Curr Neurol Neurosci Rep (2014) 14:492 DOI 10.1007/s11910-014-0492-2

DEMYELINATING DISORDERS (DN BOURDETTE AND V YADAV, SECTION EDITORS)

Gut Microbiome and Multiple Sclerosis Pavan Bhargava & Ellen M. Mowry

# Springer Science+Business Media New York 2014

Abstract The commensal flora that lives in the human gut is a unique ecosystem that has evolved over millennia with human beings. The importance of the microbiota in various bodily functions is gradually becoming more apparent. Besides the gut microbiome playing a role in bowel-related disorders, a role in metabolic and autoimmune disorders is becoming clearer. The gut bacteria play a role in educating the immune system and hence may be a player in the development of multiple sclerosis. We examine the different sources of information linking the gut microbiota to multiple sclerosis and examine the future avenues for utilizing the knowledge of the gut microbiome to potentially treat and prevent multiple sclerosis. Keywords Microbiome . Multiple sclerosis

Introduction Knowledge of the role of commensal microbes that inhabit various mucosal surfaces of the human body and their role in health and disease is growing at a tremendous rate. The term “microbiota” has been applied to the population of microbes in a given anatomical niche in the human body, whereas the term “microbiome” refers to the collective genome of all microbes in that niche [1]. It is interesting to note that the number of bacteria and their genetic material outnumbers the This article is part of the Topical Collection on Demyelinating Disorders P. Bhargava : E. M. Mowry (*) Department of Neurology, Johns Hopkins University School of Medicine, 600 N Wolfe Street, Pathology 627, Baltimore, MD 21287, USA e-mail: [email protected] P. Bhargava e-mail: [email protected]

human body’s somatic cell number and genome by a factor of 10 and 100, respectively [2]. Although until recently these commensal microbes were thought to be mere spectators, their role in health and disease is gradually being defined. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder affecting the central nervous system (CNS) [3]. The cause of this disorder is not completely understood, and both genetic and environmental factors appear to play important roles in susceptibility to the disease [3]. Although the CNS was initially thought to be an immune-privileged site, it is now clear that immune cells regularly survey the CNS, and in diseases such as MS there is an increase in the number of autoreactive immune cells targeting the CNS [4]. Since gut microbes play an important role in the education of the immune system and appear to play a role in several autoimmune and metabolic diseases, it is plausible that the gut commensal flora could be an important element in the susceptibility to MS [5]. We will attempt to review the basics of the understanding of the gut microbiome and current research linking the gut microbiota to CNS autoimmunity.

The Gut Microbiome: An Overview The “gut microbiome” refers to the collective genome of microbes (bacteria, archaea, viruses, and fungi) inhabiting the human gut. The human fetal gut is thought to be sterile; initial colonization is derived from the mother and environment at the time of birth [6]. The gut microbiota continues to expand in diversity through childhood owing to exposure to new flora through activities such as feeding and play. Several factors have been noted to affect this colonization, including mode of delivery, gestational age, diet, hygiene, and antibiotics [7, 8]. By about 1 year of age, infants have a distinct microbial profile [9]. The gut microbiota reaches maximum diversity at adolescence, and is then fairly stable [10]. The

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commonest bacterial phyla in the infant gut include Actinobacteria and Proteobacteria, with Bacteroidetes and Firmicutes becoming the predominant phyla in the adult gut [5]. Archaea, viruses, and fungi are the other classes of organisms that are important components of the gut microbiota. Initial studies suggested the existence of three distinct gut enterotypes, which were separated on the basis of variation in one of three genera—Bacteroides (enterotype 1), Prevotella (enterotype 2), and Ruminococcus (enterotype 3) [11•]. While, this concept has been challenged, although the presence of distinct enteric profiles in different human populations seems fairly clear [12]. Within the gut there is a spatial distribution of different bacterial species, with microbial diversity increasing from the stomach to the colon. The terminal ileum marks a transitional zone at which the predominant species in the microbiota change from aerobes to anaerobes [5]. Within a region of the gut, there may be a difference in the species that inhabit the mucosal surfaces and those that prefer the lumen [13, 14]. This is relevant since the species at the mucosal surface could have a greater impact on the immune system, whereas those in the lumen may be more important for energy and metabolic interactions. This spatial disparity in the microbiota represents a challenge, since most studies of the human gut microbiome use fecal samples, which may overrepresent certain bacterial populations. Functions of the Gut Microbiota The importance of the gut microbiota in normal physiology can be demonstrated using germ-free mice. In these mice, there is a lack of development of gut-associated lymphoid tissue (GALT) as well as defects in the peripheral immune system [6]. Additionally, there are defects in the development of the epithelium, musculature, and vasculature of the gut, underscoring the importance of the gut microbiome in normal development [6]. The gut microbiota plays an important role in the development of the immune system. Whereas certain commensals elicit the production of IL-10-producing regulatory T (Treg) cells, others make possible the maturation of T-helper 17 (Th17) cells [15••, 16]. The production of Treg cells may help in suppressing the response of the immune system to commensal bacteria, whereas Th17 cells could help in preparing the body for attack by pathogens. Effects on dendritic cells, natural killer T cells, and B cells have also been described. Besides modulating the immune system, the gut microbiota also seems to have significant metabolic functions. Although some of these functions have been known for a long time, such as the salvage of dietary sugars, production of shortchain fatty acids (SCFAs), deconjugation of bile acids and metabolism of drugs, new light is being shed on the importance and relevance of the gut microbiota through

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technologies such as metabolomics [5]. Metabolites produced by the gut microbiota can have effects on the immune system as well as play a role in processes such as atherosclerosis. Bacteria in the colon produce SCFAs by fermenting nondigestible carbohydrates such as pectins, hemicelluloses, and gums [17]. One of these SCFAs is butyrate, which is an important source of fuel for colonic epithelium [18]. Besides being an important fuel source, butyrate has also been shown to modulate the immune system in multiple ways. Butyrate was shown to enhance the production of IL-10 and IL-4 from CD3-stimulated monocytes, thus skewing the immune system towards a noninflammatory state [19]. Other in vitro studies have demonstrated a role for butyrate in the reduction of leukocyte adherence to the vascular endothelium, causing apoptosis of T cells and inhibiting the interferon-γ(IFN-γ)–signal transducer and activator of transcription 1 signaling axis [20–22]. More recent studies suggest that butyrate can upregulate populations of colonic Treg cells by signaling through a G-protein-coupled receptor for SCFAs [23, 24]. This evidence is consistent with demonstration of reductions in the populations of butyrate-producing organisms in ulcerative colitis and Crohn’s disease patients [25]. Reductions were seen in the population of Clostridium group IV members, specifically Faecalibacterium prausnitzii [26–28]. A group of 17 human Clostridium species that produce butyrate were able to inhibit experimental colitis by inducing colonic Treg cells [16]. Other metabolites such as choline and carnitine are now being explored for their role in inflammation as well as atherosclerosis. Mice fed diets deficient in methionine and choline develop a nonalcoholic steatohepatitis (NASH) phenotype [29]. This is mediated through the NLRP3 and NLRP6 inflammasomes. In these mice, an increase in Erysipelotrichi population was noted in the gut microbiota. In humans fed a choline-deficient diet, individuals who were susceptible to developing a NASH-like process were those with increased Erysipelotrichi and decreased Gammaproteobacteria populations in their stool at the baseline [30]. Choline is metabolized by the gut bacteria to trimethylamine and is subsequently converted to trimethylamine N-oxide (TMAO) by the human body. TMAO levels were correlated with the risk of atherosclerosis and cardiovascular disease in a cohort study [31]. It was hypothesized that the gut microbiome may play a role in producing larger amounts of trimethylamine and thus influence the risk of cardiovascular disease. With use of the same dataset it was also shown that L-carnitine levels were also associated with TMAO levels and atherosclerotic disease [32]. The Prevotella enterotype was associated with a greater risk of cardiovascular disease than the Bacteroides enterotype.

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Microbiota–Gut–Brain Axis The discovery that the gut microbiota can produce neurotransmitter molecules that can then influence the enteric nervous system and ultimately the CNS has led to the concept of the “microbiota–gut–brain axis” [33•]. Regulation of the CNS by the microbiome can occur through several mechanisms. Enteric neurons express Toll-like receptors 2, 3, 4, and 7, which can sense viral RNA and bacterial peptides [34]. In vitro studies have shown direct activation of these neurons by bacteria and bacterial products [35, 36]. Commensal bacteria also produce a range of neuroactive molecules such as serotonin, melatonin, GABA, histamine, and acetylcholine [37, 38]. Dysregulation of the production of these molecules could potentially affect the CNS. The gut microbiota also play a role in the development of the hypothalamic–pituitary–adrenal axis (HPA) [39]. The HPA axis is an essential component of the response of an individual to stress, and could serve as another means of communication between the gut and the CNS. The CNS can also influence the microbiota by control of food intake through signaling satiety. The HPA and the autonomic nervous system can also influence the microbiota by affecting the motility and secretory function of the gut [40]. This modification of the gut microenvironment may lead to modifications in the gut microbiota. The complex interplay of factors in the microbiota–gut–brain axis may be deranged in various CNS disorders, and a better understanding of these interactions is required [33•]. Methods of Characterizing the Gut Microbiome As mentioned earlier, “microbiome” refers to the entire genome of the gut microbiota. Initial approaches to study the gut microbiota consisted primarily in the culturing and isolation of microbes. Advances in DNA sequencing and reductions in the costs of these technologies have made it possible and more feasible to study the gut microbiome. Different approaches have been used, and each has its strengths and limitations, which need to be kept in mind when designing and interpreting studies related to the gut microbiome (Table 1). Metagenomic approaches are now the methods most commonly used to characterize the gut microbiome. “Metagenomics” refers to study of genetic material obtained directly from patient samples such as fecal samples, in contrast to cultivated cell cultures. Targeted approaches, such as 16S ribosomal RNA (a component of the 30S ribosomal subunit found in prokaryotes) gene sequencing, use selective amplification followed by sequencing of a gene that is found in all bacteria and archaea [41]. This method makes possible the identification of component microbes in the gut microbiome that could previously not be cultured, allowing better

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definition of the composition and diversity of the gut microbiota. Although this method provides a list of the microbes in the gut, other important information, such as the genetic makeup and functional capabilities of these different organisms, is not provided. Wholecommunity shotgun sequencing uses DNA extraction from the microbial community followed by shotgun DNA sequencing (fragmentation of the DNA followed by sequencing of the individual fragments) [41]. The sequences are then pieced together with algorithms and can be analyzed to monitor functional capabilities of the microbial community by identifying genes that are important for various processes. Advances in areas such as single-cell genomics are making possible the identification of rare organisms in the microbiota. Limitations of metagenomic methods include variability in efficacy of DNA extraction, gaps in metagenomic libraries, especially in the genomes of less abundant species, and ambiguity in assigning function to gene sequences on the basis of similarity alone [42]. Additionally, functional metagenomic screening and analysis of gene expression through metatranscriptomics (sequencing RNA from whole microbial communities) and metaproteomics (identifying proteins from whole microbial communities) are helping to better define functions of the gut microbiota that can be correlated with the genetic information derived from metagenomic approaches [43, 44]. These approaches are limited by large gaps in reference databases, thus limiting the ability to classify several identified protein or RNA functions. These advances, however, have not obviated the need for microbial culture, since molecular methods are not entirely unbiased. Although there are several limitations to using culturing to study the entire gut microbiota, certain lowabundance species have been cultured in the laboratory after having been missed by sequencing approaches [45]. Additionally, prior to using microbes as a therapeutic option, it would be necessary to culture them, isolate them, and test them in animal models.

Microbiome and Inflammatory Demyelinating Disease Studies of the Microbiome in Experimental Allergic Encephalomyelitis The Gut Microbiota Influences the Course and Severity of Experimental Allergic Encephalomyelitis Studies have investigated the role of the gut microbiota in experimental allergic encephalomyelitis (EAE), an animal model of MS.

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Table 1 Methods of characterizing the gut microbiome Method

Description

Strengths

Weaknesses

Targeted approaches

Sequencing of specific genes such as the 16S ribosomal RNA gene Sequencing of DNA from the entire microbial community

Inexpensive; widely used

Cannot identify certain classes of organisms; may miss low-abundance organisms

Can identify bacteria, archaea, viruses, and fungi in the microbiome

Variable efficiency of DNA extraction; gaps in metagenomic libraries especially for lowabundance organisms

Wholecommunity shotgun sequencing Functional approaches Microbial culture

Genomics, proteomics, or transcriptomics

Identify functions of the gut microbiota

Large gaps in reference libraries; functions of certain sequences or proteins may be unknown Can culture low-abundance species missed by Can be used to identify only a small number of other approaches; necessary before using an organisms; the ability to culture different organisms organism therapeutically in the gut microbiota is variable

First, Ochoa-Repáraz et al. [46] used a cocktail of antibiotics to alter the gut microbiota of mice prior to EAE induction. They demonstrated that oral antibiotic treatment significantly reduced bacterial populations in fecal and intestinal samples compared with intraperitoneal antibiotic treatment or no treatment. In the oral antibiotics group, there was a significant reduction in the onset and severity of EAE. This effect on the severity of EAE seemed to be mediated by an increase in the number of FoxP3+ Treg cells in the mesenteric and cervical lymph nodes. A possible role of CD103+ dendritic cells in skewing T cells towards a Treg cell phenotype was also demonstrated. In addition, adoptive transfer of CD4+ T cells from antibiotic-treated animals reduced EAE severity when compared with CD4+ T cells from naïve animals. Additional studies in mice treated with oral antibiotics suggest a possible role for IL-10-producing CD1dhighCD5+ B cells in the amelioration of EAE [47]. In a second study, germ-free mice were highly resistant to developing EAE and had a milder disease course when they did develop EAE [15••]. This resistance to EAE was found to be secondary to a reduced infiltration of inflammatory cells into the CNS. In the germ-free mice, there was a reduction in the populations of IFN-γ-producing CD4+ type 1 T-helper (Th1) cells and IL-17A-producing CD4+ Th17 cells, both of which play a crucial role in the development of EAE. The Treg cell populations were increased in germ-free mice. It was also demonstrated that dendritic cells derived from mesenteric lymph nodes of the germ-free mice were deficient in promoting the development of Th1 and Th17 cellular responses. The colonization of the germ-free mice with segmented filamentous bacteria led to a marked increase in the severity of EAE, and examination of nervous tissues from these mice showed increased invasion of the CNS by Th1 and Th17 cell populations. These observations helped establish that the gut microbiota could have an impact on immune responses in extraintestinal tissues, even the CNS. A third study used SJL/J mice with a transgenic T cell receptor for myelin oligodendrocyte glycoprotein 92–105

peptide [48••]. These mice developed spontaneous EAE. When the mice were reared in germ-free conditions, however, none of them developed EAE. In these mice, there were fewer Th17 cells in the GALT and reduced IL-17 and IFN-γ production from splenic immune cells. Recolonization of the germ-free mice led to reversal of these effects. Antibiotic treatment, which depleted the gut microbiota, led to a decreased proliferation of T cells in the GALT. Microbial Products Can Influence the Course of EAE Following the recognition of the importance of the gut microbiota in EAE susceptibility and severity , the effect of commensal microbes or their products on the course of EAE was evaluated. Ochoa-Repáraz et al. [49] treated mice with orally administered capsular polysaccharide A (PSA) of Bacteroides fragilis. EAE severity and cumulative scores were decreased in the PSA group compared with untreated mice. In the EAE brains of mice treated with PSA compared with untreated mice, there were decreased numbers of Th1 and Th17 cells. Lymphocytes isolated from the cervical lymph nodes of the PSA-treated mic produced less IL-17 and IFN-γ when stimulated with myelin oligodendrocyte glycoprotein and produced larger quantities of IL-10. PSA treatment ameliorated EAE even when it was begun 3–7 days after the induction of EAE. PSA enhanced the conversion of naïve T cells to FoxP3+ Treg cells by CD103+ dendritic cells. Consistent with this theory was the demonstration of abrogation of the PSA effect in IL-10-/- mice. Round et al. [50] showed that PSA stimulates Toll-like receptor 2 on T cells and causes increased production of inducible Treg cells, which secrete IL-10, even in the absence of antigen-presenting cells. Studies of the gut microbiome in EAE have shown us that the gut microbiome plays an important role in the development of Th1 and Th17 cells, which play a crucial role in EAE. In addition to these findings the possibility that certain bacteria/bacterial products could skew the immune system to a more regulatory phenotype has also been demonstrated.

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These studies point to the importance of studying the gut microbiome in MS. The Microbiome in MS Studies in MS patients looking at the gut microbiome are still in the early stages. Vartanian et al. [51•] recently demonstrated the first instance of human gut colonization by Clostridium perfringens type B in a patient with her first relapse of MS. This was exciting since epsilon toxin produced by this pathogen can lead to a microangiopathy, which can cause blood– brain barrier disruption, leading to neuronal and oligodendrocyte damage [52–55]. This could thus perhaps serve as a trigger for future autoimmune demyelinating events in susceptible individuals. There was also an increased prevalence of antibodies against epsilon toxin in sera from patients with MS compared with controls. Intriguingly, a mixture of Clostridium species was used to enhance Treg cell populations in a previous study [16]. Thus, besides an effect of a toxin, it is possible that the findings may point to an imbalance within the particular species of Clostridium in the microbiota of MS patients. Jhangi et al. [56] presented evidence for an increase in archaea (Methanobrevibacter) concentration in MS patients compared with controls. They also noted a reduction in Butyricimonas (phylum Bacteroidetes) and Lachnospiraceae (phylum Firmicutes) concentrations in MS patients compared with healthy controls. A reduction in Methanobrevibacter smithii concentrations has been noted in obese individuals, and the concentrations of this microbe vary inversely with BMI. Also, a recent study in patients with inflammatory bowel disease looking at the two predominant archaea species, Methanobrevibacter smithii and Methanosphaera stadtmanae, revealed increased Methanosphaera stadtmanae concentrations in patients with inflammatory bowel disease [57]. Treatment of human peripheral blood mononuclear cells with Methanosphaera stadtmanae produced larger quantities of proinflammatory cytokines than did treatment with Methanobrevibacter smithii. Further studies will be required to assess whether the observations of increased concentrations of archaea noted in these preliminary studies are confirmed in larger populations. In an exploratory study by our research group of fecal samples from 15 subjects (seven MS patients and eight healthy controls) a decrease in Faecalibacterium abundance in MS patients compared with controls was demonstrated [58]. Faecalibacterium prausnitzii is an important butyrateproducing organism whose abundance was noted to be reduced in the microbiome of inflammatory bowel disease patients [27]. Since butyrate production is linked to increased Treg cell populations, this could suggest a mechanism by which gut microbiome alterations would predispose individuals to developing MS. Additionally MS patients treated with

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glatiramer acetate demonstrated a difference in the abundance of Bacteroidaceae, Faecalibacterium, Ruminococcus, Lactobacillaceae, Clostridium, and other members of Clostridiales compared with untreated MS patients. Changes in the gut microbiota in response to vitamin D supplementation also differed between groups, with untreated MS patients showing an increase in abundance of Akkermansia, Faecalibacterium and Coprococcus compared with healthy controls or MS patients treated with glatiramer acetate. This study suggests a difference in the gut microbiome between MS patients and healthy controls, and additionally also suggests the possibility of an effect of disease-modifying therapy and vitamin D on the gut microbiome composition, although reverse causality cannot be excluded for the diseasemodifying therapy–microbiome link. Relation of Other Environmental/Genetic Factors Associated with MS and the Gut Microbiome Diet and the Gut Microbiome Diet has been shown to possibly play a role in EAE susceptibility and disease activity, and is postulated to play a role in MS risk as well. A low-calorie diet has been proposed to reduce disease activity in EAE [59], whereas a diet high in salt was shown to increase Th17 cell production in mice and caused increased severity of EAE [60]. Diet plays an essential role in shaping the gut microbiome. The type of feeding as an infant (breast milk vs formula) can determine the composition of the gut microbiota. In adults, a change in diet can produce a change in the gut microbiota. In one study, a mainly plant-based diet led to an increase in the population of Firmicutes (Roseburia, Ruminococcus bromii, Eubacterium rectale), which can help ferment plant polysaccharides, whereas a switch to a primarily meat-based diet led to an increase in the abundance of bile-tolerant microbes (Allstipes, Bilophila, and Bacteroides) [61]. A possible mediator between diet and MS disease activity could be the modulation of the microbiome by changes in diet, ultimately resulting in effects on the immune system. Given that data have shown a reproducible and rapid change in the microbiome with changes in diet, this could potentially be used as a means of modifying the gut microbiome in a favorable manner. BMI and the Gut Microbiome A relationship between childhood BMI and risk of developing MS has been described in females [62, 63]. The mechanism of this relationship is unknown. There is a strong relationship between BMI and gut microbiota composition. Studies have shown that obese individuals have lower microbial diversity and increased levels of the phylum Bacteroidetes [64, 65].

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Low bacterial genetic diversity was linked with insulin resistance, higher lipid levels, and inflammatory markers. Also, the individuals with lower genetic diversity had greater weight gain, and analysis of the gut microbiome revealed a reduction in the level of eight bacterial species, which were butyrate producers [64]. The elevated BMI in childhood could be associated with a reduction in the levels of butyrateproducing species, resulting in reduced production of Treg cells and ultimately a bias of the immune system towards an inflammatory state, predisposing to the development of MS.

Future Directions Humanized Mice Germ-free mice have been used as a method to study the role of certain commensal microbes in animal models of human disease. However, these mice require special facilities and include only certain genetic backgrounds. Recently, a study demonstrated a method for creating mice with a humanized gut microbiome. Using a course of broad-spectrum antibiotics and antifungals followed by repeated gavaging with donor human feces, Hintze et al. [66] were able to produce nongerm-free humanized mice whose fecal microbiome closely resembled that of their human donors. This method may help in further assessing the mechanisms by which an altered gut microbiome in MS patients impacts the development and prognosis of the disease.

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infection [67]. This involves the selection of a healthy donor, preferably one with no known transmissible disease and free of metabolic or autoimmune disorders. The recipient requires a course of antibiotics to clear the resident gut microflora. The donor feces are then diluted in water or normal saline and can be administered by different routes—nasogastric tube, endogastric tube, colonoscopy, or retention enema. In most indications, studies have shown that FMT is safe, although long-term safety studies are scarce. Studies have shown that at 6 months after FMT the recipient’s microbiota resemble that of the donor, suggesting that it is possible to produce a sustained change in the gut microbiota using FMT [68]. Studies are required, however, to assess the durability of this change over a longer time and in specific disease settings such as MS.

Conclusions Although there appears to be evidence for a role of the gut microbiome in CNS autoimmunity, substantial work needs to be done to establish what role gut commensals play in the susceptibility to and the course of MS. Future studies will need to identify microbial populations that are likely to be related to MS as well as define their functional role within the microbiome. This understanding will help devise methods for modulation of the gut microbiome to prevent or treat MS.

Modulation of the Gut Microbiome Compliance with Ethics Guidelines

Strategies to modulate the immune system, through alterations in the gut microbiome or using microbial products/metabolites, may serve as treatment options for MS in the future. As mentioned previously, several gut commensals and microbial products have now been shown to increase Treg cell populations (which have an anti-inflammatory effect). Whether these strategies will be applicable in MS remains to be determined. Better delineation of the gut microbiome alterations in MS followed by trials of different interventions targeting the gut microbiome will help answer this question. An important consideration in the design of these studies is the reciprocal interactions between the gut microbiome and the human body. Besides considering the effects of the gut microbiota on the immune system, the effects of CNS inflammation and disease-modifying therapies on the gut microbiota need to be considered.

Conflict of Interest Pavan Bhargava declares he has no conflict of interest. Ellen M. Mowry has received grants from Teva Neuroscience. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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