrimental role of LT in remyelination are not necessarily contradictory. Inflammation in MS is a twoedged sword inducing both myelin destruction and repair. Remyelination is a mechanism requiring both pro-and anti-inflammatory stimuli to be completed, and these stimuli must occur sequentially to ensure that remyelination to be completed. Therefore, if the proinflammatory micro-environment is exacerbated like when Th17 cells are grafted in a cuprizone model [START_REF] Baxi | Transfer of Myelin-Reactive Th17 Cells Impairs Endogenous Remyelination in the Central Nervous System of Cuprizone-Fed Mice[END_REF], this will prevent OPC differentiation as the M1 phenotype induced by Th17 cells can inhibit the maturation process. It is thus likely that pro-inflammatory LT can induce a strong pro-inflammatory response, preventing the regeneration steps to be performed in MS whereas anti-inflammatory LT drives an anti-inflammatory environment that do not inhibit the early steps but foster the later steps of remyelination.

Overall, LT can influence remyelination by direct effects on OPCs and by driving MIG and macrophage activation.

Modeling LT role in remyelination

Advantages of our model

Our studies are the only ones using MS patient LT to study the role of these cells in remyelination. Interestingly, the graft of these cells in a demyelinated lesion of the spinal cord was sufficient to induce a heterogeneous pattern of remyelination, with LT from some patients inducing a remyelination level comparable to HD levels, and those from other patients inducing a less efficient myelin repair process. Several studies have tried to understand why remyelination sometimes fails, but as said previously (see Chapter 1, III.4), the remyelination heterogeneity in patients was never properly modeled. . To understand why it fails in some patients, a new model was needed and we proposed a new experimental paradigm combining the advantages of focal demyelination, in which the timing and the cellular and molecular events of myelin repair are well known, and human LT from MS patients and HD, allowing to better model of remyelination in a pathological context.

A second advantage of our model is that the mouse strain (RjOrl:NMRI-Foxn1 nu/ Foxn1 nu ) in which LT were grafted is athymic, and therefore does not have any T cells, thus the specific effect observed is most likely due to grafted LT. Nude mice do however have B and natural killer cells, and we can therefore not completely rule out the hypothesis that human grafted LT could influence the murine adaptive immune system and eventual consequences on remyelination.

An advantage of using LPC-induced demyelination is that the myelin destruction event happens only before (not during nor after) remyelination, making it easy to separate the events due to demyelination and the ones due to remyelination.

The major issue with rodent remyelination models (such as cuprizone, LPC) is that myelin repair in these models is spontaneous and extremely efficient. Therefore what can be observed in experimental conditions is only acceleration or a deceleration of remyelination.

Therefore, these models are not perfectly suited for pharmacological studies as the putative effect induced by a tested compound is an acceleration of remyelination and not an overcome of remyelination failure: in other words, the tested compound might change the kinetics of remyelination but its capacity to induce myelin repair in a inhibitory micro-environement cannot be evaluated. In our model, however, we have a remyelination failure caused by LT from patients with low repair capacity. This model would therefore be more suited to highlight the effect of a compound that could overcome failure of myelin repair.

Interestingly, our model reproduces most of the characteristic of an active lesion of patients with short disease course analyzed by biopsy: we found a similar density of LT [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF][START_REF] Kuhlmann | Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time[END_REF], and LT are localized preferentially near blood vessels.

Drawbacks of the model

Our model also presents some drawbacks: grafting human LT in a mouse will not account for immune activation that is cell contact dependent as the human TCR and the mouse MHC are not compatible. Thus, a part of the cross-talk between adaptive and innate immune cells that putatively happens during remyelination is not represented in our model. In addition, the graft of human cells in a mouse does not allow a perfect cytokine communication, even though the vast majority of cytokine pathway are common between mice and human.

When the graft was performed, it contained a mixed population of T and B cells. Therefore we cannot conclude which cell type influence the most the remyelination process. It would be necessary to sort LT through FACS before grafting to test which LT subset is influencing MIG activation and OPC behavior.

The only unquestionable way to demonstrate that our model reproduces remyelination heterogeneity in patients would be to graft the LT of patients whose remyelination capacities are known, such as the patients from the study evaluating remyelination by PET imaging [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF].

We Preventing neurodegeneration is now the most interesting strategy to cure MS as axonal death is the cause of irreversible disability. There is an accumulating amount of evidence showing that remyelination is beneficial both in animal models [START_REF] Duncan | Extensive remyelination of the CNS leads to functional recovery[END_REF][START_REF] Slowik | The sphingosine 1-phosphate receptor agonist FTY720 is neuroprotective after cuprizone-induced CNS demyelination[END_REF][START_REF] Smith | Central remyelination restores secure conduction[END_REF] and MS patients [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF][START_REF] Bramow | Demyelination versus remyelination in progressive multiple sclerosis[END_REF]Kornek et al., 2000a) by restoring loss of function and providing neuroprotection. In the last decade, a vast number of studies therefore aimed to to enhance the endogenous myelin repair process in order to find new treatments for MS.

As detailed in chapter 1 III., remyelination failure in patients is most likely the consequence of OPC differentiation failure due to a deleterious micro-environment and the presence of OPC differentiation inhibitors, such as cytokines, chemokines, growth factors, microRNAs, myelin debris, and extracellular matrix proteins [START_REF] Franklin | Why does remyelination fail in multiple sclerosis?[END_REF]Franklin and Ffrench-Constant, 2008;[START_REF] Patel | Mediators of oligodendrocyte differentiation during remyelination[END_REF]. Therefore, huge efforts have been made to discover molecules directly enhancing OPC differentiation. Several screenings have been performed, using hundreds of already FDA approved treatments [START_REF] Najm | Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo[END_REF] or using thousands of compounds [START_REF] Deshmukh | A regenerative approach to the treatment of multiple sclerosis[END_REF][START_REF] Mei | Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis[END_REF], highlighting several candidates enhancing OPC differentiation in vitro and promoting remyelination in vivo (Figure 16). Several of them are under clinical trial [START_REF] Plemel | Remyelination therapies :a new direction and challenge in multiple sclerosis[END_REF].

Clemastine is one of the first pro-remyelinating agents that have gone through clinical trial. It was discovered after screening using an elegant technique utilizing micropillars around which OPC can differentiate and form myelin [START_REF] Mei | Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis[END_REF]. While the first results of clemastine were promising, the secondary endpoint of the phase II clinical trial (NCT02040298) showed a mild enhancement of visual evoked potential (VEP, a value measuring the speed of conduction through the visual pathway, often slowed down in MS patients because of demyelination of the optic nerve), but no improvement in EDSS and a worsening of fatigue in patients receiving the treatment.

An anti-LINGO-1 antibody treatment was also tested in a clinical trial. LINGO-1 is a transmembrane signaling protein expressed by OPCs inhibiting their differentiation [START_REF] Jepson | LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions[END_REF]. Anti-LINGO-1 treatment was shown to have beneficial effects on EAE and to promote remyelination [START_REF] Mi | LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis[END_REF][START_REF] Mi | Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells[END_REF]. The results of the clinical trial were globally disappointing at first as the treatment did not reach the first end-point (VEP-latency) but a significant difference was found at a second end-point [START_REF] Cadavid | Safety and efficacy of opicinumab in acute optic neuritis (RENEW): a randomised, placebocontrolled, phase 2 trial[END_REF]. Another clinical trial is ongoing, with an augmented number of patients to demonstrate the potential beneficial effect of anti-LINGO-1 treatment.

Limitations

A. Choosing the right MS population to demonstrate the efficacy of a pro-remyelination treatment

Theoretically, patients with RRMS and who are at an early stage in the disease course seem to be the ideal cohort, as their axons are not yet undergoing extensive neurodegeneration and are therefore available for remyelination. The best window of action for a pro-remyelinating treatment is still debated: If the treatment is given too early after a relapse, OPC recruitment will not have occurred, and therefore giving a pro-differentiating drug would not favor remyelination. If it is too late after a relapse, at that moment the inflammatory process would have calmed down, and their beneficial effect could be lost.

Patients with progressive forms of MS would theoretically be less sensitive to proremyelinating drugs, as a massive axonal degeneration would have started, making remyelination less extended.

Patients with progressive forms of MS would theoretically less sensitive to pro-remyelinating drugs, as a massive axonal degeneration would have started, making remyelination less extensive.

B. The dual role of inflammation

Promoting OPC differentiation in MS patients could potentially enhance remyelination in patients. However, one of the major flaws of this strategy is that inflammation would not be modulated.

Chapter IV: Discussion and conclusion

125

Even if OPC differentiation is boosted by a pro-remyelinating drug, it is not known how much this will counteract the presence of a deleterious micro-environment induced by inflammatory processes. For instance, in the presence of inhibitory molecules, molecules known to induce OPC differentiation have only a limited beneficial effect [START_REF] Keough | An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous system remyelination[END_REF]. One could argue that these pro-remyelinating treatments acting directly on OPCs could be given in addition to Chapter IV: Discussion and conclusion an anti-inflammatory drug. This strategy would raise several issues: inflammation is necessary for remyelination and for its first steps: proliferation and migration. Even if many experimental observations argue for a defect of OPC differentiation as the major cause of remyelination failure in patients [START_REF] Chang | Premyelinating oligodenrocytes in chronic lesions of multiple sclerosis[END_REF][START_REF] Kuhlmann | Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis[END_REF], some plaques analyzed postmortem show few or no OPCs around the lesion [START_REF] Boyd | Insufficient OPC migration into demyelinated lesions is a cause of poor remyelination in MS and mouse models[END_REF]. Promoting OPC differentiation in chronic plaques in which OPC recruitment did not occur would be inefficient.

Moreover, the potential deleterious interactions between drugs would have to be taken into account.

C. The stage of OPC differentiation failure may be different between patients

In patients, OPC differentiation can be blocked at different time-point of the process. The different markers of OPC maturation show that this process can be blocked early on in some patients, or at a pre-myelinating stage [START_REF] Chang | Premyelinating oligodenrocytes in chronic lesions of multiple sclerosis[END_REF][START_REF] Kuhlmann | Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis[END_REF]Wolswijk, 1998a). We have data concurring with this observation as the differentiation block in patients with low repair capacities [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF]) could occur at early, late of final stage of differentiation (Figure 17) . Even if the mechanism of action of molecule promoting OPC differentiation are not fully known, they can be efficient only at certain time points of the maturation process and be inactive at others, making the drug inefficient for some patients.

In other words, this approach does not take into account the heterogeneity of remyelination efficacy in patients.

D. Lack of appropriate models for validation

The major flaw of OPC pro-differentiation molecules in the way they were validated: The different molecules tested were validated using toxic demyelination, in which there is not a failure of remyelination or were validated using EAE. EAE is not an accurate model of demyelination [START_REF] Behan | EAE is not a useful model for demyelinating disease[END_REF], and as neuronal death is occurring very rapidly, the experimental evidences demonstrating a pro-remyelinating effect of a molecule as the cause for a diminished clinical score has to be taken with caution.

As mentioned earlier, models of toxic demyelination can only prove an acceleration of remyelination and not rescue a rescue of myelin repair failure. Like most of the pathophysiological features of MS, remyelination extent is highly variable from one patient to another, leading to more or less severe symptoms [START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF][START_REF] Patani | Remyelination can be extensive in multiple sclerosis despite a long disease course[END_REF][START_REF] Patrikios | Remyelination is extensive in a subset of multiple sclerosis patients[END_REF] Our data demonstrate that LT could be at least partially responsible for inducing this heterogeneity [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF]. Following a simple stimulation with antigen-presenting mimicking beads, MS patient LT had a distinctly different secretory profile compared to HD, and strong inter-individual differences between patients with high repair capacities compared to patients with a low one were also highlighted (Figure 18). This secretory profile could potentially be responsible for setting the micro-environment in a lesion and notably drive MIG activation, leading to successful or failed remyelination.

Interestingly, we found several cytokines correlated with either high or low repair capacities.

Knowing that remyelination inversely correlates with disease severity, the level of these cytokines could potentially be used as biomarkers to predict disease evolution. Of course, a lot of experimental validation and replication of our data would be needed before a clinical use but this could be a promising noninvasive technique as the measurement of the levels of cytokines secreted by patient LT only requires a blood sample. Of note, a few biomarkers are promising in MS: The level of neurofilament, released after axonal damage, dosed in the CSF of patients, can predict severity progression and initiation of the SP phase in a small cohort of patients [START_REF] Salzer | Neurofilament light as a prognostic marker in multiple sclerosis[END_REF]. GFAP and MBP could also be used to a certain extent to predict disease evolution (Housley et al., 2015a).

Capitalizing on patients with high repair capacities to develop innovative therapeutic targets

The ideal treatment would have both immunomodulatory and pro-remyelinating effects. That is potentially the case of targeting CCL19. High level of CCL19 were produced by patients inducing low OPC differentiation [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF] and this cytokine is found in MS actives and inactive plaques [START_REF] Krumbholz | CCL19 is constitutively expressed in the CNS, up-regulated in neuroinflammation, active and also inactive multiple sclerosis lesions[END_REF]. This cytokine was driving MIG activation towards a pro-inflammatory phenotype leading to inhibition of OPC differentiation [START_REF] Behi | Adaptive human immunity drives remyelination in a mouse model of demyelination[END_REF]. Using a compound that would either inhibit CCL19 or its receptor CCR7 could potentially restore the pro versus anti-inflammatory balance in MIG leading to a successful remyelination. Another interesting feature of CCL19 is that this cytokine is known to be involved in B and T cell migration and homeostasis [START_REF] Förster | CCR7 and its ligands: balancing immunity and tolerance[END_REF]. For instance, ectopic expression of CCR7 leads to the creation of tertiary lymphoid structures, similar to the ones found in the meninges of some patients with progressive MS. Inhibiting the receptor could also theoretically be beneficial by limiting B migration to the brain. Inhibiting the creation of ectopic follicle in the brain would prevent grey matter damage induced by B cells.

On the other hand, CCR7 is also critical for proper Tregs function, as CCR7 -/-mouse Tregs are unable to migrate to lymph nodes and suppress the proliferation of antigen-specific T cells Chapter IV: Discussion and conclusion [START_REF] Schneider | CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells[END_REF]. It is not known if this mechanism is CCL19 dependent (CCR7 has at least one known other ligand, CCL21), but in order to develop a drug targeting CCR7, one must be careful not to target Tregs. Of course, in order to deeper explore the potential beneficial effect of CCL19 in MS, a further testing is necessary: We must determine whether an anti-CCL19 antibody is enough to restore a beneficial MIG activation and OPC differentiation, and test if a treatment targeting CCL19 can foster remyelination in vivo.

Intriguingly, CCL19 was also highly expressed by HD LT. This result argues that the molecular chain of events leading to a successful remyelination are specific to pathological conditions.

A molecular actor having a beneficial role in physiological conditions can be deleterious in a pathological context. Therefore, we argue for a new approach in myelin repair research by focusing on patients with high remyelination capacities to find new therapeutic targets.

Genetic variants as the root cause

A. Rational of the genetic stratification

The questions left unresolved by our first study is why, between patients and HD and inbetween patients, are LT responding differently to a same stimulus. In other words, their secretion pattern can be extremely different in response to the same stimulation. As we studied a global LT population, one might argue that this differential response is just the consequence of an over-representation of a particular cell type. However, from a patient to another, the cell count for each of LT subtype evaluated (B cell, Th1, Th17, Treg) was similar.

Moreover, the number of cells for each cell type was not different between HD and MS patients. This is concurring with the literature as it is consensual that MS patients display no or very minor changes of the proportions of LT subtypes in their blood compared to HD [START_REF] Legroux | Multiple Sclerosis and T Lymphocytes: An Entangled Story[END_REF] Therefore, it is more likely that the differences in LT response are the consequences of inner characteristics of LT that could consequently induce an abnormal activation. Genetic variants responsible for MS susceptibility influencing the capacity of LT to respond to a stimuli is the most likely hypothesis. The variants are located mostly in regulatory DNA [START_REF] Sawcer | Multiple sclerosis genetics[END_REF] and can influence LT response by modifying the expression of key inflammatory genes. (Housley et al., 2015b).

The cellular events occurring during myelin repair are finely regulated mechanisms alternating between pro-inflammatory and anti-inflammatory micro-environment. If this inflammatory balance is disrupted, that could lead to remyelination failure. Since, Th cells activation can both lead to pro-inflammatory (Th1, Th17) and anti-inflammatory phenotypes (Th2, Treg), we hypothesized that genetic variants accumulated in genes involved in a specific Th phenotype could modify the global inflammatory balance leading to a modification of remyelination outcome.

We had access to a cohort of French MS patients for whom we had access to complete clinical data (clinical score, disease form, years of evolution, treatment etc) and their genotyping profile for all the MS susceptibility SNPs. We decided to focus on the SNPs associated with

C. Limitations

In this study we focused on the SNPs responsible for MS susceptibility. However, as the mechanisms driving MS triggering and disease evolution are believed to be different, it is likely that other genetic factors could drive disease evolution. It has been demonstrated for instance that variants independent of MS susceptibility can drive an heterogeneous immune response in a cohort of HD [START_REF] Li | Inter-individual variability and genetic influences on cytokine responses to bacteria and fungi[END_REF].

The SNPs kept for the genetic stratification were chosen because they have a role in Th pathways. However, several other SNP could influence remyelination: For instance, a genetic variant is associated with a gene involved in OPC biology and is therefore likely to influence the myelin repair process independently from Th cells [START_REF] Scott-Hewitt | Heterozygote galactocerebrosidase (GALC) mutants have reduced remyelination and impaired myelin debris clearance following demyelinating injury[END_REF]. More globally, the principal weakness of the approach is to consider only SNPs involved in LT functions. Even if it has been demonstrated that SNPs involved in MS susceptibility have consequences on LT [START_REF] Farh | Genetic and epigenetic fine mapping of causal autoimmune disease variants[END_REF][START_REF] Maurano | Systematic Localization of Common Disease-Associate Variation in Regulatorty DNA[END_REF][START_REF] Sawcer | Multiple sclerosis genetics[END_REF][START_REF] Simeonov | Discovery of stimulation-responsive immune enhancers with CRISPR activation[END_REF], susceptibility SNPs can also influence macrophages functions [START_REF] Raj | Polarization of the Effects of Autoimmune and Neurodegenerative Risk Alleles in Leukocytes[END_REF] and potentially others cell types involved in remyelination, like MIG and astrocytes [START_REF] Domingues | Oligodendrocyte, Astrocyte, and Microglia Crosstalk in Myelin Development, Damage, and Repair[END_REF][START_REF] Hammond | Astrocyte-Derived Endothelin-1 Inhibits Remyelination through Notch Activation[END_REF], in which the potential functional consequences of the genetic variants have not been studied.

We also did not take into account the role of environmental factors that could have a role in modulating directly or via epigenetic mechanism, the immune response and the remyelination process.

More globally, even if we highlighted two genetic subgroups having LT inducing poor remyelination, we could also notice that patients having LT inducing high remyelination were coming from different subgroups. Therefore, we believe that implementing the stratification strategy by studying the effect of the presence of SNP on MS patient's macrophages could unravel new reasons why remyelination fails or succeed.

IV. Conclusion

The era of therapeutics having pro-remyelinating and/or neuroprotective effects is at its start.

As the end goal of the treatment is changing from suppressing inflammation to promote repair, a shift in the routinely used model in MS research is absolutely necessary. In the quest of understanding remyelination failure in some patients, we provided a novel tool. By grafting MS patients LT in a demyelinated lesion, we reproduced at least partially the heterogeneity of myelin repair efficacy, providing new insights into the role of adaptive immune cells in this regenerative process.

In a follow-up study, we took advantage of our model to gather clues on understanding how the genetic background of a patient can influence the functionality of his immune cells during remyelination.

MS is a complex disease in which a lot of parameters can influence disease severity and evolution. Therefore, personalized therapy is probably the only approach that will lead to a cure.

Having the proper tools to understand patients' heterogeneity in their pathophysiological mechanisms should be the focus of MS research, and we provided a tool that unveiled a small part of the reasons of this variability.

Figure 1 :

 1 Figure 1: Representation of myelin structure and organization in the CNS and the PNS. In the CNS, myelin is formed by OL that can wrap multiple axonal segments. In the PNS, SC wrap only one axon segment.

Figure 3 :

 3 Figure 3: Molecular organization of myelin allowing fast saltatory conduction. (A) Diagram of a myelinated axon. (B) Ion current occurring during saltatory conduction. The depolarization of the axonal membrane only occurs in nodes Ranvier in which myelin is absent and voltage dependent channel are clustered resulting in an accelerated velocity of axonal conduction. The lower panel represent the changes in the axonal membrane potential during the propagation of the action potential. From (Purves et al. 2001).

Figure 4 :

 4 Figure 4: Risk to develop MS in family's age-adjusted recurrence risks for different relatives. These data represent pooled studies from population-based survey. Modified from[START_REF] Compston | Multiple sclerosis[END_REF]

Figure 5 :

 5 Figure 5: MS genetic maps with all known predisposition SNPs and associated genes and their location in the genome. In 2014, 110 SNPs were correlated with MS susceptibility. From P. De Jager.

Figure 6 :

 6 Figure 6: World map of the prevalence of MS in 2014. A clear north-south gradient can be observed.

Figure 7 :

 7 Figure 7: Frequency and spectrum of symptoms affecting MS patients. From the national multiple sclerosis society.

Figure 8 :

 8 Figure 8: Representation of disability progression in the RR (A), SP (B) and PP (C) forms of MS. Modified from (Lublin et al. 2014).

  are involved in T cell function (The international multiple sclerosis genetics Consortium (IMSGC), 2013). In addition, T cells are able to infiltrate the CNS, and MS patients have a higher proportion on myelin-reactive T cells in their blood. T cells are divided into two groups depending on the expression of CD4 or CD8 surface receptors. From a functional point of view, CD4+ cells are cells coordinating the adaptive immune response: they induce and drive the most adaptive immune answer to fight the pathogen encountered. Naïve CD4+ cells, or Th0 cells, can be polarized in different functional phenotypes according to the costimulation received during antigen presentation (Figure10): They are activated into the Th1 pro-inflammatory phenotype in response to intracellular bacteria or protozoa and into the Th17 pro-inflammatory phenotype in case of cancer or fungus. After this inflammatory phase aiming at destroying the pathogen, Th2 cells inhibit Th1 activation. Of note, Th2 have also a role in pathogen neutralization, in case of extracellular parasites. Treg are extremely efficient at calming inflammation, by suppressing T cell proliferation. Finally, T follicular helper cells (TFH) are activated and proliferate to help B cell maturation in case of a humoral immune response. CD8+ cells (or effector T cells) are soldiers of the immune response: they kill infected cells by lysing their membrane. CD4+ cells are believed to play a critical role in MS, by inducing the immune response against myelin leading to its destruction and not correctly suppressing the inflammatory attacks. CD8+ cells are suspected to be one of the main effectors of myelin destruction (Figure9).

Figure 9 :

 9 Figure 9: Simplified view of the mechanisms leading to myelin destruction in MS. A peripheral mis-activation leads to the proliferation of myelin-reactive T and B cells. The dysfunction of the peripheral tolerance induce their survival. Invading Th1, Th17 and B cells induce the first demyelination, leading to the invasion of the CNS by macrophages and to MIG activation. MIG and macrophages, in response to demyelination, induce a pro-inflammatory environment inducing a secondary myelin damage. Invading cells can be reactivated by MIG leading to the chronicity of demyelinating attacks in MS. From (Macmillan et al. 2008)

Figure 10 :

 10 Figure 10: CD4+ T helper cells differentiation pathways. After antigen presentation and co-stimulation, naïve Th0 cells can differentiate into different phenotypes : The Th1 or Th17 pro -inflammatory phenotypes, involved in anti microbian response, The Th2 or Treg anti-inflammatory and immunoregulatory phenotypes that can regulate and calm the inflammatory response and finally, the TFH phenotype, involved in B cell maturation

B

  cells are LT specialized in the humoral response: They secrete antibodies to neutralize extracellular pathogens. The role of B cells in MS has for a long time been underestimated. In the last decade, several roles in pathogenicity of these cells have been established and an FDAapproved treatment targeting B cells, efficient in PPMS, has been commercialized. B cells are found in the brain undergoing neuroinflammation. Several molecules secreted by the innate immune system are known to attract B cells into the CNS: C-C motif chemokine ligand 20 (CCL20) (Kalinowska-Łyszczarz et al., 2011), C-X-C motif chemokine ligand 13 (CXCL13)

Figure 11 :

 11 Figure 11: Representation of B-cells follicles in the meninges of patients with progressive form of MS. CD20+ B cells are infiltrating the along and in the depth of the cerebral sulci, from where they induce a strong inflammatory response leading to grey matter damage.

  but until recently, none of them can label myelin specifically. A longitudinal study was published recently: using a compound labelling myelin specifically ([ 11 C]PiB) and the combination of multiple MRI and PET-scans (Figure 12B-B'), they provided new insight about remyelination efficacy in MS patients[START_REF] Bodini | Dynamic Imaging of Individual Remyelination Profiles in Multiple Sclerosis[END_REF].

Figure 12 :

 12 Figure 12: Remyelination is occurring in the CNS of MS patients and can be studied using focal demyelination animal models. In Patients, remyelination can be visualized on brain slices. After luxol fast blue staining, demyelinated area (green arrows) and area with ongoing remyelination (red arrows) can be noticed (A). From the Wolfe Medical Publications Ltd, 1989. Remyelination can also be visualized by PET-SCAN and the specific marker [ 11 C]PiB. Here is represented the myelin baseline content of a patient (B), the demyelinated (red) and remyelinated area (blue) following a longitudinal imaging session three month after (B'). From Bodini et al. 2016. Remyelination can also be studied in animal models (C): here are shown axons in the rat cerebellum before demyelination (left panel), following the injection of the demyelinating agent ethidium bromide (center panel), and remyelination of axons four weeks post demyelination (right panel).

Figure 13 :

 13 Figure13: Sequential event in remyelination. After a demyelination injury (A), MIG and macrophages areactivated toward a pro-inflammatory M1 phenotype. Both cell types will phagocytose myelin debris even if some experimental evidence suggests a better efficacy of macrophages for this process (B). M1 macrophages and MIG will then secrete proliferation and migration factors that will trigger OPC recruitment toward the lesion (C). A switch to an M2 anti-inflammatory status occurs in MIG and macrophages , triggering the secretion of pro-differentiation and trophic factors leading to the start of the differentiation process in OPC (D) and finally to a complete remyelination (E).

  behavior during myelin repair, this data could explain why OPCs do not get the right signal at the right time to enter recruitment and/or differentiation. The differential cytokine secretion panel could be explained by a misactivation of old macrophages. Another experiment of heterochronic parabiosis model demonstrated that young macrophages are more activated Chapter I : Introduction

Figure 14 :

 14 Figure 14: Fate of remyelination and consequences on clinical course of patients. After a demyelinating injury (A), the endogenous myelin repair process can either succeed (B) or fail (B') Remyelination can fail for different reasons: Poor myelin debris phagocytosis, failure of OPC recruitment, failure of OPC differentiation. In patients, the latest hypothesis is the most documented and demonstrated.

  established a major role of LT in driving remyelination but we cannot exclude that the individual capacities of patients to instruct remyelination could also be the consequence of other cellular actors and molecular mechanisms that are independent from any LT involvement or that the LT sampled in the blood of patients are not a representative population of the LT invading the CNS in those patients.

Figure 16 :

 16 Figure 16: Pro-remyelinating compounds under clinical trial and their putative mechanisms of action. Some of them target acetylcholine or histamine receptors like benzotropine, clemastine, quetiapine and GSK239512 and induce OPC differentiation. Other intracellular receptor can be targeted by Clobetasol, Vitamine D, Liothyronine or IRX4204 to promote survival, differentiation or proliferation of OPCs. Finally, monoclonal antibodies inducing OPC differentiation are being tested (LINGO1, rHigM22). From (Plemel et al., 2017).

  human and murine OPCs do not have exactly the same biological pathways activated during differentiation, therefore candidate molecule must be tested on human OPCs prior further investigations. For instance, clemastine was only tested and validated using murine models.

Figure 17 :

 17 Figure 17: Representation heterogeneity of OPC differentiation blocking step in MS patients. The conditioned media of MIG pre-exposed to HD (n=12) or MS (n=27) LT supernatant was put on a primary culture of OPCs. The state of maturation was evaluated 72h later using 3 chronologically expressed markers: O4 (A), GalC (B) and CNPase (C). In A, B and C, the patients inducing a level of expression of one of the marker statistically inferior (2 standard deviation) are represented in pink, the one inducing a level of expression not statistically inferior are represented in purple. The percentage of patients inducing Low level of O4, GalC or CNPase compared to the total MS population is represented in D. Representative examples of patients inducing an early (O4 stage), a late (GalC stage) or a final differentiation defect (CNPase stage) compared to HD are represented in E.

Figure

  Figure 18: Heatmap representation of the LT cytokine secretion pattern of patients with high repair capacities, low repair capacities and HD. After in vitro activation, the level of expression for 72 cytokines by HD, MS HIGH or MS low patients was evaluated by luminex. Mean values for each tested cytokine were calculated. A heatmap was generated with a color coding representing in green the values lower than, in red the values higher than and in grey values close to the mean for each cytokine in HD and multiple sclerosis HIGH and LOW conditions.

  genes having a role in Th differentiation pathways. We took advantage of the model developed during the previous study to analyze the potential effect of SNPs concentrated in a specific immune pathway on remyelination.Interestingly, it was the LT of the patients who have a strong MSGB in variants concentrated in pathways responsible for antigen presenting/naïve Th0 cells and TFH cells that were inducing remyelination failure. This suggests that the first step of naïve Th0 cells activation is critical for the success of the process. If this first step of the adaptive immune system is disturbing the pro/anti-inflammatory balance, this could lead to a systematic remyelination failure. Our data suggest also that a dysregulation of TFH cells differentiation might lead to LT detrimental effect on remyelination. TFH cells are a cell type indispensable for the generation and selection of high-affinity memory B cells and plasma cells in germinal centers and are suspected to help the formation of ectopic lymphoid structure in the CNS of MS patients[START_REF] Crotty | T Follicular Helper Cell Differentiation, Function, and Roles in Disease[END_REF]. However, their role is myelin repair is totally unknown. could be tested as a biomarker predictive of remyelination capacity and/or disease evolution.ii)Highlights of customized therapeutical targetsThe genetic background is likely to induce remyelination failure as different steps. In other words, LT of patients with low repair capacities are inducing a failure of myelin repair at a step depending on the combination of genetic variants carried by an individual.Ideally, we would detect and validate molecule responsible of a change in the cellular or molecular landscape occurring during myelin repair in the context of a specific genetic background and develop a customized treatment approach.

  Chapter I : Introduction disease susceptibility. Several thousands of SNPs can be analyzed at the same time. The largest GWAS study of MS was the fruit of an international collaboration between members of the International Multiple Sclerosis Consortium (IMSGC). By comparing 475 806 SNPs in the genome of 9 772 MS patients and 17 376 healthy donors (HD), the study highlighted 34 new

	susceptibility variants and confirmed 23 others (The international multiple sclerosis genetics
	Consortium (IMSGC), 2012). Shortly after, a large meta-analysis called Immunochip was
	performed by analyzing GWAS data from MS and other auto-immune diseases and new
	variants were discovered, carrying the total to 110 susceptibility variants for MS (The
	international multiple sclerosis genetics Consortium (IMSGC), 2013) (Figure

Table 1

 1

	).. Natalizumab is a monoclonal antibody

Table 1 :

 1 Characteristics of FDA-approved treatments for MS

	Molecule	Name Company	Class	Administration	MS form	ARR relative reduction	EDSS relative reduction	Severe side effects
	Interferon β1a	Avonex ® Biogen	1 st Line	i.m	RR	18%	37%
	Interferon β1a	Rebif ® Merck	1 st Line	s.c	RR	32%	32%
	Interferon β1b	Extavia ® Novartis	1 st Line	s.c	RR	34%	29% (n.s)
	Interferon β1b	Betaferon ® Bayer	1 st Line	s.c	RR	34%	29% (n.s)
	Glatiramer Acetate	Copaxone ® Teva	1 st Line	s.c	RR	29%	12% (n.s)
	Dimethyl Fumarate	Tecdifera ® Biogen	1 st Line	oral	RR	53%	41%	PML Lymphopenia
	Teriflunomide	Aubagio ® Sanofi	1 st Line	oral	RR	36%	24%	Breathing problems
	Fingolimod	Gylenya ® Novartis	2 nd Line	oral	RR	55%	28%	Bradycardia Skin cancer PML
	Natalizumab	Tysabri ® Biogen	2 nd Line	i.v	RR	68%	42%	PML
	Alemtuzumab	Campath ® Sanofi	2 nd Line	i.v	RR	49% vs IFN β1a s.c	42% vs IFN β1a s.c	Secondary autoimmune diseases
	Mitoxantrone	Novantrone ® Mylan	2 nd Line	i.v	RR, SP	66%	64%	Cardiomyopathy Leukopenia
	Ocrelizumab	Ocrevus ® Roche	1 st Line	i.v	PP		24%	Infections

ARR: Annual relapsing rate, i.m: intramuscular, s.c: subcutaneous, i.v: intravenous n.s: non-significant. ARR and EDSS relative reductions are calculated vs placebo if not stated otherwise. In light blue, 1 st line treatments with immunomodulatory properties. In light grey, 2 nd line treatments with immunosuppressory properties.

  , yet only two molecules are available on the market: mitoxantrone for SPMS and Ocrelizumab for PPMS. Mitoxantrone is a molecule originally developed to treat lymphoma. It is a DNA intercalating agent that blocks topoisomerase II DNA repair. It induces systemic immunosuppression by being cytotoxic to highly proliferating cells such as T cells and macrophages. It has a moderate efficacy on disease progression, and can have irreversible side effects like cardiomyopathy and sterility. Here again, very careful monitoring of patients

is necessary. Ocrelizumab was FDA-approved in March 2017 as the first treatment against PPMS. Like Mitoxantrone, Ocrelizumab was originally developed to be used against lymphoma. It is an anti-CD20 antibody targeting B cells, a cell type that has been ignored for a long time in MS research. Ocrelizumab slowed disease progression in PPMS patients by 24%,

Chapter IV: Discussion and conclusion

Abbreviations

Chapter 2: Evaluation of the role of lymphocytes in remyelination and defining the molecular basis for an efficient myelin repair in patients. Chapter II : Evaluation of the role of lymphocytes in remyelination and defining the molecular basis for an efficient myelin repair in patients

III. Supplementary unpublished results

To evaluate the global effect on remyelination, the influence of MS LT on SC remyelination has also been evaluated using a specific marker of PNS myelin. Our results show a similar results than for OL remyelination as MS LT impede the process compare to HD LT (supplementary Figure 6).

IV. Patent

PROGNOSIS OF DEMYELINATING DISEASES PATIENTS AND TREATMENT THEREOF

Application number: EP16194192 Submitted: 17 October 2016

FIELD OF INVENTION

The present invention relates to the field of demyelinating diseases, to methods for prognosing demyelinating diseases and to methods for treating thereof.

ABSTRACT

The present invention relates to an in vitro method for the prognostic of a subject affected by a demyelinating disease comprising detecting and quantifying the level of at least one of CCL19, IL-15, ENA78 and/or TRAIL in a biological fluid from the subject. The invention also relates to the treatment of a subject affected by a demyelinating disease comprising the use of at least one modulator of CCL19, IL-15, ENA78 and/or TRAIL.

B. Perspectives and expected outcomes

Additional experiments to complete the study

As the effect of a SNP can be tenuous, we need very accurate biological readouts covering all steps of myelin repair to identify where the SNPs on LT could act. We therefore plan to complete our preliminary data for the 2 nd article to increase the numbers of biological read outs for a more thorough multivariated analysis.

We will indeed maximize the chances to spot any changes in the cellular of molecular landscape occurring during myelin repair by evaluating LT secretory pattern. An RNAseq analysis is also scheduled to decipher even more precisely the effect of the SNP on LT transcriptomics. MIG secretory pattern will also be assessed by Luminex to identify the molecular interplay between MIG and OPC. We will also pursue our analysis in vitro by quantifying OPC differentiation/proliferation/migration in response to MIG conditioned media pre-exposed to LT supernatant.

Compiling all the results, completing the ones presented in article 2, a bioinformatics analysis will be performed. The successful in vivo remyelination or the proper OPC proliferation/migration/differentiation will be set as an end-point value and the genetic, cellular and molecular elements needed will be analyzed. The analysis will be run both ways:

the first way to see if there is a correlation between our "Th cell based" genetic stratification and patients LT effect on inflammation and remyelination, the second way by evaluating what SNPs or association of SNPs are correlated to a high or low repair capacity.

We will be therefore able to pinpoint any subtle change in the cellular and molecular actors involved, and its consequences on myelin repair.

Expected outcomes

Several outcomes with potential clinical will be obtained: i) Correlation between the genetic profile of patients and remyelination capacity If a SNP or a combination of SNPs is strongly correlated with remyelination failure. We would then validate this SNP in vitro. We would use the CRISPR technique, that is used to study biological consequences of a genetic variant on LT functionality and validated [START_REF] Simeonov | Discovery of stimulation-responsive immune enhancers with CRISPR activation[END_REF]. If this SNP or this combination of SNPs is validated biologically, it

Chapter IV: Discussion and conclusion