Abstract

Multiple sclerosis (MS) is a complex progressive, disabling neurological disease that involves both genetic and environmental factors with the pivotal role of immune cells. MS lesion is also a crucial fact that is triggered by the disruption of the blood-brain barrier and is involved in its pathobiology. Several risk factors including environmental, epigenetic, and genetic are implied and play an important role in the causation of MS e.g., environmental (sun exposure, ultraviolet rays, and different latitudes), gender-based (hormonal effects), exposure to infection (EBV), the role of vitamins (Vitamin D), familial predisposition (siblings and first-degree relatives) and genetic factors (MHC and HLA). MS has long been considered T lymphocytes disease because of an important genetic risk factor Major histocompatibility complex (MHC) class II locus. Infact, the T cytotoxic (CD8+) and T helper (CD4+) cells have been the main target for immunomodulatory and immunosuppressive treatment in MS. The diagnosis of MS depends on the history and clinical presentation of the disease, supplemented by MRI findings of CNS as well as biochemical changes in it.  In this short communication, the pathophysiology of disease and the role of T lymphocytes in the causation of MS has been discussed followed by the understanding of the mechanism of several factors and their interaction that may help in designing or formulating combinational drugs or therapy for the benefits of patients of MS. This article has also elaborated various promoting factors for the causation of MS, its progression to understand the complex mechanism of the disease. It emphasizes the need for specific or multiple biomarkers to scan and confirm MS along with better non-invasive techniques such as MRI.

Keywords:    Multiple Sclerosis, Common Disease Common Variant (CDCV), Genome-Wide Association Study (GWAS), Single nucleotide polymorphism (SNP), major histocompatibility complex (MHC), Matrix Metalloproteinases (MMPs)

Introduction

Multiple sclerosis is a chronic autoimmune, inflammatory, demyelinating neurological disease of the central nervous system (CNS), which is mediated by lymphocytes and affects people differently¹⁾. According to the National Multiple Sclerosis Society, more than 2.3 million people are identified worldwide with MS²⁾. The disease affects people between the ages of 20 to 40 years and shows mild (no disability) to chronic symptoms (increased disability with time). Women are at higher risk than men. Various factors, either alone or in an association, are responsible for the disease including environmental, genetic, and nutritional.^(3,4) The lesion in MS involves T lymphocytes with activation of microglia and is hallmark pathophysiology. In this mini-review article, we are focusing on the complexity of MS to understand multifactorial aspects with its complex interaction for a better understanding of the disease process and its application for future therapies.

Role of Immune cells in MS

Multiple sclerosis (MS) causation is the result of unregulated immune cells, which are converted from physiological monitor to pathological marker due to various pathological reasons. The T immune cells react against basic myelin protein antigens.⁵In normal circumstances, an intact blood-brain barrier prevents entry of damaged cells or pathogen into the central nervous system (CNS). The T lymphocytes recognize myelin derived antigen over microglia as antigen-presenting cells (APC) and undergo clonal proliferation. The disease is initiated by TH1 and TH17 (CD 4+) cells that react against myelin antigens and secrete cytokines. Researchers have identified the role of T helper 17 (Th17 or CD4+) cells. The TH1 cells secrete IFN-γ, which activates macrophages. TH17 cells also promote the recruitment of leukocytes. The demyelination is caused by these activated cells and their cytokines IL-23, IL-17, and IL-6^(6,7). The resulting inflammatory reaction causes destruction of the oligodendrocytes-myelin sheath. The characteristic demyelinated lesion and the infiltrate in plaques with surrounding regions of the brain consists of T cells (mainly CD4+, some CD8+) and macrophages. The lesion has CD8+ T cells and CD4+ T immune cells in the ratios of 100: 1 to 50: 1. The other characteristic feature of MS includes the continuous synthesis of immunoglobulins (oligoclonal IgG) in cerebrospinal fluid (CSF)^8). 

Experimental Autoimmune Encephalomyelitis (EAE) animal model of MS has shown demyelination and inflammation of axons after myelin proteins injections. The major concepts and understanding of MS pathogenesis have been derived from these model studies only. The experimental disorder can passively be transferred to non-immunized animal models. In the experimental mice model, Th17 (CD4+) cell population was found responsible for EAE. The similarity was confirmed with the presence of CD4+ and CD8+ cells expressing IL-17in MS^(5,9). The Th17 cells express chemokine receptor 6 (CCR6), which are transported to choroid plexus into CSF and perivascular space. The chemokine subfamilies CCR4, CCR5, CCR6, and CXCR3 from both peripheral blood and CSF had a similar type of structure and use CCR5 or CCR6 to enter the brain. The reduction in chemokine receptor expression, except CCR4, was observed after T cell activation. The transport mechanism into the brain in MS and activation of pathological initiation may provide new targets for drug therapy. The chemokines expressed on venule endothelial walls are responsible for T cell migration into the brain. CCR6 interacts with CCL20/MIP-3α expressed on vascular endothelium for transport⁸⁾. These chemokines bind T cell chemokine receptors for extravasation.

The blood-brain barrier (epithelial layer) has tight junctions. The cytokines like IL-17 and IL-22 from Th17 cells lose these tight close junctions. These cytokines enhance recruitment and adhesion of T cells, which initiates a cascade of inflammation in the brain, perivascular infiltration, and glial cell destruction⁹⁾. Entry of immune cells into brain parenchyma is supported by the interaction between perivascular Antigen-presenting cells (APC) and macrophages secreting matrix metalloproteinases (MMP2 and-9). The MMPs degrade the transmembrane receptors, which are anchoring astrocytes (Dystroglycan) to the basal parenchyma membrane^(10,11). Matrix Metalloproteinases (MMPs) are zinc-dependent endopeptidase enzymes having a role in inflammation, invasiveness of the tumor, metastasis, and angiogenesis. The MMP9 (Gelatinase B) is observed to degrade extracellular matrix (ECM) and myelin basic protein and facilitate inflammatory cells recruitment in CNS^(12,15).

CD8+ T cell receptors also express different CD8+ clones with conserved recognition region for an antigen. T cells secrete various Cytokines IL17, granzymes, and perforin. The perforin plays an important role in axonal injury and neurological disability in the MS lesion. The perforins released as granules from CD8+ cells form holes in the target cell membrane, which causes intracellular molecule leakage and results in cell death⁽¹⁶⁾. CD8+ T cells secrete lymphotoxin and other cytokines and are responsible for early acute and progressive phase of neuron degeneration in MS^(17).

CD4+ self-reactive T-cell proliferation inhibition is mediated by cytokine IL-10 and transforming growth factor-beta (TGF-beta), which are regulated by CD8+ cells. The antigen presentation cell (APC) presents antigens to CD4+ T cell receptors. The antigen-binding to receptor enhances CD4+T cell differentiation into various subtypes like   IFN-γ, secreting TNF-α, typeTh1 (CD4+) T cells, TGF-β, and IL-10 secreting Th2 CD4+ cells.  Regulatory CD4+T regulatory (TREG) cells express FoxP3 and other transcription factors for proliferation^(18). All of these mentioned subtypes of T cells down-regulate immune cells⁽¹⁹⁾. CD4+ self-reactive T-cell proliferation inhibition is mediated by secretory cytokine IL10 and transforming growth factor-beta (TGF-β), which are secreted by CD8+ regulatory cells. Any attempt to delete CD8+T cells from the MS lesion could potentially worsen the disease by eliminating CD8+ T regulatory cells. Therefore, the maintenance of the CD8+ T cell population may control MS progression.

Environmental factors

Multiple sclerosis is a chronic acquired neuron inflammatory disease. Amongst its causative factors, both environmental and genetic factors are responsible²⁾. Family clustering of disease and variable frequency are two identified epidemiological factors in the world⁽¹⁹⁾. Prevalence of MS amongst people-living in equator zones are lesser and increases amongst people living in the temperate zone.²While family clustering is decided by genetic factors, the regional differences are usually influenced by both genetic and environmental factors. The environmental factors like viral infection e.g., exposure of population with Epstein-Barr virus (EBV), Vitamin D deficiency, use of tobacco, environmental pollution; or epigenetic factors may cause gene silencing or alter gene expression through histone remodeling complexes and DNA methyltransferase⁽²⁰⁾. High salt intake is also associated with risk factors in autoimmune disorders because of the induction of pathological T-helper 17 cells^(16).

Gender-based Susceptibility

Environmental factors like ultraviolet exposure or viral infections during pregnancy may affect the risk of MS development later in life.²¹ The genetic role in MS development with a higher risk in first degree relatives and decreasing with distant relations are discussed in this paper. The hormone and environmental factors also influence epigenetic DNA modifications differently in men and women. MS is observed to be more prevalent in women than men across the globe as indicated from the MS prevalence ratio of women to men 2.3-3.5:1 in the last decades⁽²²⁾. Predominance in women is also affected by latitude⁽²³⁾. The reason may be that expressed genes in humans might be from paternal or maternal alleles due to methylation and histone modifications. The numbers of imprinted genes in the developed brain are reported to be sex-specific parental allelic bias⁽²¹⁾.T he more prevalence of MS in females can be associated with hormone levels also. The 20 papers’ systematic review has shown multiple sclerosis relapses reduction during pregnancy. The relapses were increased after delivery⁽²⁴⁾

The hormone level like estriol, progesterone, prolactin, α fetoprotein, early pregnancy factor (EPF), and leptin change during pregnancy. The early pregnancy factor (EPF) in an animal model study has shown a positive effect in encephalitis (EAE) in recovery⁽²⁵⁾

Regulatory T cells and T helper cells (Th2) were increased in contrast to Th1 and Th17 helper cells which were reduced in several studies. Though MS susceptibility regions have not been confirmed on the X chromosome, the X chromosome has a definite role in autoimmunity. The women need only one active X chromosome to avoid an excess of active genes for normal growth. Sometimes the other X chromosome genes are also expressed and might contribute to risk factors. The one overexpressed gene in the T cell was found to be the Kdm6a gene to code for a regulatory protein. This protein modifies the structure of DNA and modulates gene expression in the cell. As seen in the experimental mice model, the Kdm6a gene is associated with a higher risk of MS.

As lifestyle disorder contribution, some of the studies suggest diabetes mellitus type II association as a risk factor in multiple sclerosis but more research is needed to confirm and explain this⁽²⁶⁾

Epstein Barr human virus (EBV)

Role of B cell-tropic Epstein–Barr virus (EBV) nuclear antigen 2 (EBNA2) are well known in MS, systemic lupus erythematosus, rheumatoid arthritis, and Sjogren’s syndrome. EBV infection reduces EBV-specific CD8+ T cells ability to act in Multiple sclerosis^(27,28).

The Epstein Barr human virus (EBV) infects naïve memory B cells in germinal centers (GC), where these infected B cells (latent) stay, proliferate, and then enter the brain through circulation in MS. These infected B memory cells in CNS produce oligoclonal IgG bands and pathogenic autoantibodies. The B cell receptor (BCR) and EBV proteins are expressed on latently infected auto-reactive memory B cells. The EBV (human virus) modulates MS relevant immune responses of both memory B cells and memory T cells by controlling their differentiation pathways. The EBV infection to immune cells generates auto-reactive T cells in lymphoid tissues, which migrate into CNS. The survival co-stimulatory signals from EBV-infected B cells mediate antigen presentation to CD28 receptors expressed on auto-reactive T cells to stimulate apoptosis inhibition and inflammation induction. The localized CNS auto-reactive T cells are reactivated by infected memory cells presenting antigens bound to MHC molecules. These autoreactive T cells produce IL2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-beta cytokines. These cells also recruit other inflammatory cytotoxic T cells to disrupt oligodendrocytes, myelin, and neurons. The infected memory B cells enhance the auto proliferation of type 1 T helper (Th1) cells and CD4+ T cells. The type 1 T helper (Th1) cell and CD4+ T cells recognize auto-antigens and cause inflammation in both B cells and MS lesions. These cells interact with HLA-DR and the RAS guanyl-releasing protein 2 (RASGPR2) for inflammation^(29,30).

Role of Vitamin D

The role of vitamin D is well established in many cancers, cardiovascular diseases, type 2 diabetes mellitus, infectious diseases, mental disorders, and autoimmune disorders⁽³¹⁾. Active form of Vitamin D (1, 25-dihydroxy vitamin D or Calcitriol) is synthesized in the body from UV (290–315-nm) rays. Vitamin D is essential for activation and proliferation and differentiation of immune cells including T helper cells, B cells for antibody secretion, and tissue-specific immune cells.^32).

Sun exposure and vitamin D are independent risk factors for CNS demyelination. The greater prevalence of MS is associated with higher latitude and low UV exposure in Epidemiological studies. A lower level of 1, 25(OH)D increases the risk of demyelination at latitude gradient. The higher sun exposure and a high level of vitamin D have shown a decrease in demyelination⁽²¹⁾. In mice, the expérimental auto-immune encéphalites (EAE) was controlled with UV light exposure⁽¹³⁾. A genetic defect in the vitamin D synthesis pathway increases the MS susceptibility by decreasing the blood 1,25(OH)D level. An increase of 1,25(OH)D levels by decreased the odds of getting MS is approximately 50%^(14,5). The primary generation of Th1-type T-cell responses is also inhibited by 1,25(OH)D both in vitro and in vivo.

Familial genetics

Genetic factors appeared to play an important role in the causation of MS. The risk of MS is about 15% higher in a first-degree relative and roughly 150-fold higher in affected monozygotic twins^5).

Genetic studies in twin and familial clustering suggest a genetic contribution to MS, where monozygotic twins had a higher clinical relationship (3% to 7%) than fraternal twins⁽³⁴⁾. In another study, a 30% higher risk was reported in the monozygotic twins than the normal population^(34,35). Children of both parents diagnosed with multiple sclerosis, had a higher risk than half-siblings or step-siblings⁽³⁶⁾. More than 500 meta-analysis studies have shown 18.2% recurrence risk in monozygotic twins, 2.7% in their siblings, and  16.8%  in sibling relatives⁽³⁷⁾. In another study of 216 twins from a population of 50 million people in Italy had shown  0.48 inheritable factors, 0.29 for shared environmental factors, and 0.23 for individual-specific environmental factors⁽³⁸⁾. The other Swedish population study of 28,396 people suggested sibling recurrence risk (λs = 7.1) and heritability estimated recurrence of 0.64. The data is based on 348 proband twins with MS without any shared environmental component. They found 0.35 individual-specific environmental factors⁽³⁹⁾. However, this twin heritability based meta-analysis methodology have their limitations, because of size and ascertainment. Biometric multigroup analysis under liability threshold model has tried to explain role of genetic factor to some extent but environmental factors contribution to the disease also needs to be considered for these studies^(40,41).

Genetic factors associated with MS

Identification of genes responsible for MS aret specifically well defined and appeared polygenic.²The association of MS with the human leukocyte antigen (HLA) gene in major histocompatibility complex (MHC) has been studied for over three decades^(42,43). HLA gene cluster on chromosome 6p21 is strongly and consistently linked to disease and confirmed by using microsatellite markers. HLA genes are located within the highly polymorphic major histocompatibility complex (MHC) region and code for polymorphic cell surface glycoproteins. These glycoproteins interact with class I non-self-intracellular or class II extracellular proteins executing immune response⁽⁴⁴⁾. Initially the susceptible marker for MS was understood in HLA class I genes and later found to be in the class II region (HLA-DR2)⁽⁴⁵⁾. The classical class II MHC molecules like HLA-DR, -DQ, and DP consist of an alpha and beta chain. These are receptors for processed peptides (e.g., bacterial peptides), which present these peptides primarily to CD4+ T-lymphocytes. The DR2 type has two molecular types DR*15 and DR*16.

The role of DRB1*15(DRB1*15:01) is found in MS causation with a new advanced genotyping technique⁽⁴⁵⁾. HLA-DRB1*15 and HLA DRB1*21 alleles are associated with major risk haplotype in MS. These haplotypes increase 3 times more risk of MS. The class I MHC molecules regulated by HLA-A*02:01 allele have shown a protective role against MS⁽¹⁴⁾. The findings of Genome-Wide Association Studies (GWAS) have significantly improved the understanding of MS concerning MHC and identified additional associations with the interleukin (IL) genes^(46,47). Approximately 50 susceptible loci are identified with GWAS, which is involved in the various immune response like the gene for interleukin (IL)-2 and IL-7 receptor⁽⁴⁸⁾ C type Lectin domain family member16 A (CLEC16A) and CD226 genes, which might influence MS inheritance^2).

GWAS is based on the common disease/common variant hypothesis (CDCV). According to the CDCV hypothesis, the susceptibility to common disease is decided by similar variants with lower permeation in contrast to other multiple rare variants (MRV) hypotheses. The MRV theory supports susceptibility determination by many rare variants with higher inclusion.

Numbers of identified genes, coding soluble proteins in the MS immune response, are cytokines and receptors, co-stimulatory molecules, and cytoplasmic signaling molecules. The GWAS reported 23 Single Nucleotide Polymorphism associations, 34 new associated variants (29 with genome significance), and 05 variant suggestions⁽⁴⁸⁾. Out of these 57 SNPs variants, 81% variant were localized in exon mRNA and 21 SNPs were involved in other autoimmune disorders.  A gene in close neighborhood to these 57 SNPs code for signaling molecule is responsible for immune cell activation and proliferation⁽⁴⁹⁾. Multiple sclerosis linkage studies could not answer all quarries for multiple sclerosis risk. The reason might be i) nonreproducible results and ii) non-mendelian inheritance⁽⁴⁹⁾. According to non-mendelian inheritance or common disease common variant (CDCV)  theory, small common genetic variations with high allele frequency in the population may be responsible for MS⁽⁴⁸⁾. The segregation analysis in familial studies suggested the role of one locus with moderate effect ((HLADRB1 15:01) and many others loci with small effects⁽³⁶⁾. The Genome-Wide Association Study (GWAS) has identified rs12722489 single nucleotide polymorphism in the first intron of IL 2 Receptor A (IL2RA) to justify  CDCV theory by using the Chip Array technique. This SNP is different from the MHC region linked to MS. The IL2RA gene encodes the α chain of the interleukin-2 receptor involved in various immune response⁽⁵⁰⁾. Numbers of genome-wide variants were discovered in other autoimmune disorders also by GWAS and some common in MS.  TAGAP gene locus is associated with MS, type I diabetes, and rheumatoid arthritis in the same direction and celiac disease in opposite direction⁽⁵¹⁾. These susceptible alleles in multiple sclerosis are associated with an increase in risk factors. The three associated alleles “rs6897932 in IL7R, 46 rs2104286 in IL2R, and 82 in rs1800693” variants increase soluble receptor concentration to inhibit signaling on the membrane. The risk allele rs6677309 is found to reduced CD58 and FOXP3 expression by modulating regulatory T lymphocytes⁽⁵²⁾. Due to the polygenic nature of gene variants in MS, it is difficult to predict susceptibility because of one single factor⁽⁵³⁾. The aggregated risk score of multiple genetic variants was associated with clinical course and sub phenotype, which may be important for clinical infomation^(54).

Pathology of the lesion in MS

Lesions in MS histopathologically are characterized by inflammatory demyelination plaque in the brain most commonly in the periventricular region, spinal cord, and optic nerves. The lesions initially begin with demyelination of myelin sheath, loss of oligodendrocyte and inflammatory infiltrates of activated T lymphocytes and macrophages⁽⁵³⁾. The comparative preservation of axons and neurons in these early lesions differentiate MS from other pathology showing local inflammation⁽⁵⁵⁾. After the initial attack, healing occurs with gliosis leaving a shrunken scar²⁾. The inflammatory T cells in the perivascular space and parenchyma enhance more movement of T cells, B cells, dendritic cells, and natural killer cells. The cytokines destroy neighborhood cells⁽⁶⁶⁾. Demyelination starts because of complement depositions, opsonization, and microglia. The macrophages activation causes neural cell death^(57).

Diagnosis of MS

There are no specific markers or laboratory tests for the diagnosis of MS; various investigations need to be combined with the clinical picture for final diagnosis.  The diagnosis is based on the demonstration of two or more lesions separated in time and space i.e., two or more disease relapses affecting different parts of the CNS. The diagnosis depends on patient history, anatomical and biochemical changes in CNS.  MRI is the most important diagnostic investigation and is a very sensitive tool for brain and spinal cord lesions. The characteristics of plaque lesions in two or more separate areas of the brain, spinal cord, and optic nerves are observed by MRI with intravenous contrast agent such as gadolinium.

The cerebrospinal fluid (CSF) examination may show increased lymphocytic population in the acute phase and oligoclonal bands of immunoglobulin (IgG) in between attacks. Although oligoclonal bands are found in 70-90% of patients but are not specific for MS and indicate only intrathecal inflammation of CNS.

The acute lesions are found in white matter, which contains variable perivascular, interstitial lymphocytes, plasma cells, and macrophage infiltration with loss of myelin most commonly in the periventricular region, optic nerves, and subpial region of the spinal cord. This is followed by loss of oligodendrocytes and increased reactive astrocytes. The comparative preservation of axons and neurons in these lesions differentiate MS from other pathology showing local inflammation⁽⁴⁸⁾. The subacute form of the disease is characterized by demyelinating lesions in CNS with bands of relatively intact white matter present alternatively. After the acute attack in MS gliosis follows and a shrunken scar is formed.

Clinically the disease progression in MS   patients is characterized either into four forms:

(a) Relapsing-Remitting MS: marked by flare-ups of symptoms (relapses or exacerbations of disease symptoms) followed by periods of remission (clinical conditions or symptoms improve or disappear completely),

(b) Secondary progressive MS:(relapsing-remitting disease) The disease continues to worsen with or without periods of remission of symptom severity (plateaus),

 (c) Primary progressive MS: symptoms continue to worsen gradually without relapses or remissions. These patients may experience occasional plateaus of disease severity. The primary progressive MS is more resistant to traditional drugs,

(d) Progressive-relapsing MS: a rare form affecting less than 5% MS population. It is progressive from the beginning with intermittent flare-ups with worsening of symptoms and without remission phase.

Conclusion

MS is a multifactorial disease involving genetic and environmental factors in its causation, with the pivotal role of immune cells. Many therapeutic agents and drugs are implicated as modalities to reduce the severity of the disease. The developed drugs are mostly immune cell modulators, which suppress MS relapses, disease progression, and control disability. Currently, there is no single complete therapeutic regime available for the disease. Simultaneously, strategies for the development of new therapy or combinational approach, targeting multiple factors need to be advocated. The multiple biomarkers need to be developed to scan and confirm MS along with better noninvasive techniques, such as MRI. Vitamin D and hormonal therapy could be tested along with present therapies. The KDM6A in T-cell modulation is a new therapeutic target in autoimmune disorders with a female preponderance, but it should be further studied in detail.

Acknowledgment: Academic support of Shivaji College, University of Delhi, Delhi under DBT star scheme, Govt. of India.