The Immunological Role of Genetics, Environment, and Pathogens in Multiple Sclerosis Autoimmunity
Author: Jenny M. Willis
The following is a term paper the founder wrote in grad school during a semester of Independent Study in Immunology (Spring 2019).
INTRODUCTION
Multiple sclerosis (MS) is a disease that has been confounding scientists since it was first systematically characterized by Jean-Martin Charcot in 1868 as 'la sclérose en plaques,' which is French for “multiple sclerosis,” and translated from Latin, means, “many scars” (Orrell, 2005). Charcot’s work set the puzzle before us, and we researchers have been trying to understand it ever since. We have been searching for the cause of MS rigorously long before 1981 when the first magnetic resonance images (MRIs) of brain lesions were captured (NMSS, 2018). As we have learned since, MS is a multi-faceted disease with several potential triggers relating to immune, environmental, epidemiological, genetics, and several infectious agents such as viruses and bacteria (Getts et al, 2014), (Hassani et al, 2018), (NMSS, 2018), (Rosche et al, 2004), (Willis et al, 2009).
Etiology
Yet still, ~151 years since Charcot’s work, the exact cause of the self-directed onslaught of myelin and neuronal axons eludes us. Some of the hallmark characteristics of MS are demyelination, axonal damage, and inflammation that results in disability (Hassani et al, 2018), (Kouwenhoven et al, 2001), (Ramasamy et al, 2017), (Willis et al, 2009). We have learned that MS is a chronic, neuro-inflammatory condition of the central nervous system (CNS), and there are several different forms of the disease ranging from relapsing-remitting to chronic progressive (Hassani et al, 2018). One of the things that makes MS so complex is the fact that it is not only a disease of the CNS, but it is also autoimmune in nature (Orrell, 2005), (Kouwenhoven et al, 2001), (Coico & Sunshine, 2015). There are also other factors dealing with epigenetic mechanisms that have also shown a role in the development of MS, specifically miRNA, DNA methylation and histone modification (N’daiye, 2018). There are additional factors in the etiology and pathogenesis of MS that are major players in the disease process, but everything takes place within the CNS and is the result of an autoimmune response.
Having said all of that, I would like to hone in on a few specific things for this research paper. The focus of this paper is to survey some literature surrounding the immunological aspects of MS that may give clues about its cause, and how the autoimmune process initiates and continues. I will discuss the key features of MS (genetics, environment, pathogens and inflammation) as they relate to the immune system and what I have learned in Immunology this semester. For pathogens, I will explore the role viruses may play in MS, including a tidbit about intracellular viruses and a smidgeon on c-type lectin receptors in autoimmunity. This is important to learn because determining the roles of the etiology and progression of MS may enable us to effectively treat and cure this disabling disease.
MS: Neurological vs Autoimmune
The neurological aspect of MS comes from the fact that the origination of and actual tissue damage takes place within the CNS. But the more I learn about the mechanisms of the disease and the disease process, it seems clearer that the immune system is the main player and the CNS is just sort of where the story takes place. Some of the tissues within the CNS such as the brain have some pretty unique features which makes them behave in certain ways when the immune system goes haywire, and this deals with a term that I recently learned about and will discuss later called “immune “privilege” (Coico & Sunshine, 2015), (MMD, 2009), (N’diaye, 2018). With autoimmunity in MS, Th1 cells somehow infiltrate the blood-brain barrier and wreak havoc to tissues within the CNS (Coico & Sunshine, 2015). What we have yet to figure out is why certain components of the immune system leak into the CNS and initiate such a destructive path. The main point is that there may be significant neurodegeneration that occurs in MS, but it only happens as a result of the continual autoimmune response that is involved in the disease process of MS.
When the entire system as a whole that deals with immune responses just plain malfunctions and starts to attack its own healthy cells and tissues, that’s called an autoimmune response. Unfortunately, if a person’s body has already developed this type of response to self, the process continues because it is fooled in thinking that is the normal and proper function. The result is a disease where the immune system is in constant destructive hyper-drive towards a part of itself (Coico & Sunshine, 2015). Why would the body attack its own vital tissues?
AUTOIMMUNE DISEASES
Some of the things about autoimmunity I have learned this semester in Immunology are probably old hat news and boring to you, but new to me, so please bear with me as I go through them. I did not previously know that all autoimmune diseases are classified as B- or T-cell mediated diseases. I also did not know that that Immunologists consider MS a T-cell mediated disease pretty much without debate. As it turns out, most B cell responses are really mediated by T cells, so T cells must be really important! Both B and T cells, even though they are made in different parts of the body, play vital roles working together within the immune system as a whole, and B cells are the actual APCs that activate the T cells (Coico & Sunshine, 2015). Another fascinating thing about autoimmune diseases that I learned is that they are classified by the mechanism that seems most responsible for causing the organ damage in the body, which is either antibody or T cells (Coico & Sunshine, 2015). So it now makes sense that MS is classified as a T-cell mediated disease because it is the T cells that play the major role in organ damage within the CNS.
MS is T-cell Mediated
There seems to be a surmounting stockpile of immunology research that identifies MS as being classified as a T cell disease. One of the main reasons is that some of those susceptibility genes have to do with the expression of some HLA (Human Leukocyte Antigen) Class II alleles. Immunopaedia.org (2019) describes HLA as being part of the Major Histocompatibility Complex (MHC), which is actually an entire set of genes that help to manage and direct T-cell mediated immunity. There are two main classes of MHC involved with T cell immunity. The Class I MHC do the job of presenting the peptides to the CD8+ T cells and the Class II MHC genes present peptides to the CD4+ T cells (Immunopaedia.org, 2019), (Coico & Sunshine, 2015). The actual peptides they present are made of either ‘self’ proteins or invading pathogenic proteins (Immunopaedia.org, 2019). Because the HLA genes are so polymorphic, immune responses are unique in every individual person and they are associated with different diseases. Once learning about HLAs, it was not a surprising to hear that they are involved in MS. The HLA complex is the human version of the MHC, and it’s associated with autoimmunity in general, not just in MS, which makes sense, because most autoantigens are really just proteins that activate T cells, and HLA molecules are important in presenting the peptides to the T cell receptors (Coico & Sunshine, 2015). In research, MS has shown to have an intermediate association with HLA Class II DR2 allele, because this is the area of the molecule that forms the peptide binding pocket and leads to autoreactivity (Coico & Sunshine, 2015).
Another reason MS is considered a T-cell-mediated disease is that the brain lesions resemble cellular infiltrates that are associated with Th1 cells, which imitates delayed-type hypersensitivity (Coico & Sunshine, 2015). Cellular infiltration happens when cells leave their original place because there is some sort of abnormal growth occurring. Th1 cells are the body’s main cell-mediated defense against infection, and they secrete IFN-γ, which enables them to kill intracellular microbes and intracellular pathogens, and present antigens to T lymphocytes (Hohl, 2015). Th1 cells support our antimicrobial defenses, giving us defense against intracellular bacterial infections like Salmonella typhumurium or Mycobacterium tuberculosis, and intracellular parasites like Leishmania major (Hohl, 2015). Th1 cells are a subset of CD4+ T cells whose main jobs are to synthesize the cytokines IL-2, IFN-γ, and TNF-β, and they mainly activate cells for cell-mediated immunity and stimulate B cells to produce IgG3 antibody (Coico & Sunshine, 2015). Th1 cells help us defend against several different intracellular pathogens and viruses, even including deep fungal infections, so they are pretty important!
This information contributes to the mounting evidence already in my mind that MS is intimately related to chronic infection. Just like brain lesions take several days to form in the MS brain, it takes several days to develop delayed-type hypersensitivity, aka Type IV hypersensitivity, because it is a cell-mediated response that involves T-cells, monocytes, and macrophages responding to antigen (de Weck, 1998), (Hohl, 2015). Within the lesions of MS are activated, blood-borne monocytes, so they circulate and can be picked up on a simple blood test. Blood monocytes are just now becoming more common to use in MS diagnostic criteria and they can be used as a diagnostic measure of immune status, whereas in the past, brain Magnetic Resonance Imaging (MRI) and Cerebrospinal Fluid (CSF) tests are the Gold Standard for confirming the presence of demyelinating disease in MS. A study by Kouwenhoven et al (2001) used flow cytometry to measure various monocytes that secrete cytokines relating to inflammation and infection, and certain MHC molecules. The researchers found that the levels of blood monocytes that secrete IL-6 and IL-12 were higher in patients with untreated MS, while there were higher percentages of certain monocytes in MS patients who had longer disease duration and higher disease severity (Kouwenhoven et al, 2001). It became clear from this experiment that monocyte aberrations occur in MS and may change over the chronic disease course (Kouwenhoven et al, 2001). Delayed-type hypersensitivity reactions are very common in chronic diseases (which MS is), because most chronic diseases are caused by pathogens that are continually giving off an antigenic stimulus (de Weck, 1998). In order for delayed-type sensitivity to occur, there must be pathogens present in the system for a period of time.
The next main point linking MS to being a T-cell mediated disease is the fact that cytokines and T-lymphocytes have been found in CNS plaques. Autoimmunity develops when our body’s tolerance mechanisms go crazy and the autoreactive lymphocytes escape regulatory checkpoints. If they get activated, autoimmunity can happen. In MS, T lymphocytes become activated, cross the blood-brain barrier, and do their thing. Recent studies also point to the proinflammatory cytokine IL-17 is made in MS lesions, and thus that CD4+ TH17 cells may play a role in MS (Coico & Sunshine, 2015). All of these types of cells contribute to tissue injury. There is a study by N’diaye (2018) that does some current research on IL-17 and CD4+ Th cells that I get into later on, so be on the lookout for that in the section of this paper called “C-type Lectin Receptors”.
Usually, the blood-brain barrier prevents cells and other macromolecules from entering the CNS, but when T cells latch onto blood vessel walls, particularly during viral infections, the blood-brain barrier becomes compromised, and the T cells go into the brain. The textbook gives a description of the autoimmune process that I have never heard before, from the immunologist’s perspective. It describes the process of how T cells become activated and produce metalloproteinases, which allows T cells to accumulate in the CNS; then they get stimulated by antigens and become autoreactive, releasing the inflammatory cytokines mentioned earlier like IFN-γ and TNF-α, which activate macrophages (Coico & Sunshine, 2015). When the chemokines and cytokines are releases, this attracts more inflammatory cells, and more T cells and other cell types also end up accumulating. The inflammation process causes Fas expression to be upregulated on the oligodendrocytes, which then become the targets for T cells and microglia that express FasL, and then apoptosis begins in the oligodendrocytes (Coico & Sunshine, 2015). Wow, what a very complex and detailed chain of events! I am going to zoom slightly ‘out’ of that in-depth explanation and just discuss the concept of autoimmunity as it relates to the three main factors that are most suspected to play a role in causing it in MS.
AUTOIMMUNITY IN A NUTSHELL: Genetics, Environment, Pathogens
The complexity of multiple sclerosis comes, largely, because autoimmunity is still not fully understood. We do know that in order for an autoimmune response to happen, there are three main factors that play a role: genetics, environment, and pathogens. And within the scope of these three main determining factors, there are other highly suspected mechanisms involved in MS pathogenesis, such as inflammatory factors, gene expression and overexpression, free radicals’ overproduction, the blood brain barrier (BBB) breakdown, neuroinflammation, vitamin D deficiency, and mitochondrial dysfunction (Mousavi et al, 2017). So far, there is not just one, but we have identified several genes or segments of genes within 12 specific regions of the human genome that might play a role in whether or not a person is predisposed to developing MS (Coico & Sunshine, 2015). But even though a person may have all of the predisposing genes, that factor alone is not enough to make a person develop MS. There has to be something in the environment that provokes the immune system to go either haywire or rogue and initiate the autoimmune response against seemingly otherwise healthy tissue.
The Role of Environment in MS Autoimmune Development
An example of an environmental factor that people who are deeply nested in bureaucracy seem to dismiss is the concept of environmental pollution. Usually in autoimmunity, when we refer to the environment, we think of anything other than the actual environment. However, a study by Mousavi et al (2017) revealed the association between neurodegenerative diseases and environmental exposures, in particular air pollution, and how it relates to the pathogenesis of MS. Their findings point to air pollutants as the initiating factor for the destructive mechanisms that lead to inflammatory-oxidative cascades, reduction of immunological self-tolerance and neurodegeneration, and leading to brain autoimmunity (Mousavi et al, 2017). The World Health Organization has made the statement that air pollution leads to more than 3 million deaths per year, and it supposedly happens because particulate matter (PM), heavy metals, and airborne biological pollutants such as lipopolysaccharide (LPS) are able to reach the brain, promote inflammation, and start the immune responses (Mousavi et al, 2017).
ENVIRONMENTAL TRIGGERS: VIRUSES – Demyelination & Inflammation
Viruses are quite the definition of an environmental trigger, not quite as literal as the first example I gave earlier. Viruses are considered major environmental triggers because not only are they literally floating around in our external environment and must enter into us in order to infect us, but they also cannot survive or thrive unless they also have the proper host environment. Viruses can infect cells outside (peripheral) or inside the CNS, and they are associated with demyelination (Hassani et al, 2018), (Libbey and Fujinami, 2010). MS sufferers are commonly infected with several viruses such as peripheral Torque Teno virus (TTV), peripheral and internal CNS EBV, and internal CNS infections of HHV-6 (Human Herpesvirus-6) (Libbey and Fujinami, 2010), (Miner and Diamond, 2016), (Borosky et al, 2012).
If the environment is just right and the CNS becomes infected with multiple viruses, this can prime the immune system and trigger the disease process to initiate, but if the infections are outside of the CNS, this leads to inflammation within the CNS, and the result is demyelination (Libbey and Fujinami, 2010). If neurotropic viruses get into the CNS by crossing the BBB, inflammation will occur because there are too many cytokines, and the BBB breaks down (Miner and Diamond, 2016). The BBB is made of several different types of specialized cells, and they all work together to prevent pathogens, immune cells, and soluble molecules from getting into the CNS (Miner and Diamond, 2016).
If pathogens do get through the BBB, they can infect the CNS, causing inflammation and demyelination (Miner and Diamond, 2016). A study investigated EBV and TTV as possible candidates involved in the pathogenesis of MS (Borkosky et al, 2012). They measured viral replication of two different TTV isolates from MS brains in both EBV-positive and negative cell lines (Borkosky et al, 2012). Their findings suggest that there is a helper relationship between the two viruses: EBV infections help TTV replicate, which suggests that these two viruses may be involved in the etiology and progression of MS (Borkosky et al, 2012). While all of these pathways lead to the inflammatory process via an immune response, I would like to point out that inflammation has just recently been shown to precede demyelination in MS (Pyka-Fosciak et al, 2018). What stands out is that even though inflammation precedes demyelination, that is the second phase of the innate immune response, which I will discuss later (Rodriguez, 2010).
How Can Autoimmunity Begin?
Another study dealing with environmental factors involvement in MS that I was fascinated by this semester is actually a PhD dissertation from Sweden. This must have been a huge research project, because she wrote three separate papers for the different research projects she studied, all about MS. The main gist of all three research projects is that in order for autoimmunity of the CNS in MS to happen, there are a few things that all have to take place. First, self-reactive immune cells must be activated, then self-antigen must be presented by mature antigen presenting cells (APCs) (N’daiye, 2018). In the experiments by N’daiye (2018), the researchers studied genetic and environmental mechanisms that affect APCs and CD4+ T cells which regulate susceptibility to Experimental Autoimmune Encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein (MOG) in the rat. EAE is the most common animal model of MS that is used to study the disease mechanisms of MS because it produces nearly identical symptomology in the animals as in humans, including demyelination and autoimmunity cellular infiltration in the myelin sheaths of the CNS, which leads to demyelination and eventual paralysis. T cells (and Th17 cells) play a major role in EAE because injecting CD4+ T-cell clones specific for one of the myelin sheath antigens can also induce disease in the EAE model (Coico & Sunshine, 2015).
In a normal-functioning immune system, lymphocytes are the immune cells that respond to foreign antigens but are tolerant to the organism’s own tissues. The specific type of lymphocytes that are involved in MS are T cells, or T lymphocytes. When the T cells are developing in the thymus, they learn how to distinguish self from non-self. Simply put, if they malfunction and start being self-reactive, they are usually eliminated (Kronenberg & Rudensky, 2005), (Coico & Sunshine, 2015). But we have now learned that some of these malfunctioning self-reactive cells that should be destroyed somehow avoid this and develop into specialized regulatory cells (Kronenberg & Rudensky, 2005). This can actually be beneficial in some cases, but the fact remains that self-reactive cells pose an immediate threat of autoimmunity. As humans, we have multiple mechanisms in place that are derived from our immune system’s innate function of either tolerating, inactivating, or eliminating the lymphocytes that have receptors for autoantigens (Kronenberg & Rudensky, 2005), (Coico & Sunshine, 2015). One way the auto-reactive clones prevent them from becoming pathogenic is through regulatory T cells (TR) (Coico & Sunchine, Kronenberg & Rudensky, 2005).
In the third paper of N’daiye’s (2018) dissertation, they describe their research experiment linking the environmental factors like lack of sun exposure and vitamin D deficiency to increasing a person’s chance of developing MS. In the EAE study they did, their experiment showed that vitamin D had an affect on activating CD4+ T cells via epigenetic mechanisms (N’daiye, 2018). There was a reduced pathogenic potential of miRNA, DNA methylation and histone modification (N’daiye, 2018). They used both animal models and human samples to find out how genetic and environmental factors that are relevant to MS could regulate susceptibility to EAE by modulating the development of pathogenic Th17 cells (N’daiye, 2018).
ENVIRONMENTAL FACTORS: Sequestered Antigens
Having one or some of the 12 identified gene regions may make one genetically predisposed to developing MS, but it’s actually the environmental factors that will get things kicked off as far as activating those genes and getting the autoimmune response going (Coico & Sunshine), (Orrell, 2005), (Pyka-Fosciak et al, 2018), (Rodriguez, 2010). Environmental triggers can be either infectious or noninfectious. In the context of air-borne environmental pollutants, they may or may not be infectious. One thing that is clear, however, is that there are three mechanisms the environmental triggers use most often to activate the immune response: inducing the release of sequestered antigens, molecular mimicry, and polyclonal activation (Coico & Sunshine).
The sequestered antigens hypothesis proposes an explanation for autoimmunity that focuses on the relationship between antigen exposure, immunogenic cells, and body cells (MMD, 2009). It says that in order for immunological tolerance to occur, there must be contact between different immunological or body cells and be exposed to antigen (MMD, 2009). Supposedly, sequestered antigens are protected if they are in the brain, eye lens, or sperm; because they are isolated from blood and lymph circulation, they don’t have any contact with the immune system. So when body tissues are damaged, the sequestered antigens are suddenly exposed to the immune system, which thinks they are foreign, and this triggers the autoimmune response (MMD, 2009). Mosby’s Medical Dictionary (2009) says that some autoantigens are protected from the immune system because they are found in ‘privileged sites,’ and some neuronal antigens are examples of sequestered antigens.
Immune Privilege
It was fascinating to learn that there is such a thing called ‘immune privilege,’ where several places in the body just plain do not react with immune responses to pathogens or tumor cells (Coico & Sunshine, 2015). The immune privileged sites include the eye, testis, brain, ovary, and placenta. Up until a few years ago, we believed that these areas were protected because they don’t have lymphatic drainage or the blood barrier, which prevents inflammatory cells from reaching the antigens that are located in privileged sites; however, we have learned that there are other factors that have roles in setting up immune privilege (Coico & Sunshine, 2015). Some of the major players in immune privilege that we have identified are the immunosuppressive cytokines IL-10 and TGF-β, and expression in privileged sites of FasL (Coico & Sunshine, 2015).
However, I am a bit confused because after having just learned about the concept of immune privilege and wrapping my brain about that, I have uncovered some newer information that may render the information in our textbook obsolete. In the dissertation by N’daiye (2018), they discuss how the CNS was considered to be immune privileged for a long time because it can’t reject grafts and because of the blood-brain and blood-CSF barriers, but we have learned that macrophages, dendritic cells, and other innate immune cells populate different areas of the CNS and both memory and activated T cells patrol the CNS during homeostasis (N’daiye, 2018). They also say that the CNS is actually capable of sensing and responding to inflammation that activates cells and cytokine production (N’daiye, 2018). Also, they point out that CNS antigens are found in deep cervical lymph nodes traveling through the lymphatic system (N’daiye, 2018). So basically, the sequestered antigens hypothesis / concept of immune privilege should not hold true because all of the necessary factors are already in place to allow both innate and adaptive immune responses within the CNS to happen. And this leads right into the next interesting part of the environmental mechanism suspected to lead to autoimmunity in MS: molecular mimicry.
ENVIRONMENTAL SUSCEPTIBILITY: Molecular Mimicry
Autoimmunity may also arise by the phenomenon known as molecular mimicry. This happens when an epitope on a microbial or pathogenic antigen is very similar to the epitope on the self-antigen of the host cell, and they end up cross-reacting; the antibodies interact with each other because they are so similar (Coico & Sunshine, 2015). Viral infections can sometimes be the starter for initiating an autoimmune response because of T-cell cross-reactivity. A T cell specific for a viral peptide can cross-react with a peptide derived from an autoantigen (Coico & Sunshine, 2015). There are literally dozens of viruses somehow linked to people with MS providing the proper environment for MS to initiate, but there have only been a couple of instances where specific viruses or pathogens have been directly implicated in molecular mimicry with MS.
The inflammatory process that is mediated by the T cells in the autoimmune process is what causes the demyelination and axonal destruction in the CNS, and the result is lesions in the white matter of the brain (Coico & Sunshine, 2015), (Ramasamey et al, 2017). That much is clear. What is not yet clear is whether this autoimmune response is due to the release of the sequestered myelin antigens after some sort of trauma that happens to the CNS, or molecular mimicry to a neuroepitope after a viral infection. I will come back to molecular mimicry, but let’s delve into viruses a little more first, since most molecular mimicry mechanisms with MS deal with viruses.
VIRUSES IN GENERAL AS CAUSATIVE AGENT FOR MS
There are numerous viruses that have been implicated in MS in some form or another, and I will name some of them and discuss a couple, but there is one virus that has been the most highly-debated over the years and finally very recently has been given the most attention as having some real connection to MS in some way, whether in pathogenesis or initiating the autoimmune response, is the Epstein Barr Virus (EBV) (Hassani et al, 2018). While researching, it became overwhelmingly clear that people with MS have internal conditions welcoming to viruses and provide the proper environment for initiating autoimmunity. Some of the viruses that have been associated with MS are: coronavirus, cytomegalovirus (CMV), herpes-simplex viruses 1 and 2 (HSV-1, HSV-2), Epstein-Barr virus (EBV), human herpes-virus-6 (HHV-6), measles, rubella, torque teno virus (TTV), human endogenous retroviruses (HERVs), human T cell lymphotropic virus type I (HTLV-I), John Cunningham virus (JCV), Polio virus, progressive multifocal leukoencephalopathy (PML), and varicella zoster virus (VZV) (Antony et al, 2004), (Banki et al, 1994), (Borkosky et al, 2012), (Owens and Bennett, 2012). It has been debated for years whether or not viruses play a part in actually causing MS, and even though several viruses are present and/or seem to be involved in the pathogenesis of MS, no single virus has been identified as the specific culprit (Borkosky et al, 2012). However, one virus recently found in inflammatory brain tissues may be enough to establish a connection with the etiology of MS: the Epstein Barr virus (Hassani et al, 2018).
VIRUSES AS THE START OF MS: Epstein Barr virus
Research seems to be pointing the start of the autoimmune process in MS to genetically susceptible people after they are exposed to one or more infectious agents (Hassani et al, 2018), (Owens and Bennett, 2012). When doing research for this paper, EBV came up the most. EBV is a human herpes virus that infects B cells in ~95% of the human population and remains in the memory B cells throughout life (Owens and Bennett, 2012). In several studies over the past 20 years, EBV has been identified as an environmental trigger and a direct causative agent of CNS immunopathology (Angelini et al, 2013), (Casiraghi and Horowitz, 2013), (Hassani et al, 2018), (Owens and Bennett, 2012).
However, the EBV has been the focus of much controversy for several years because published studies have shown pretty convincing evidence both for and against the involvement of the EBV in MS. It was not until very recently that we have figured out some of the underlying mechanisms that could link the EBV to the pathogenesis of MS. In a large study, DNA samples and brain tissues tested for EBV revealed EBV in 90% of MS cases and only 24% of non-MS samples; none of the common herpesviruses were detected in the PCR reactions (Hassani et al, 2018). In qPCR reactions, EBV was low-moderate in most cases, but in 18% of MS cases, EBV was very widespread but scattered (Hassani et al, 2018). Further analysis of heavily-infected samples revealed an EBV latent protein and an early lytic EBV protein expressed (Hassani et al, 2018). By 2013, we already knew that EBV can infect and establish itself in B cells, infect other cell types, and activate the immune system by causing pro-inflammatory mediators to be produced (Casiraghi and Horowitz, 2013). But the link may finally be established because for the first time, when they used double-staining, they were able to see that both astrocytes and microglia (in addition to B cells) were infected (Hassani et al, 2018). This demonstrates EBV is present and transcriptionally active in the brains of most cases of MS and support EBV’s role in MS pathogenesis (Hassani et al, 2018).
VIRUSES: Autoimmunity, Molecular Mimicry, Retroviruses
Although the etiology of multiple sclerosis is not very well understood, molecular mimicry between myelin proteins and internal retroviral proteins has been proposed (Banki et al, 1994), (Morandi et al, 2015), (Ramasamy et al, 2017). Molecular mimicry is an interesting concept because it describes the possibility that two molecules, one from the body and the other from a foreign pathogen, are so similar that the body gets confused, and so the immune system reacts (Banki et al, 1994), (Coico & Sunshine, 2015). When the immune response happens in MS, it targets specific molecules like myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein (Ramasamy et al, 2017).
One earlier attempt to connect MS with molecular mimicry deals with the human T cell lymphotropic virus type I (HTLV-I), which is a human retrotransposon (Banki et al, 1994). They cloned it and found it plays an important role in the coding sequence of the human transaldolase gene (TAL-H), which is involved in lipid biosynthesis (Banki et al, 1994). Analysis of human brain sections and cell cultures showed that TAL-H is possibly linked to production of large amounts of lipids as a major component of myelin and protecting the myelin sheath from oxygen radicals (Banki et al, 1994). They also linked similar sequences between TAL-H and core human retrovirus proteins, suggesting that molecular mimicry between viral core proteins and TAL-H can lead to infection and destruction of oligodendrocytes in MS (Banki et al, 1994).
More recently, two alleles, DRB1*1501 and DRB5*0101, combined with the expression of human internal retroviral envelope proteins, have been linked to MS but the molecular mechanisms that explain how was unclear, so a follow-up study performed sequence homologies of everything: the two alleles, the retroviral envelope, the myelin proteins, and the in silico predictions of peptides derived from them that can bind to the two alleles (Ramasamy et al, 2017). They found that there was molecular mimicry taking place between the peptide epitopes from the envelope proteins of the Human Endogenous Retrovirus W (HERV-W) family of endogenous retroviruses and myelin proteins (Ramsamy et al, 2017). Specifically, mimicry between myelin protein and syscytin-1, a HERV-W envelope protein only expressed during pregnancy, which could explain why more females are affected by MS ; they think this could be a possible trigger for multiple sclerosis (Miner and Diamond, 2012), (Ramasamy et al, 2017). Syncytin is upregulated in glial cells located in lesions of MS patients (Antony et al, 2004). In a previous study, syncytins in astrocytes caused redox reactants to be released, which caused neuroinflammation, which caused oligodendrocytes to die, suggesting that an endogenous retrovirus protein is involved in the demyelination process (Antony et al, 2004).
VIRUSES AS DECOY OF THE IMMUNE SYSTEM: More on Autoimmunity
The precise trigger for autoimmunity is unknown, and although genetics clearly plays a significant role, it is likely a combination of several factors, including infection (Getts et al, 2014), (Hassani et al, 2018), (NMSS, 2018), (Rosche et al, 2004), (Willis et al, 2009). There are a couple of mechanisms identified that might explain how infection sets off the autoimmune response: cross-reactive T cell recognition (see the sections about molecular mimicry), and T cell activation that causes epitope spreading (Getts et al, 2014). In people with MS, the EBV is present in high amounts in serum, and if a person ever becomes infected with mononucleosis, they are at a higher risk of developing MS (Angelini et al, 2013), (Casiraghi and Horowitz, 2013). Other scientists suspect that the aggressive autoimmune responses in MS happen because of an imbalance between immune cells and cytokine production during infections, so they did a study to try and address this in Sardinia, Italy, which is an area where MS is rampant (Sanna et al, 2008). They tested the mononuclear cells of MS patients and controls to measure specific antigen expressions and inflammatory markers before and after infection with HSV-1 (Sanna et al, 2008). Their results agreed with Owens and Bennet (2012) because they showed that when people with MS are infected with viruses, not only do they have reduced immune responses, but their immune cells are altered (Senna et al, 2008).
I have named a few different mechanisms where we have found viruses implicated in the autoimmune process of MS. However, keep in mind that viruses have been found to be involved in pretty much every aspect of the disease. For the sake of length, I will not go into details and add all of the citations for these unless you want them (let me know and I will get you my research on this if you are interested). But I will mention why I have come to the conclusion that autoimmunity in MS is absolutely initiated and propagated by a pathogenic component, very likely viruses. Here is a summary of some specific areas where I have found published, peer-reviewed research articles on the connection of pathogenic viruses to autoimmunity and MS:
- Viruses play a general role in causing MS
- Viruses most likely to be involved at the start of MS are EBV, and Torque Teno Virus
- During MS relapses, Varicella Zoster Virus and EBV have been found in people with MS
- Viruses involved in diagnosing MS are Varicella Zoster, Measles, and Rubella
- There has been much controversy over Varicella Zoster Virus, Cytomegalovirus, and EBV but they have been shown to be active in several MS studies
- Viruses involved in reactions to medications are the varicella zoster virus and the JCV, which causes PML, a rare and fatal viral disease in the brains of certain patients who take Tysabri and other disease-modifying therapies (DMTs) to treat their MS
- Retroviruses and genetics seem to be the potential trigger for developing MS
- Viruses have been proposed to be used as a decoy of the immune system in autoimmunity
- Some viral genetics have connected herpes virus, polio, and EBV to MS autoimmunity
- The coronavirus, much debated, but still connected to MS autoimmunity
ENVIRONMENTAL SUSCEPTIBILITY: Polyclonal activation
The third and final likely candidate that points to environmental mechanisms leading to autoimmunity in MS is polyclonal activation. The textbook uses the term ‘polyclonal activation,’ but when doing a Google search for it, the definition is nowhere to be found. Our textbook even only uses the word in passing when it makes the statement about the three leading environmental factors that can contribute to autoimmunity in MS (Coico & Sunshine, 2015). The textbook does give the definition of a polyclonal activator as, “a substance that induces activation of many individual clones of either T or B cells… see also mitogen…” and the definition our book give of ‘mitogen’ is, “a substance that stimulates the proliferation of many different clones of lymphocytes” (Coico & Sunshine, 2015). There is also a couple of sentences in the chapter on CD4+ T cells that talks about how there ae several natural materials that can trigger the proliferation and differentiation of just about all clones of T lymphocytes, and they are called polyclonal activators or mitogens because they have the ability to induce mitosis in the cells (Coico & Sunshine, 2015). So it has been very difficult for me to figure out what exactly polyclonal activators are!
One article I found that was pretty interesting and talked about the differences between monoclonal and polyclonal antibodies in general. In research applications and drug therapies, there are monoclonal antibodies and polyclonal antibodies that are the two main types of antibodies, and they both interact with the same antigen. The main difference between monoclonal and polyclonal antibodies is that monoclonal antibodies are produced by the same clone of plasma B cells, and they bind to a unique epitope whereas polyclonal antibodies are produced by different clones of plasma B cells, and they bind to the different epitopes in the same antigen (Panawala, 2017). Antibodies are only made by the B or T cells, and antibodies are only made in response to a specific antigen. The antigen interacts with the cells of the immune system to trigger an immune response.
VIRUSES: The Normal Immune System
Usually, when the body comes in contact with a virus or virus particle, the immune system attacks them either before they enter our cells or when they become released after replication (Rodriguez, 2010). Sometimes, the body attacks infected cells when it is producing protein or assembling more viral particles, and in this case, if there are antigens in the membrane of the infected cell, this can initiate an immune response (Rodriguez, 2010). In other cases, such as in the case of the herpes virus, infection can last for long periods of time where there are no viral particles or surface antigens being expressed in the membranes of the infected cells. This situation puts the body into the situation where it is not able to fight the infection, because there are no viral peptides to put up the markers and tell the immune system know there is an invader. But eventually, and we still do not fully understand why, the infection reactivates and new infectious virions are released (Rodriguez, 2010). There is another, alternative pathway of complement activation that can activate the immune system to destroy the virus particle, and it is part of the adaptive immune response (Rodriguez, 2010). This process is mediated by CD8+ T cells and antibodies, which is a very effective method of getting rid of viral infections. But there are alternative pathways that the body uses to take care of viruses and other pathogenic molecules if they persist in the body.
C-type Lectin Pathway
One method I want to delve into a bit here is part of the natural or innate immune response, which is also called the rapid action pathway because it usually happens anywhere from 4 minutes to 4 hours. This rapid action response is the non-specific barrier of our immune system that uses both humoral and cellular mechanisms to activate cytokines and macrophages, and it is mediated by an alternative pathway of complement activation (Rodriguez, 2010).
I may be going off on a bit of a rabbit trail here but for some reason, the lectin pathway stood out to me when we came across it very early in the semester. (Because there is a medium and slow action response of the immune system that takes anywhere from 4 hours to 4 days, and it is mediated by inflammation, activation of NK cells, and production and release of Interferon, which all seem pretty important and relevant to MS). But that is not what I am referring to here. I am talking about complement activation that has three specific pathways by which the effector functions of complement can be activated: the classical, alternative, or lectin pathway. This activation that happens by the alternative and lectin pathways happens as a direct response to foreign particles and its part of the first line of defense in the natural response (Rodriguez, 2010). This alternative pathway is triggered by the fragment C3b. This pathway is activated by a lectin that is present in the membrane of MBL (Mannose Binding Lectin), which can activate the attack complex and destroy the membranes of several microorganisms without activating antibodies (Rodriguez, 2010). In my undergrad research on the relationship of sucrose and glucose to pathogens, MBL and several other forms of ‘mannose’ and things that have to do with inflammation and infection in MS kept coming up over and over. So I am trying to figure out what is the relationship of that here with the C-type lectin receptors, because it may be that people with MS have an autoimmune response that is perpetuated by some type of hidden allergy to something structurally similar to glucose or mannose or sucrose, etc. Or perhaps molecular mimicry occurs to set off the autoimmune response due to cross-reaction.
C-type Lectin Receptors
In an article by Bermejo-Jambrina et al (2018), they describe C-type lectin receptors (CLRs) as important pattern recognition receptors that are involved in recognizing and inducing adaptive immune responses to pathogens. Many CLRs play an important role in viral infections because they are primed to be able to interact with viruses efficiently. But there are some very deadly viruses that are able to promote infection because they somehow escape antiviral immunity (Bermejo-Jambrina et al, 2018). It seems that viruses target CLRs so that they can suppress or regulate certain interferons that have a role in the innate and adaptive defense against viruses (Bermejo-Jambrina et al, 2018). CLRs can bind to enveloped viruses like HIV-1 and Dengue viruses, and they effect infection by the signaling cascades they induce (Bermejo-Jambrina et al, 2018).
In the second paper of the dissertation by N'diaye (2018), the researchers explored the role of two specific CLRs named MCL and Mincle. They characterized how MCL and Mincle made the rats susceptible to EAE because they re-activated the CD4+ T cells in the CNS towards the pathogenic IL-17-producing Th17 phenotype (N’diaye, 2018). The reason this happened was because of an endogenous ligand of MCL and Mincle called SAP130, which is an alarmin, and I am not familiar with yet, but will be learning more about later. I do know that a ligand is a protein that binds to a receptor (another protein). And I understand that receptor proteins have specific sites where the ligands fit, sort of like a key fits into a lock. So an endogenous ligand would be one made in the body, not outside of it.
In this second paper, they also found out that the MCL/MINCLE signaling pathway was hyperactive in MS blood monocytes, so this might contribute to disease severity in EAE models of MS (N’diaye, 2018). Part of the mechanisms they addressed in the second (and also the third) papers dealt with regulating T cell activation and EAE development, which I am interested in because I am doing an EAE study here at IUP. In their experiments, they used interesting strategies to address their biological questions and gave new insight into the immunological aspects of autoimmune neuroinflammation, so I will likely be referring back to their research to look for more clues.
Another reason CLRs stood out to me is because of their link to viruses that have been shown to play a role in MS, most notably the HERVs (see the section above on HERVs). HERVs are actually endogenized exogenous retroviruses that were integrated into the human genome during primate evolution or earlier, and so they represent a molecular link between the host genome and infectious intracellular viruses (Bermejo-Jambrine et al, 2018). Current research with active MS patients revealed that monocytes had increased expression of certain HERV epitopes, and also that the humoral immune response associated with it was HERV-directed (Bermejo-Jambrine et al, 2018). This was noted in the peripheral and CNS inflammatory areas of MS patients, so it is yet another viral link to MS, in particular an intracellular viral link. Their research also presented a detailed analysis of the circulating monocytes and their subsets in both newly diagnosed MS patients and during disease progression. They analyzed the HERV epitope expression and also expression of surface-expressed markers of activation, inflammation, and neurodegeneration. I will have to go back and read about these more carefully over the summer because I am way past the space and time limit for getting this paper turned into you!
CONCLUSION
One of the overarching themes throughout this study is that the autoimmune response in MS is very complex! There seem to be several key factors required in order for a person to be able to actually develop the disease. First, the person must have the correct set of susceptibility genes that are found in one of the 12 regions of the genome. Second, the genes must be activated by something in the environment. This something is likely microbial or pathogenic in nature, and in my researched opinion, it is viruses that play the greatest role as environmental triggers to initiate and propagate the autoimmune response in MS. Molecular mimicry seems to be a pretty guilty suspect as having a role in intracellular viral molecular mimicry and cross-reaction mechanisms because when people with MS are infected with viruses, not only do they have haywire immune responses, their immune cells are altered and they are generally susceptible to viral infections. There seem to be two main viruses that play a major role in MS etiology relapses: EBV and Varicella Zoster Virus. The most data pointing to a causal link has been on the EBV, and it is believed that if we can pinpoint EBV as a trigger, it’s possible that we could potentially prevent the condition by treating the virus (Hassani et al, 2018). Since EBV is present and transcriptionally active in the brain of most cases of MS, this could support a role for the virus in MS pathogenesis (Hassani et al, 2018). Also, since gastrointestinal microbiota modulate BBB function, improving gastrointestinal microbiota health (like taking a probiotic) may be beneficial for people with MS (Miner and Diamond, 2016), (NMSS, 2018).
In summary, the main points of this research are: MS is a complex, multi-faceted, elusive disease; we still don’t fully understand the cause of have a cure for MS; there are many different forms of MS; it seems like the main player in MS is the immune system and the CNS is just the setting of the story; both B and T cells are involved in MS but MS is a T-cell-mediated disease; environmental pollution is a current hot spot for MS pathogenesis regarding brain autoimmunity; in order for MS autoimmunity to develop, self-reactive immune cells must be activated, then self-antigen must be presented by mature APCs; there are three mechanisms the environmental triggers use most often to activate the immune response: inducing the release of sequestered antigens, molecular mimicry, and polyclonal activation; CLRs are pattern recognition receptors that help recognize and induce adaptive immune responses to pathogens- CLRs play an important role in viral infections; and there are several viruses involved in MS- people with MS provide perfect conditions for viruses to thrive (Casiraghi and Horowitz, 2013), (Getts et al, 2014), (Ramasamy et al, 2017), (Rosche et al, 2004).
After analyzing all of this information, I think the best course of action is to continue doing research to piece together the clues. If we study which genes are involved in the all of the factors that are the key players in MS, we can develop genetic testing techniques to determine which medicines are good for which subsets of people with MS and thereby avoid the undesirable side effects of the current medications. Also, while scientists are still piecing together the MS puzzle to find a cure, I think the best option for people who either have MS or are predisposed to developing it would be to use nutrition and supplements that help create an unfavorable host environment in the body so viruses cannot coexist inside of them. I didn’t mention nutrition and supplements in this paper, but I have been studying those things in relation to MS for 18 years and so I have lots of information if you or anyone else is interested. Also, my research project here is using diet as a treatment for EAE, and I am excited to see what happens.
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