Pathophysiology and Immunopathology of MS

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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) (Compston and Coles, 2008; Dendrou et al., 2015) 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

Structure and physiological myelination 

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 (Aggarwal et al., 2011; Kursula, 2008). 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).

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 (Bercury and Macklin, 2015). 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 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 (Jessen and Mirsky, 2005). 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).
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 (Zalc et al., 2008). 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 voltage-dependant 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) (Hartline and Colman, 2007). 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 (Fünfschilling et al., 2012; Simons and Nave, 2016). 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 (Rasband et al., 2005). 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 (Byravan et al., 1994; Dai et al., 2001, 2003; Wilkins et al., 2001) . 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.
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).

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.

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 % 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) (Compston and Coles, 2008; Hansen et al., 2005), 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 (Strominger, 1986). 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) (Hollenbach and Oksenberg, 2015).

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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 (Gregory et al., 2007; Munoz-Culla et al., 2013). 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 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 5) . The vast majority of the SNPs are closely associated with genes having a role in immune pathways (Sawcer et al., 2014).

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 (Okuda et al., 2009). 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 (O’Brien et al., 2010).
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 (Piédavent-Salomon et al., 2015). 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 pro-inflammatory 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 (Esposito et al., 2015). 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 (Bieber et al., 2010). 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 (Olsson et al., 2016)) and the fact that the loci discovered for MS susceptibility only explain half of the genetic predisposition risk for MS (Zheleznyakova et al., 2017). 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 (Xiang et al., 2017).
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 (Baranzini et al., 2010). 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 (Maltby et al., 2015, 2017).
Multiple other putative epigenetic mechanisms have been proposed to be responsible for MS susceptibility and severity (Küçükali et al., 2015), 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.

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 (Ascherio et al., 2010; Lucas et al., 2015): 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 (Duan et al., 2014; Martinelli et al., 2014). 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 (Gale C.R., 1995).
Vitamin D has potent immunomodulatory effects that could explain its protective role for MS (Ascherio et al., 2010; Koch et al., 2013; Prietl et al., 2013). 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.

Table of contents :

Chapter I: Introduction
I. Multiple sclerosis: Etiology and treatments
1. Foreword
2. Myelin
3. Etiology
4. Clinical description & existing treatments
II. Pathophysiology and Immunopathology of MS
1. Inflammation, demyelination and neurodegeneration
2. Role of T cells in MS and animal models
3. Role of B cells in MS and animal models
4. Role of Macrophages and Microglia in MS and animal models
5. MS Lesions
III. Remyelination
1. Forewords
2. Histological description and clinical relevance
3. Mechanisms of remyelination
4. Remyelination heterogeneity: causes of remyelination failure
IV. Aims of the project
Chapter 2: Evaluation of the role of lymphocytes in remyelination and defining the molecular basis for an efficient myelin repair in patients.
I. Introduction
II. Article 1 and contribution
III. Supplementary unpublished results
IV. Patent
Chapter 3: Linking MS susceptibility variants to remyelination capacity
Chapter 4: Discussion and conclusion
I. Role of lymphocytes in remyelination
1. Experimental evidence
2. Cellular mechanisms involved
3. Modeling LT role in remyelination
II. Enhancing endogenous remyelination: acting directly on OPCs
1. Rational
2. Limitations
III. Deciphering patient’s remyelination heterogeneity to determine the prerequisite for efficient myelin repair in MS
1. Involvement of LT in heterogeneity.
2. Capitalizing on patients with high repair capacities to develop innovative therapeutic targets
3. Genetic variants as the root cause
IV. Conclusion
Bibliography

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