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. 2016 Jun 1;36(6):347–357. doi: 10.1089/jir.2015.0177

Emerging Concepts on the Gut Microbiome and Multiple Sclerosis

Justin D Glenn 1,✉, Ellen M Mowry 1
PMCID: PMC5118957  PMID: 27145057

Abstract

Microbiota of the human body perform fundamental tasks that contribute to normal development, health, and homeostasis and are intimately associated with numerous organ systems, including the gut. Microbes begin gut inhabitance immediately following birth and promote proper gut epithelial construction and function, metabolism and nutrition, and immune system development. Inappropriate immune recognition of self-tissue can lead to autoimmune disease, including conditions such as multiple sclerosis (MS), in which the immune system recognizes and attacks central nervous system tissue. Preclinical studies have demonstrated a requirement of gut microbiota for neuroinflammatory autoimmune disease in animal models, and a growing number of clinical investigations are finding associations between MS status and the composition of the gut microbiota. In this review, we examine current undertakings into better understanding the role of gut bacteria and their phages in MS development, review associations of the gut microbiota makeup and MS, and discuss potential mechanisms by which the gut microbiota may be manipulated for therapeutic benefit.

Introduction

The human body is an exquisite composition of cells derived from every taxonomic domain. Over time, human eukaryotic cells, and those of most other higher-level mammals, have evolved to acquire diverse structural and functional phenotypes that are grouped into higher-level organs and systems that work in concert to promote a stable well-being (homeostasis) and survival of the organism. These cell-based systems vary in anatomical space, and as such may be relatively sessile (ie, central nervous system) or dispersed throughout the organism (ie, the immune system). Interspersed on and within various human cell-based systems are also collections of diverse nonhuman microbial organisms and entities.

The total microbial collection within a specific anatomical niche is known as microbiota, while the entirety of genes derived from the microbiota comprises its microbiome. It is estimated that microbes of the collective microbiota outnumber human somatic and germline cells by a factor of 10 and inhabit vast stretches of human skin and mucosa, especially of the lung and genital microenvironments, though the largest collection (∼70%) resides in the gut (Savage 1977; Backhed and others 2005). Of the different microbial domains composing the microbiome, we will focus on bacteria and viruses, as these are the most numerous and highly investigated members. Current methods to study bacteria in the gut consist of collecting DNA from stool and using high-throughput gene sequencing of bacterial 16S rRNA and bioinformatics analyses (Backhed and others 2005; Jandhyala and others 2015). Bacteriophages, which are the most abundant viral entities of the gut, are studied by viral particle concentration from stool, elimination of contaminating cells and nucleic acids, amplifying viral sequences and metagenomics analyses (Dalmasso and others 2014).

The gut microbiota lies in close association with the host immune system and both play crucial roles in regulating gut development and immunity. Altered host immunogenetics and environmental factors can result in disease pathogenesis as in the case of autoimmune disease. A growing number of studies are exploring the contributions of the gut microbiota to the development of multiple sclerosis (MS) and suggest this field as a potential novel target for MS therapeutics and prevention.

The Gut Microbiome

Bacteria

The adult human intestine is populated by an astonishing 100 trillion microbes, and its microbiome contains at least 100 times the number of genes of the human genome (Savage 1977; Backhed and others 2005). Indeed, bacteria within this environment reach the highest cellular densities of any ecosystem studied (Whitman and others 1998). Given the enormity of this population, it is surprising that at the division level, bacterial diversity diminishes. Only 8 of 55 known bacterial divisions have been observed in the gut (Hugenholtz and others 1998; Eckburg and others 2005). Those of the divisions cytophaga-flavobacterium-bacteroides and Firmicutes reach considerably large percentages at ∼30% of bacteria each, while Proteobacteria and Actinobacteria are observed but at low abundance; it is this bacteria divisional makeup that suggests a healthy human gut (Backhed and others 2005; Eckburg and others 2005; Jandhyala and others 2015).

Infants are rapidly colonized by bacteria shortly after birth from the mother and environment, and infant mode of delivery is thought to impact bacterial colonization. Babies born by vaginal delivery (Andersch-Bjorkman and others 2007) are exposed to maternal vaginal and intestinal microbiota, while those born via caesarian section delivery (CS) lack these contacts (Neu and Rushing 2011). Studies have shown that infants born by CS have delayed bacterial colonization, which may even last for 6 months (Gronlund and others 1999). Furthermore, delivery-based differences in the intestinal microbial makeup has been observed in 7-year old children, with those by vaginal delivery having significantly higher numbers of Clostridia (Salminen and others 2004). Long term, those delivered via CS may exhibit an abnormal development of the immune system due to abnormal infant GI tract colonization (Neu and Rushing 2011).

Bacteria of the gut microbiota contribute tremendously to maintaining healthy human physiology, with many functions that can only be achieved by bacterial intervention. These functions largely consist of (a) complex carbohydrate metabolism and nutrient extraction; (b) xenobiotic metabolism; (c) antimicrobial protection; (d) promoting gut barrier function; and (e) contributing to shape gut immunity (Fig. 1).

FIG. 1.

FIG. 1.

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Functions of bacteria in the human intestinal tract. Gut bacteria perform a range of functions in the human gut, including participating in metabolism of complex biopolymers, metabolism of drugs and foreign agents, promoting the production of AMPs, and stimulating proper development of intestinal structures and parenchymal functions. AMPs, antimicrobial molecules.

Viruses

In addition to bacteria, viruses are even more bountiful in the human gut and comprise both eukaryotic and prokaryotic types. We will highlight prokaryotic viruses, particularly the bacteriophages, as these are the most numerous biological entities of the human gut and engage in complex interactions with gut bacteria of the microbiome. Bacteriophages (phages) can be extracted from human feces at 109 virus-like particles (VLP) per gram and may inhabit up to 1014 bacterial cells in the gut (Rohwer 2003; Breitbart and others 2008). Phages vary in the types of bacterial surface molecules that they target for binding, and thus viral tropisms exist for certain strains of bacteria for a given bacterial species. Generally, phages exhibit 2 lifecycles: the lytic phase/lifecycle, in which a phage infects the bacterial cell and kills it upon replication and viral release, and the lysogenic phase/lifecycle, in which the phage infects the bacterial cell and integrates its viral genome into the host bacterial genome without inducing lysis and simultaneously maintains its genome in a latent form (prophage) (Dalmasso and others 2014). Under a range of stimuli, the prophage may exit latency and enter the lytic phase.

The principle intestinal phages consist of (1) the tailed, double-stranded DNA viruses of the order Caudovirales (Podoviridae, Siphoviridae, and Myoviridae) and (2) the tail-less, cubic or filamentous, single-stranded DNA viruses of Microviridae (Ackermann 2009; Minot and others 2011; Foca and others 2015). Phages appear in the newborn human gut as early as 1 week after birth at 108 VLP per gram of feces and exhibit low diversity, as in the case of bacteria (Breitbart and others 2008). However, it is possible that more viral genotypes remain to be discovered, as a large number of assembled contigs from a recent metagenomics investigation matched no known public sequences (Reyes and others 2010; Minot and others 2011; Wagner and others 2013).

As these are viruses that infect bacteria, most of their effects are manifested in changes to bacterial phenotype, and they thus represent an extra dimension of regulation on bacterial dynamism within the gut microenvironment. Bacteria may utilize a form of immunity known as CRISPR (clustered regularly interspaced short palindromic repeats) to evade phage infection (Gasiunas and others 2014). Here, the bacterial genome contains a region of short DNA repeats that are regularly spaced and contain variable, foreign phage DNA between them. Upon recognition of foreign nucleic acid, the CRISPR system matches the foreign nucleic acid to its complementary sequence saved between 2 spacers and proceeds to cleave the intruding sequence, thus thwarting infection. However, phages have also been shown to counter bacterial immunity (Seed and others 2013; Villion and Moineau 2013).

Regulation of the Gut Microbiome

Bacteria–bacteriophage interaction

Most human gut phages exhibit a temperate lifecycle in which the phage is able to undergo lytic or lysogenic lifecycles (Dalmasso and others 2014). When phages incorporate viral DNA into bacterial chromosomes, they may transfer exogenous segments or whole gene clusters into the host bacterial chromosome by gene transfer (horizontal gene transfer if exogenous bacterial DNA is introduced), which may occur via transduction or lysogenic conversion (Brussow and others 2004).

In transduction, a phage introduces DNA from previous bacteria into a new bacterial chromosome, as exhibited in some Streptomyces spp. (Burke and others 2001). Importantly, lysogenic conversion can wield crucial power in determining bacterial pathogenicity through the introduction of genes that increase bacterial fitness, such as preventing lytic infection and endowing bacteria with toxins. In some cases, phage DNA integration may even disrupt host bacterial genes. The outcome of phage-mediated gene transfer in bacteria is dynamic and heavily dependent on environmental stress. In a recent example, antibiotic treatment led to an enrichment of phage-encoded resistance genes in gut bacteria, which promoted host colonization and growth adaptation, ultimately strengthening connections between the phageome and gut bacteria (Reyes and others 2013); such an observation may represent 1 mechanism of bacterial adaptation and potential drug resistance.

Bacteriophages are also thought to exert beneficial influences on the human host outside of the bacterium, as they are populous in the gut mucus layer and bind to mucin glycoproteins with immunoglobulin-like domains on their capsids (Barr and others 2013). This interaction contributes to gut epithelial protection from bacteria.

The bacteria and their phages are in constant interaction within the gut, and each strives to regulate the other. However, it is possible that phages may negatively affect bacterial communities in the host by promoting dysbiosis, in which the balance of bacterial symbionts and pathogens has been skewed, which may ultimately translate into disease (Foca and others 2015). Though there is scant investigation into the contribution of gut phages to bacterial dysbiosis, several rational scenarios have been proposed (De Paepe and others 2014).

In the “Kill the winner” mechanism, phages target and kill only the dominant bacterial species, thus indicating viral predation based on bacterial density. This has been explored in a study of GF mice colonized with 15 known human symbiotic bacteria and subsequently infected with a combination of phages (Reyes and others 2013). Changes in the community structure were observed though transient. The “biological weapon” model has also been suggested in which commensal bacteria would use its own phage to kill competitive bacteria by inducing lysis (Bossi and others 2003; Brown and others 2006). “Community shuffling” holds that stressful scenarios such as antibiotic treatment, inflammation, and oxidative stress may lead to the induction of prophages and eventually alter the symbiont/pathobiont relationship (De Paepe and others 2014). Evidence for this model comes from studies in Escherichia coli (Zhang and others 2000) and Clostridium difficile (Meessen-Pinard and others 2012). As discussed earlier, lateral gene transfer is potentially a critical mechanism of dysbiosis, in which viral virulent genes are endowed to bacteria, as in the case of E. coli strain O104:H4, in which an E. coli strain acquired Shiga-toxin via lysogenic conversion (Muniesa and others 2012).

Host gut immunity and microbiome

Commensal bacteria and their phages perform indispensable tasks for the human organism, yet their very foreign identity, along with their potential virulence and the existence of pathogenic bacteria and viruses, necessitates a fully functional immune system that continuously interacts with this microbiome (Fig. 2). This interaction is bidirectional; the gut microbiota elicits a specific immune microenvironment in the gut for the sake of gut homeostasis and optimal parenchymal function, while the immune system controls the species composition of the gut microbiota for optimal growth and nutritional benefit. Dysfunction on either end of this transaction can lead to disease.

FIG. 2.

FIG. 2.

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Cross-regulation among bacteriophages, bacteria, and immune system in the gut. Bacteria, their phages, and the gut epithelia and immune system engage in complex cross-regulations to maintain gut homeostasis. Bacteriophages infect bacteria and through a largely temperate lysogeny can horizontally transfer genes or disrupt genes to affect bacterial colonization and growth. Bacteria also limit phage infection through CRISPR immunity. Bacteria promote proper gut lymphoid tissue development, AMP production, and induce the development of immunosuppressive and proinflammatory immune cell types, dependent on the bacterial species. These very cell types then work together with AMPs to limit bacterial contact to gut epithelia. Bacteriophages are known to bind mucin and also aid in limiting bacteria-epithelial adhesion but how gut immune cells and epithelia directly affect bacteriophages remains largely unknown. As each component is situated as an intermediary among the other 2, and all are connected, indirect regulation among the 3 components also occurs. ↑, promote, ↓, suppress.

First, bacteria (and presumably their phages) are absolutely required for the development of the intestinal immune system, as documented by studies in axenic (GF) mice (Smith and others 2007). Axenic mice exhibit a range of intestinal immune structural and cellular defects, including general reduction in lymphoid tissue size, smaller mesenteric lymph nodes, which contain fewer cells and no germinal centers; reduction in the number and size of Peyer's patches; and a reduction in the number of intraepithelial lymphocytes, all in combination with defects to aforementioned epithelial cellular defects. Interestingly, these defects can be reversed with bacterial colonization and touts the potential therapeutic use of commensal administration for corrective defects in intestinal immune defects (Smith and others 2007; Palm and others 2015).

Furthermore, bacteria can be classified into 2 distinct categories, each of which is dependent on the response they elicit from the immune system: immunosuppression or proinflammation.

Extensive investigation into the proinflammatory effects of the immune system in the gut have been done with a limited group of bacteria. Studies using segmented filamentous bacteria (SFB) have by far revealed much about induction of inflammatory responses (Schnupf and others 2013). SFB (Candidatus Arthromatis) represent a unique clade of commensals in mice, and perhaps in humans, that are most related to the Type I Clostridia (Kuwahara and others 2011). They are intimately associated with the gut epithelium and are primarily recognized as strong drivers of immune responses. Studies in mice mono-colonized with SFB demonstrated that they dramatically induce the number of intestinal IgA+ B cells and increase the concentration of secretory IgA in the intestinal lumen (Klaasen and others 1993). Underlying this observation was a short-lived but strong activation of germinal centers in Peyer's patches by SFB (Talham and others 1999). SFB also strongly expand the number of intraepithelial lymphocytes, including γδ T cells and CD8+ T cells (Schnupf and others 2013). Of all proinflammatory immune cells stimulated by SFB, TH17 cells have garnered the most attention. Intestinal dendritic cell presentation of SFB antigen is required for TH17 development in the gut mucosa (Gaboriau-Routhiau and others 2009; Ivanov and others 2009). SFB colonization also protects against pathogenic infection by Citrobacter rodentium (Ivanov and others 2009); this is a prime example of the importance of indigenous bacterial colonization as a means of combating pathogenic bacterial infection.

TH17 cells mainly produce the cytokines IL-17A, IL-17F, IL-21, and IL-22, which are critical to controlling fungal and bacterial infections and maintaining antimicrobial gut function. Both IL-17 cytokines recruit neutrophils to the infection site for bacterial phagocytosis and induce epithelial production of the chemokine CCL20, which recruits even more TH17 cells to the site via binding to the chemokine receptor CCR6 on the TH17 cell surface (Huber and others 2012). These cytokines also increase the production of β-defensin by epithelial cells (Liang and others 2006; Ishigame and others 2009). In addition to the IL-17 cytokines, TH17-produced IL-22 promotes epithelial layer integrity and antimicrobial homeostasis by inducing the expression of antimicrobial molecules (AMPs) from the epithelium, promoting epithelial cell proliferation, survival, and tissue repair (Huber and others 2012).

In addition to SFB, the Gram-negative bacterium Helicobacter hepaticus has been shown to promote TH17 responses and those of TH1 cells, which are marked by production of the proinflammatory cytokine interferon-γ (IFNγ) and are important for defense against intracellular pathogens such as intracellular bacteria and viruses (Palm and others 2015).

As bacteria of the microbiome do represent a foreign yet beneficial entity to the host, their presence also elicits immunosuppressive immune responses to tolerate commensal gut microbes. Mechanisms of immunosuppression include induction of immune cell subsets that dampen immune responses and the suppression of proinflammatory immune cells. One of the most prominent immunosuppressive cells of the intestine is the regulatory CD4+ T cell (Treg), which is induced by the activation of CD4+ T cells in the presence of the immunosuppressive cytokine transforming growth factor-β (TGF-β). These cells produce the anti-inflammatory cytokines IL-10 and TGF-β, which potently decrease proinflammatory cytokine production and cell proliferation. GF mice colonized with a microbiota known as Altered Schaedler's Flora induce Treg to the intestine and are required for homeostasis and prevention of colitis (Geuking and others 2011). Furthermore, a consortium of bacteria from the genus Clostridium from conventionally reared mice was shown to induce mobilization of Treg to the intestine (Atarashi and others 2011). A rational selection of 17 Clostridia from human fecal samples that enhance Treg abundance in the gut was shown to attenuate mouse models of colitis and allergic diarrhea after oral administration (Atarashi and others 2013). Furthermore, Clostridia are major producers of short chain fatty acids (SCFAs), which have been demonstrated to induce Tregs (Palm and others 2015). SCFAs, especially butyrate, induce Treg formation via epigenetic regulation in the form of increased acetylation of the locus of Foxp3, which is the master transcription factor of TGF-β production (Arpaia and others 2013; Furusawa and others 2013; Smith and others 2013). In addition to bacteria of the genus Clostridia, Bacteroides fragilis induces Treg development by its production of capsular polysaccharide A (PSA), which is recognized by TLR2 on Tregs (Round and Mazmanian 2009; Round and others 2011).

Other immune cell types that are affected by intestinal microbiota include invariant natural killer T cells (iNKT cells) and innate lymphoid cells (ILCs). iNKT cells are a T-cell subset that expresses the invariant T-cell receptor α-chain (Sommer and Backhed 2013). These cells are proinflammatory, as following activation they produce the cytokines IFNγ, IL-2, IL-17A, and tumor necrosis factor (TNF) among others, which promote TH1 and TH17 immune responses. Importantly, GF mice have increased numbers of these cells in the intestine, which suggests that the gut microbiota serve to limit their potentially exacerbated proinflammatory presence (Olszak and others 2012). ILCs rapidly respond to inflammatory cues and develop along effector pathways parallel to those of CD4+ T cells; for instance, Type 3 ILCs express the transcription factor retinoid-related orphan receptor γ t (RORγt, which promotes TH17 development) and produce IL-17A and IL-22 (Jandhyala and others 2015). Studies have shown that the bacterial metabolite indole-3-aldehyde stimulates ILC production of IL-22 via the aryl hydrocarbon receptor, which is expressed in type 3 ILCs (Zelante and others 2013). Interestingly, loss of MHC-II on ILC3 induces TH17 development independently of SFB, which may indicate a regulatory role of ILC3 on gut TH17 differentiation (Goto and others 2014).

The interplay of the gut bacterial microenvironment with the gut immune system is further compounded by the crosstalk of bacteria-bacteriophage and direct interactions of bacteriophages and the immune system. It is conceivable that bacteriophages could interact indirectly with the gut immune system by manipulating gut bacteria via the aforementioned mechanisms discussed, and bacteriophages are presumed to be strong drivers of intestinal bacterial biodiversity. Although the phages predominate over other microbes of the gut, how they directly interact with the intestinal immune system is very poorly understood and understudied.

In nonimmunized humans and animal sera, a low level of phage antibodies are detectable, and most are neutralizing against the phage tail (Jerne 1956; Kamme 1973; Kucharewicz-Krukowska and Slopek 1987). Few studies have addressed the effects of phages on T cells; Srivastava and others (2004) reported no significant difference in phage clearance between WT and T cell-deficient mice, thus suggesting an insignificant T cell-mediated role in antiphage immune responses. However, phages can exert an immunosuppressive effect on T-cell function, as purified T4 phage has been shown to suppress human T-cell proliferation (Gorski and others 2006). Studies investigating bacteriophage treatment in tuberculous guinea pigs showed that mycobacteriophages suppressed phytohemagglutinin-induced activation of T lymphocytes in a dose-dependent fashion (Mankiewicz and others 1974). Unpublished data from Gorski and others suggest that phages inhibit some dendritic cell functions and NFκB activation. Phagocytosis and generation of reactive oxygen species (ROS) are critical functions of innate immune cells that maintain appropriate bacterial densities in the gut, though excessive activity can also drive pathology. Phagocytes, including neutrophils and monocytes, preincubated with T4 and F8 (a Pseudomonas aeruginosa phage) exhibited a reduction in phagocytosis of E. coli (Przerwa and others 2006). However, in vivo phage-affected neutrophil phagocytosis varied depended on pretreatment with bacteria. ROS can be quickly generated by respiratory bursts in phagocytes. T4 phages can suppress E. coli-induced respiratory burst with preincubation with the bacteria (Przerwa and others 2006). Other reports suggest that staphylococcal A3/R purified phage or lysates may have no effect on phagocytic respiratory burst, thus suggesting that this effect may be bacteriophage dependent (Borysowski and others 2010).

In the context of cytokine production, phages display largely mixed results, which may reflect the differential effects of bacteriophage cell tropism and/or mode of phage preparation. While purified Staphylococcus aureus phage can increase IL-6 production in activated splenocytes, it may also decrease it, along with decreasing TNFα production (Zimecki and others 2003). The preparation staphage lysate induced the cytokines TNFα, IL-1β, IFNγ, and IL-10 in human mononuclear cultures (Gorski and others 2012). Moreover, purified preparations of the T4 phage decrease IL-2 and IL-6 production in these cells, but its lysate increases that of IL-6 (Gorski and others 2012). These observations show the complexity of phage effects on immune cells and the potential confounding effects phage use may have in treatment depending on the therapeutic outcome desired. Purified phage preparations appear to elicit more immunosuppressive outcomes, which could depend on direct phage immunomodulation or reducing the burden of bacteria and thus driving down bacterial antigen load. On the contrary, lytic phage infection could cause an increase in bacterial antigen and exposure to the immune system.

The Gut Microbiome, Autoimmune Disease, and MS

The gut microbiota is a direct product of the environment and is situated topologically outside of the host; as such, it is in constant contact with external environmental factors against which it must maintain equilibrium. However, it must also maintain normal, physiologic associations with the host gut stroma, parenchyma, and immune system. Numerous metagenomic investigations show that there exists limited physiologically acceptable temporo-spatial changes in the dynamics of the gut microbiota (including microbial composition, specific bacterial densities and ratios, and phenotypes) that are compatible with overall healthy intestinal function. On the other side, the immune system, in concert with gut epithelia, must keep commensal bacteria and other microbiota at acceptable distance from the epithelium, contribute to bacterial homeostasis, and preserve the epithelial border from insult while allowing proper nutritional absorption. Alterations to various parameters on either side of the gut host-microbiome microcosm can result in serious complications, from improper nutrition to autoimmune disease.

Multiple sclerosis

Broadly, autoimmune pathogenesis defines conditions in which the host immune system is activated to mount inappropriate inflammatory immune responses against host cells, and includes conditions such as systemic lupus erythematosus, type-1 diabetes, rheumatoid arthritis, and MS. MS is a debilitating, inflammatory autoimmune disease of the central nervous system characterized by axonal demyelination and neurodegeneration (Wekerle and Kurchus 2006). It is thought to be mainly a disease of the Western world and, like most autoimmune diseases, has an increased female: male disease ratio (Trojano and others 2012). Although its etiology remains unknown, MS development is multifactorial, involving both heritable, genetic components and environmental components.

Epidemiological and genome-wide association studies have identified common MS risk-associated loci, mostly immune related, including 2 human leukocyte antigen (HLA) alleles DRB1 × 1501 and DQ6, IL2RA, IL7R, IRF8, TNFRSF1A, and CD40 (Bush and others 2010). In addition, defects in tolerance mechanisms have been identified in MS (Gonsette 2012). It is possible that the associated HLA molecules may be more apt at presenting antigen to T cells in the process of MS development, there is change in the activation threshold of certain components of the overall immune response associated with certain immune susceptibility loci, and there is an abnormal escape of self-antigen-specific T cells from the thymus in MS. These factors in some combination could greatly contribute to MS development.

Environmental associations with the risk of MS development are greatly varied; those confirmed include lower vitamin D levels, cigarette smoking, viral infection (Epstein-Barr virus), and obesity, although this list is suspected to be incomplete (Hedstrom and others 2012; Summerday and others 2012; Wingerchuk 2012; Pender and Burrows 2014). Since the incidence of MS has increased in many locations throughout the world, there is increased attention focused on identifying other environmental risk factors for MS. Given its association with the risk of other autoimmune diseases, studies of the gut microbiota are critical (Ho and others 2015).

The gut microbiome and MS

The gut microbiome and models of MS

The impact of the gut microbiome on MS development has its roots in several seminal preclinical investigations on the role of bacteria in experimental autoimmune encephalomyelitis (EAE). EAE is a murine model of MS induced by immunization with a myelin protein/peptide in combination with complete freund's adjuvant with/out pertussis toxin (Stromnes and Goverman 2006). This immunization activates myelin-reactive CD4+ T cells and leads to their differentiation into pathogenic, encephalitogenic TH1 and TH17 cells that mediate neuropathology and ascending paralysis.

It was first observed that GF mice are resistant to spontaneous and inducible EAE and fail to generate pathogenic T cells, especially TH17 cells (Berer and others 2011; Lee and others 2011). This was mechanistically attributed to a failure of gut DC activation and production of cytokines crucial for T-cell polarization. The failure to generate pathogenic T cells was concomitant with an increase in Treg development. Furthermore, EAE induction could be restored with colonization with SFB, which drove TH17 differentiation in the gut and T-cell encephalitogenicity. Gut bacteria also produce extracellular adenosine 5′- triphosphate, which induces CD11c+ DC in the gut lamina propria to produce the cytokines IL-6 and IL-23, both important for TH17 differentiation (Atarashi and others 2008); this mechanism of TH17 induction is thought to also contribute to T-cell encephalogenicity in EAE. Gut microbiota can also affect blood-brain barrier permeability, as GF mice exhibit disrupted tight junctions (Braniste and others 2014).

In the context of antibiotic treatment, mice also exhibit suppression of EAE induction, which is marked by an increase in Treg formation and IL-10-producing B cells (Ochoa-Reparaz and others 2010b; Mielcarz and Kasper 2015). Specifically, it has been shown that the product PSA by B. fragilis exerts a protective effect against EAE and involves a decrease in IL-17 production and increase in IL-10-producing CD4+ T cells, which was dependent on TLR2 signaling (Wang and others 2014). Treatment of EAE with bacterial formations has also been done. Bifidobacterium bifidum, Lactobacillus, and Streptococcus thermophiles treatment reduces EAE severity with decreases in TH1 and TH17 cells and Treg induction (Kwon and others 2013).

Importantly, these studies portray the immunomodulatory effects of bacteria and speak to the necessity of gut bacterial presence in EAE. All of these bacteria are represented in the phyla that are most commonly observed in the healthy mammalian gut, and these investigations hearken to the potential outcomes for EAE induction and possibly contribution to MS development when there is imbalance of bacterial species in the gut. It would be interesting to evaluate the outcome of EAE induction with mice harboring similar bacterial phyla and species compositions as the human gut with controlled alterations to specific bacterial populations within the microflora to more reflectively query the role of bacterial dysbiosis on EAE development.

Bacteriophages have not been evaluated for association with MS/EAE, though there has been postulation about the association of bacteriophages and autoimmunity (Riley 2004; Dalmasso and others 2014; Ogilvie and Jones 2015). As dysbiosis has been hypothesized as a risk factor for many autoimmune diseases, one method of causing underlying dysbiosis could be bacteriophage infection in which destabilization of bacterial populations occurs.

To our knowledge, there has been one investigation thus far to demonstrate a difference in bacteriophage abundance and autoimmune disease. Crohn's disease is an autoimmune disease of the digestive tract. Lepage and others (2008) showed that CD patients harbored significantly more bacteriophage VLP in colonic mucosa than non-CD counterparts, and these phages were of morphotypes consistent with the order Caudovirales. Whether this is due to increased bacterial burden or persistence of these phages in CD versus non-CD persons is unknown but warrant further investigation into the role of phages on bacterial dysbiosis and autoimmune disease.

The gut microbiome and MS

To date few, but highly informative, studies have examined the possible link between gut microbiota and MS. First, CS as a mode of infant delivery perturbs normal gut bacterial colonization dynamics and could possibly result in abnormal immune system development (Neu and Rushing 2011). Case-controlled studies reveal that CS delivery may be associated with increased risk of MS development. In the context of infant feeding, human milk is a rich source of bacteria, including staphylococci, streptococci, lactic acid bacteria, propionic bacteria, and bifidobacteria, and mother-to-infant transfer of strains belonging to Lactobacillus, Staphylococcus, Enterococcus, and Bifidobacterium through breastfeeding has been shown (Fernandez and others 2013). Breastfed bacteria are thus an important early source of infant gut bacteria, and these bacteria may contribute to normal immune system development by bacterial competitive exclusion, enhancing AMP production, and improving intestinal barrier function, among other mechanisms. Interestingly, Pisacane and others (1994) reported that persons with MS on average were breastfed for significantly shorter duration than their unaffected counterparts, and suggests a possible link between breastfed bacteria and risk of MS development that merits further inquiry. An exploratory investigation by Cantarel and others (2015) revealed overall decreased levels of the bacterial family Bacteroidaceae (phylum Bacteroidetes) and those of the genera Faecalibacterium and Ruminococcus (both of Firmicutes phylum) in persons with MS compared to Non-MS Counterparts (Table 1). In this same study, some MS patients were placed on vitamin D treatment, which led to increases in Firmicutes phylum members Faecalibacterium and Coprococcus. Importantly, Faecalibacterium is thought to be immunosuppressive via production of the SCFA butyrate and may be protective in MS. As also mentioned previously, Bacteroides, such as B. fragilis, are also known to be immunosuppressive, though via PSA production.

Table 1.

Completed Studies Investigating Gut Microbiota in Multiple Sclerosis Subjects

Study design Findings Reference
Case-controlled Overall ↓ Bacteroidaceae levels; ↓ Faecalibacterium, Ruminococcus; Vitamin D treatment ↑ Faecalibacterium and Coprococcus Cantarel and others, 2015
Case-controlled ↑ in Archaea (Methanobacter smithii) Jhangi and others, 2014
Case-controlled ↑ in Actinobacteria; ↓ in Bacteroidetes, Firmicutes; ↓ in Bacteroides, ↓Faecalibacterium, ↓ Prevoltella, ↓ Anaerostipes; ↑ in Bifidobacterium, ↑ Streptococcus Miyake and other, 2015
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↑, increased; ↓, decreased.

Archaea, which form a kingdom separate from prokaryotes and eukaryotes and compose 10% on colonic anaerobes, were found at higher levels in the gut of MS versus control persons, with specificity to the species Methanobacter smithii (Jhangi and others 2014). It is thought that the structural components of this Archaea species make it especially immunogenic. In a very recent report, Miyake and others (2015) investigated the gut microbiota of Japanese MS and healthy control cohorts and found an overall difference in the gut microbiota structure and greater inter-individual gut microbiota variability with MS versus healthy control comparison. They noted more prevalent Actinobacteria and less prevalent Bacteroidetes and Firmicutes phyla. At the genera echelon, MS subjects harbored lower levels of Bacteroides, Faecalibacterium, Prevotella, and Anaerostipes, though increased Bifidobacterium and Streptococcus. There were great differences in the levels of 21 species, 19 of which depletion was exhibited and 14 from the Clostridia clusters XIVa and IV, which are distinguished as heavy producers of SCFAs. Streptococcus thermophilus and Eggerthella lenta were more prevalent in MS versus healthy control subjects. Thus far, it appears from the current studies that there is gut microbial dysbiosis associated with MS development, with common findings including decreases in Bacteroidetes and Firmicutes phyla. These bacterial agents are crucial for immunoregulation and SCFA production. However, these studies have all been small and typically involve prevalent cases, making it difficult to know if the results are reproducible across various populations or to know if the differences are a cause or a result of MS.

Bacterial toxins may also be associated with MS development, according to a recent finding from Rumah and others (2013). Here, they report identification of Clostridium perfrigens type B from an MS patient, which was the first reported identification of this strain from humans. This strain produces its unique epsilon toxin, which has known ability to cross the blood-brain barrier and bind myelin and oligodendrocytes. Sera and cerebrospinal fluid analyses from a well-studied MS cohort showed higher immunoreactivity to epsilon toxin than healthy controls, suggesting prior exposure to epsilon toxin in these MS patients and the potential of a bacterial toxin to contribute to MS pathogenesis, especially in the absence of inflammation.

MS Gut Microbiome Manipulation for Therapeutic Benefit

There are currently no approved treatments for MS through manipulating the gut microbiome, though preclinical studies and ongoing clinical trials show great promise for therapeutic modulation of the gut microflora in MS. Vitamin D supplementation has extensively been shown to ameliorate EAE, presumably via downregulating DC and B-cell activation and Treg induction (Correale and Gaitan 2015). Investigative clinical studies show association of vitamin D treatment with MS patients and changes in the gut microbiota, specifically those of the phylum Firmicutes which exhibited increased abundance (Cantarel and others 2015). There are now several ongoing randomized controlled trials of vitamin D supplementation for MS treatment, though many small studies have been done. Positive outcomes include reports of fewer relapse events, decreases in the number of Gadolinium-enhancing regions per patient, suppression of T-cell proliferation, and increases in the percentage of peripheral IL-10+CD4+ T cells and decreases in the ratio of the cytokines IFNγ: IL-4 in T cells (Kimball and others 2007; Burton and others 2010; Smolders and others 2010). Overall, it is conceivable that vitamin D may affect the gut microbiome by rectifying dysbiosis and enforcing the immunosuppressive properties exerted by the microflora.

Probiotics are classified as live microorganisms that exert beneficial health on the host, and are heavily used in the management of intestinal illness (Jandhyala and others 2015). Their use in EAE has yielded mixed results, with some groups showing increased TH1 responses associated with exacerbated EAE, some showing lack of effects on disease symptomology, and 1 showing a protective effect (von Geldern and Mowry 2012). Specific species of bacteria have resulted in beneficial outcomes in EAE, including oral B. fragilis administration, which associated with IL-17 reduction and IL-10 induction, and Lactobacillus casei in which the mechanism of action is thought to be strain specific and enhance IL-10 production (Maassen and others 2000; Ochoa-Reparaz and others 2010a).

To date, there are no published clinical studies of bacterial probiotic administration for MS treatment, but several factors must be kept in mind for future studies. It is possible that the particular bacterial combination and amount must be patient-tailored since inter-individual variability in the gut microbiota may exist. Preexisting gut pathobionts may also prevent proper colonization of administered probiotics and present a null effect (Ventura and others 2011). Generally, any strain that increases its numbers relative to that of other bacteria will be eliminated by phage predation, which thus highlights the potential importance of phages in bacterial therapeutic intervention. As patients most likely already exhibit gut dysbiosis, a potential testable intervention may include the introduction of a combination of the potentially beneficial species of Bacteriodetes and Firmicutes, along with a tailored concoction of lysogenic phages that have specific tropism to these bacterial species and confer a survival advantage to these bacterial hosts. Moreover, administration of phages with tropisms specific for certain bacterial species already in the gut may also be considered, for instance, in conferring survival and proliferation of potentially beneficial bacteria and lysis of pathogenic bacteria, and may especially be important for antibiotic-resistant bacterial strains. So far there has been one phase II clinical trial to show safety and efficacy of phage administration for disease-Biophage-PA bacteriophage preparation for antibiotic-resistant P. aeruginosa in chronic otitis patients (Wright and others 2009).

Moreover, diet is known to play a major role in gut microbiota makeup (Mielcarz and Kasper 2015). Many studies show differences in the gut microbial composition of lean versus obese individuals, and this is highly associated with the adoption of the “Western diet,” which is based on proteins, animal fat, large sugar intake, and refined carbohydrates (Le Chatelier and others 2013; Manzel and others 2014; Cresci and Bawden 2015). This diet is associated with an increase in proinflammatory fatty acids and increased EAE severity (Mielcarz and Kasper 2015). Contrarily, calorie restriction is associated with decreased EAE severity; lowered inflammation, demyelination, and axon injury; increased corticosterone and adiponectin; and lowered IL-6 and leptin (Piccio and others 2008). A high ratio of eubiotic Bacteroidetes: Firmicutes is thought to be eubiotic, as Bacteroidetes including Prevotella heavily consume complex glycans and produce butyrate, which again is linked to increased Treg formation and proinflammatory NFκB downregulation (Jandhyala and others 2015). A fascinating recent study by Haghikia and others (2015) found that dietary fatty acids exert profound effects of T cells in the gut. Middle- and long-chain fatty acids (LCFAs) promoted TH1 and TH17 development, while SCFAs promoted Treg differentiation. Furthermore, LCFAs worsened EAE while SCFAs ameliorated this disease. This study in particular demonstrates the fundamental importance of gut bacterial composition and influence on EAE and possibly MS development, and suggests that dietary changes that favor gut SCFA production may have beneficial outcomes for MS therapy.

In sum, early small studies have already shown associations of gut bacterial makeup and MS, but larger, confirmatory studies are needed. Further, more research will need to be undertaken to also determine the gut virome makeup in these persons and how the overall microbiota changes with current therapeutics. Probiotics may give only partial coverage as an add-on treatment to some individuals, and phage therapy may also need to be explored. Thus, more investigation should also be invested in the bacteria–bacteriophage interaction in the gut and how that may factor into MS development and its importance in treatment. Additionally, changes in diet may also have beneficial effects on ameliorating the MS course, which may involve increasing intake of consumables that promote SCFA production (especially butyrate) by gut bacteria and immunosuppression. Therefore, the avenues of gut bacterial modulation and phage therapy are open for further exploration in MS therapy.

Author Disclosure Statement

Dr. Mowry receives funding from Biogen Idec for investigator-initiated research; she is also site PI of 2 Biogen trials, as well as of a trial sponsored by Sun Pharma. She receives free medication for a clinical trial from Teva Neuroscience. Dr. Glenn declares no competing financial interests exist.

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