Curr Treat Options Neurol (2015) 17:18 DOI 10.1007/s11940-015-0344-7
Multiple Sclerosis and Related Disorders (P Villoslada, Section Editor)
The Gut Microbiome in Multiple Sclerosis Daniel W. Mielcarz, PhD* Lloyd H. Kasper, MD Address * Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 1 Medical Center Drive, Lebanon, NH 03756, USA Email: [email protected]
* Springer Science+Business Media New York 2015
This article is part of the Topical Collection on Multiple Sclerosis and Related Disorders Keywords Gut microbiome I Multiple sclerosis I MS I Vitamin D deficiency I Hygiene hypothesis I Immunity I Inflammation
Opinion statement The gut microbiome is made up of a wide range of (chiefly) bacterial species that colonize the small and large intestine. The human gut microbiome contains a subset of thousands of bacterial species, with up to 1014 total bacteria. Studies examining this bacterial content have shown wide variations in which species are present between individuals. The gut microbiome has been shown to have profound effects on the development and maintenance of immune system in both animal models and in humans. A growing body of evidence has implicated the human gut microbiome in a range of disorders, including obesity, inflammatory bowel diseases, and cardiovascular disease. Animal studies present compelling evidence that the gut microbiome plays a significant role in the progression of demyelinating disease, and that modulation of the microbiome can lead to either exacerbation or amelioration of symptoms. Differences in diet, vitamin D insufficiency, smoking, and alcohol use have all been implicated as risk factors in MS, and all have the ability to affect the composition of the gut microbiota. Preliminary clinical trials aimed at modulating the gut microbiota in MS patients are underway and may prove to be a promising and lower-risk treatment option in the future.
Introduction Multiple sclerosis Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination and neurodegeneration [1]. Symptoms of MS arising from demyelinated lesions include fatigue, numbness, loss of coordination, vertigo, vision loss, dizziness, pain, bladder and bowel dysfunction, and
depression. MS presents clinically as a relapsing/ remitting or a progressive disease. Auto-reactive cells attack CNS tissue and cause demyelination and axonal damage; myelin-specific CD4+ cells are thought to be the main pathogenic cells in RRMS [2]. However, recent studies provide strong evidence that B cells play an important role in the pathology of RRMS [3].
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The etiology of MS is not fully appreciated, although strong evidence points to both genetic and environmental factors. The female-to-male ratio of MS has increased over the past decades to a current incidence of 3:1, indicating a potential role for intrinsic factors such as differences in endocrine function and perhaps hormone exposure such as from hormonal birth control [4]. Familial studies have shown a recurrence rate of MS of 20 %. However, MS is not fully genetic, as monozygotic twins only show a 25–30 % lifetime MS risk when the other twin has been diagnosed [5]. Genetic studies have revealed that two HLA alleles, DRB1*1501 and DQ6 are more common in MS patients [6, 7]. These MHC class II genes are involved in the presentation of antigen to CD4+ T cells, and these particular alleles may be more effective at presenting self-antigen in a context that leads to autoimmunity. More recently, genome-wide association studies (GWAS) have demonstrated a role for several other genes involved in the immune response, including MHC class I, supporting the notion that much of the pathology of MS is immune mediated [8•]. Geographical location influences disease incidence as well, with a higher disease incidence at higher latitudes although this distinction has become less apparent in recent years as the world has become progressively more globalized [9]. Hypotheses for the reason behind this difference include differences in sun exposure leading to different levels of Vitamin D and differing regional diets [10, 11]. Vitamin D deficiency irrespective of geography is associated with increased symptoms in MS, and high levels of 25-OH Vitamin D are correlated with reduced MS risk in Caucasians [12]. Vitamin D has been shown to be important for immune function, including promoting T regulatory cell function, which can be important for controlling autoimmunity [13•]. Other dietary factors have also been shown to play a role in MS. Leptin, a hormone produced by adipose tissue, regulates food intake. Leptin deficient mice, while obese and exhibiting immune system alterations, do not develop EAE, and neutralizing leptin during active EAE disease reduces symptoms [14, 15]. In MS, leptin is found in high levels in CNS lesions, sera, and CSF, and shows a correlation with reduced numbers of T regulatory cells, indicating a potential role for this hormone in human disease [16, 17]. Indeed, fasting mice with less circulating leptin show lower EAE disease activity [18]. Another explanation for the geographical differences seen in MS incidence is the hygiene hypothesis. The hygiene hypothesis, first formulated by Strachan in 1989 states that as people experience less microbial
Curr Treat Options Neurol (2015) 17:18 exposure, they are more likely to experience autoimmune disorders such as allergy and MS [19]. In MS studies, much of the research into the relation between the hygiene hypothesis and the geographical variation of MS incidence has focused on helminth infections. Helminth infections typically require a Th2 immune response to control parasite growth, and the hypothesis states that this Th2 response will prevent or counterbalance a self-directed Th1 response. Extensively reviewed in this journal by Jorge Correale, the balance of animal studies have shown significant reductions in EAE clinical scores when mice are infected with one of several different parasites, typically showing a reduction in Th1 response and an increase in Th2 response [20•]. Early clinical trials are underway utilizing helminth infection in MS patients, and while study sizes have been small, treatment appears safe, and improved clinical outcome measures suggest that this approach may prove beneficial. Recently, Pedrini et al. explored the relation of Helicobacter pylori infection and multiple sclerosis, potentially providing further evidence for the hygiene hypothesis [21]. Female MS patients had a significantly lower rate of H. pylori infection than healthy controls, and seropositive MS patients had a lower disability score than seronegative patients. However, in male subjects, there was no relation between infection and MS incidence or disability score, indicating that there may be complex interactions with endocrine or genetic factors.
The gut microbiome and animal models of MS Beyond parasitic infections, differences in gut colonization by normal, non-pathogenic intestinal microflora among individuals and populations may be key mediators in a wide range of altered physiologic conditions including autoimmunity and other degenerative conditions. The gut microbiome is considered to be the entire genome of the host and the extensive genome of the microflora within the gut and consists of an extremely heterogeneous range of microflora that has evolved with the host to be critical for digestion, immune system development, and protection from pathogenic microorganisms [22•, 23]. The gut microbiota represents an important boundary between the environment and the immune system, and a major site for exposure to a wide range of both pathologic and intrinsic antigen. The total number of gut microbiota associated with a given human is 100 trillion cells, 100 times larger than the number of
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Bself^ cells making up the person [24•] and over a million genes compared to the human 28,000. It thus represents a critical and essential organ comprised of non-self genetic material that is involved in our health, well-being, and perhaps susceptibility to a wide range of disease states. Given the complex etiology of MS, with both environmental and genetically influenced immune factors both playing a role, many studies have focused efforts on defining the role of the gut microbiota in animal models of MS (Table 1). The gut-associated lymphoid tissue (GALT) represents the largest immune reservoir in our body containing nearly 80 % of our immune compartment. Gnotobiotic mice (raised in germ-free conditions) show significant defects in both their gut-associated and systemic lymphoid tissue [43••]. Peyer’s patches in these animals
are hypoplasic, and they have greatly reduced numbers of plasma cells that produce IgA and lamina propria resident CD4+ T cells [44]. Systemically, the internal structures of the spleen and lymph nodes are malformed, and the serum is hypergammaglobulinemic [44]. Colonization of germ-free mice with normal flora can reverse these phenotypes. As the gut of a neonatal mouse is sterile, colonization with commensals is a critical part of immune system development [45]. Given the extreme defects in immune system development, it is perhaps not surprising that germ-free mice show resistance to EAE induction, in both spontaneous and inducible models [25, 26], clearly showing an essential role for commensal-mediated immune system development in this disease. Of further note, recent studies demonstrate that gut microbiota influence blood-brain barrier
Table 1. Studies examining the role of the gut microbiome in experimental autoimmune encephalomyelitis Animal model
Intervention
Clinical score
Immune response
Reference
C57Bl6, MOG; SJL MOG-TCR tg
Germ-free housing
Decreased
[25, 26]
C57Bl/6, MOG; SJL, PLP
Broad-spectrum antibiotic treatment
Decrease
C57Bl/6, MOG
Oral administration of wild type B. fragilis
Decrease
C57Bl/6, MOG
Oral administration of PSA B. fragilis Oral treatment with PSA from B. fragilis Oral treatment with S. typhimurium expressing CFA/I fimbriae from E. coli Oral administration of Bifidobacterium animalis Oral administration of Lactobacillus spp. or other lactic acid bacteria Oral administration of Lactobacillus spp. with Bifidobacterium bifidum, and Streptococcus thermophilus Oral administration of Hsp65-tg Lactobacillus lactis
Normal
Reduced Th1 and Th17, increased Treg frequency Increased Treg frequency, decreased inflammatory cytokines Increased Treg frequency, increased Breg frequency Normal Increased CD39+ Treg frequency Increased Treg frequency, decreased Th1 and Th17 (Not examined)
[32, 33]
Decreased Th1 and Th17, increased Treg, IL-10 dependent Decreased Th1 and Th17, increased Treg
[38–40]
Decreased IL-17, increased IL-10, increased Treg and CD4+LAP+ cells
[42]
C57Bl/6, MOG SJL, PLP
Lewis rat, MBP C57Bl/6, MOG; SJL, PLP C57BL/6 MOG; Lewis rat, MBP
C57Bl/6, MOG
Decrease Decrease (both prophylactic and therapeutic) Decreased duration Decreased
Decreased
Decreased
[27••, 28]
[29••, 30, 31]
[29••]
[34–36]
[37]
[41]
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(BBB) permeability in mice. Germ-free mice have disrupted BBB tight junction when compared to pathogen-free adult mice. The permeability is overcome by colonization of the germ free mice with conventional gut flora. This observation suggests that early colonization by microflora may be essential in the development of normal BBB function [46]. While germ-free mice are useful tools for examining the role of commensals in many disease models, the extreme deficits in immune system development create a model that is less relevant to human disease. In our laboratory and others, oral antibiotics have been used to significantly deplete the commensal burden in the gut [27••, 28]. In these depleted animals, EAE severity is significantly reduced, and an increase in FoxP3+ Treg cells was seen in both the mesenteric and cervical lymph nodes, and adoptive transfer of these IL-10 producing Treg cells protected recipient animals [27••]. Only oral administration of the antibiotic mixture was able to confer this protection whereas intraperitoneal administration had no effect. In addition, an increase IL-10producing B cells (B regs) were also observed in antibiotic-depleted animals, a population that has shown protection upon transfer in other EAE studies [30, 47]. Studies elsewhere have demonstrated a role for iNKT cells in the antibiotic-mediated protection from EAE [28]. These studies firmly establish a complex role for gut commensals in the development of EAE beyond simply allowing the immune system to develop normally. Further studies in our laboratory and elsewhere have examined the role for specific commensal species in the pathogenesis of EAE. We have focused on the commensal Bacteroides fragilis, and specifically its capsular polysaccharide A (PSA). B. fragilis PSA is a zwitterionic polysaccharide that has shown to be able to affect the immune response in vitro, and to be sufficient to induce normal GALT development in formally germ-free mice [48–51]. Studies in colitis models show that PSA or colonization with B. fragilis is able to protect from disease, whereas mice colonized with PSA-deficient B. fragilis ( PSA) were susceptible [52, 53]. A reduction in IL-17 and increase in IL-10-producing CD4+ T cells was seen following PSA treatment, and these changes were TLR2 dependent [52, 54]. We have shown PSAdependent reduction in EAE disease when mice are colonized with B. fragilis following antibiotic treatment [29••]. This protection was associated with enhanced numbers of FoxP3+ Treg cells in the cervical lymph nodes, and naïve CD4+ T cells isolated from these mice
Curr Treat Options Neurol (2015) 17:18 more readily converted into IL-10-producing Treg cells in vitro. In contrast, mice reconstituted with PSAdeficient B. fragilis were not protected from EAE, and in fact showed increased susceptibility compared with non-reconstituted antibiotic treated mice. Recent studies in our lab demonstrated that EAE susceptible mice with normal intestinal microflora that were treated with purified PSA from B. fragilis had a profound clinical effect in reducing in EAE clinical score, and that this decrease was both IL-10 and TLR2 dependent [32]. An increased level of CD39+ CD4+ Treg cells was observed in the PSAtreated mice, and these cells are found at higher frequencies in CNS-draining lymph nodes compared to mice treated with a control polysaccharide [32, 33]. Taken together, these results point to B. fragilis PSA as being a potent regulator of EAE disease in mice, and may indicate potential utility in humans. Indeed, preliminary studies in our laboratory show the ability of PSA to induce CD39+ Treg in vitro from naïve CD4+ cells isolated from healthy controls. Studies elsewhere have demonstrated several other gut microbiota-related modification of EAE disease process. Oral treatment of mice with Salmonella typhimurium expressing the CFA/I fimbriae from E. coli leads to a decrease in EAE clinical score in both a prophylactic and therapeutic model of treatment [34–36]. This treatment leads to an increase in Treg frequency and a decrease in Th1 and Th17 cells [34–36]. Adoptive transfer of the Treg cells elicited by treatment was able to confer protection, indicating a key role for this cellular subset [35]. IL-13 and TGF-beta produced by Treg were shown to be important mediators of protection from EAE [34, 55]. This promising vaccination strategy was also shown to reduce disease in a collagen-induced arthritis model, indicating a potential role for changes in the gut microflora for other disseminated autoimmune diseases [56]. Priobiotic species of bacteria have been proposed as modulators of autoimmunity, and oral administration of several species has been tested in the EAE model. Bifidobacterium animalis decreased the duration of EAE symptoms in the Lewis rat model of EAE [37]. In mouse models of EAE, oral administration of Lactobacillus spp. or other lactic acid-producing bacteria have been shown to reduce clinical score [38–40]. Specifically, treatment with these bacteria has been shown to give rise to IL-10producing Treg, and that this IL-10 is responsible for protection [38, 39]. A probiotic mixture of Lactobacillus spp. with Bifidobacterium bifidum and Streptococcus thermophiles administered orally decreased EAE clinical score in both mouse and rat models, along with a decrease in
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Th1 and Th17 cells and an increase in Treg [41]. Lactobacillus lactis modified to express Hsp65 had a profound effect on the EAE immune response, decreasing clinical score, decreasing IL-17, and increasing IL-10, an increasing numbers of Treg and LAP+ CD4 T cells that have produced the anti-inflammatory molecule TGF-β, indicating potential for transgenically modified probiotic species in fighting inflammatory disease [42]. Taken together, these studies provide strong evidence for modulation of the immune response in EAE by probiotic species of bacteria that should inform future human studies.
Examining the gut microbiome in MS While modifications in the gut microbiome have been extensively studied in EAE, studies in MS thus far have been mostly limited to case-control examinations of gut microflora in MS patients versus healthy controls. Recent human studies related to the gut microbiome are presented in Table 2. In a small study, Mowry et al. found that RRMS patients had differing levels of Firmicutes, Bacteroidetes, and Proteobacteria, and patients treated with glatiramer acetate had further differences in Firmicutes [57]. Furthermore, Vitamin D treatment led to increases in Enterobacteria in both healthy controls and RRMS
patients, with changes in Firmicutes, Actinobacteria, and Proteobacteria in RRMS patients. A similar study but larger study was performed by Jhangi et al. in which the gut microbiomes of 53 MS patients (22 untreated, 13 GA treated, and 18 IFN-β treated) were compared with 44 healthy controls [60•]. Treatment effects from GA or IFN-β were absent, but differences between MS patients and healthy controls were observed. A significant increase in Archaea was seen, specifically Methanobrevibacter smithii, which could potentially play a role in inflammation. As in the Mowry study, levels of Firmicutes were reduced in MS patients, along with a reduction in other butyrate producers, Butyricimonas. Butyrate can have anti-inflammatory properties, indicating that the reduction in these species in MS patients may contribute to disease [63]. The gut microbiome of 20 pediatric MS patients was examined in a study by Tremlett et al. [61]. Preliminary results from the study show increases in Shigella, Escherichia, and Clostridium, all species associated with infection and inflammation, and decreases in Eubacterium rectale and Corynebacterium. These observational studies have been somewhat limited in patient number, but show that differences do exist, and that these differences may play a role in the inflammatory processes of MS. A study is underway by the MS Microbiome Consortium which aims to examine much larger numbers of MS patients for microbiome content.
Table 2. Studies examining the role of the gut microbiome in multiple sclerosis patients Study type
Subjects
Details
Reference
Case-control
7 RRMS patients
[57, 58]
Case-control
26 RRMS patients, 4 SPMS patients
Case-control
53 MS patients
Case-control
20 pediatric RRMS
Some differences in Firmicutes, Bacteroidetes, and Proteobacteria. Vitamin D affected levels of Firmicutes, Actinobacteria, and Proteobacteria Clostridium perfringens type B epsilon toxin found in CSF of MS patients/C. perfingens type A was found at lower levels in MS patient stool than in healthy controls Increased Archaea in MS patients compared with healthy controls; decreased Butyricimonas and Lachnospiraceae Increased Shigella, Escherichia, Clostridium; decreased Eubacterium rectale and Corynebacterium PSA from B. fragilis was able to induce suppressive CD39+ Treg in vitro in both MS patients and healthy controls The MS Microbiome Consortium is currently analyzing hundreds of samples from MS patients by 16s ribosomal subunit sequencing
In vitro
Population
Enrolling
[59]
[60•]
[61] [62]
N/A
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In study focused on one specific commensal, prevalence of Clostridium perfringens was examined, and the commensal type A strain found to be less common in the GI tract of MS patients versus healthy controls [59]. Consistent with this, a significant subset of MS patient antibody against C. perfringens epsilon toxin—secreted by type B and D strains—in both serum and CSF, indicating a potential role for this toxin in MS disease process. Given the promising results from the EAE model, our laboratory has begun preliminary studies examining the
effects of B. fragilis PSA on human immune cells. Peripheral blood mononuclear cells were isolated from RRMS patients and healthy controls, and immune subsets sorted and exposed to PSA in vitro. PSA was able to induce the differentiation of T regulatory cells expressing CD39 and HLA-DR [62]. These Treg had increased suppressive capacity and IL-10 production, and were able to suppress monocyte production of TNF-α. This in vitro data—combined with the extensive EAE studies—point to PSA and potentially other microbiome-derived molecules as promising future therapeutics in MS.
Treatment The clinical data on the microbiome in MS is chiefly descriptive thus far, and strategies focused on directly modulating it are not yet supported. However, treatments and lifestyle changes that have been shown to affect MS may be acting through changes in the microbiome. Vitamin D has been shown to correlate with and promote the differentiation of Treg [12, 13•]. Latitudinal differences in MS incidence have been proposed to be due to vitamin D deficiency at higher latitudes [9, 64]. Vitamin D levels have been linked to changes in the gut microbiome, indicating that maintaining healthy Vitamin D levels may be important in maintaining a microbiome/immune system balance [65, 66]. With this evidence, vitamin D supplementation has been tested in several clinical trials for MS treatment. A systemic analysis of five double-blind trials showed one with positive results in a reduction of T1 lesions and four others with no advantage over placebo [67], but given the relative safety of Vitamin D supplementation, it is prudent to recommend screening and supplementation for MS patients, particularly those in higher latitudes. Beyond vitamin D, changes in diet can rapidly alter the composition of the microbiome [68]. Obese individuals have a significantly different gut microbiota than lean individuals [69]. Studies in mice examining the effect of a high-fat Westernized diet have shown alterations in the gut flora, an increase in proinflammatory plasma free fatty acids, and an increase in EAE severity [69–71]. Obesity in humans can be related to reduced diversity of the microbiome and a lower amount of Bacteroidetes [69]. These changes related to obesity and Western diet may have implications for MS pathology, although more study is needed. Experiential information suggests that there are a variety of diets including the Swank and Wahls that have been proposed to be of benefit to those with MS. To date, however, there have been no confirmatory clinical trials performed to demonstrate efficacy in either relapsing or progressive MS. As more information is gathered on the critical role of the gut microbiome in CNS demyelination, it is expected that clinical trials focused on diet with clearly defined outcome measures will be undertaken. Research examining the gut microbiome and EAE/MS has already provided strong evidence for an immune system link between the gut and the CNS. While it is too early to directly proscribe modulation of the gut microbiota for
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treatment of MS, it is clear that said modulation can have profound effects in animal models of MS. We hypothesize that future studies will show the gut microbiome to be an important environmental risk factor in MS, and that treatments targeted at or derived from the microbiome will be safe and effective ways to treat the disease.
Diet and lifestyle & &
No specific diets have been yet been linked to improved outcomes in MS, although a high fat Western diet has been linked to increased severity of EAE in the mouse model. Obesity has been linked to reduced diversity of the gut microbiome and specifically lower prevalence of Bacteroidetes. Given the potential antiinflammatory role of these bacteria, patients should be counseled to maintain a healthy BMI.
Compliance with Ethics Guidelines Conflict of Interest Daniel W. Mielcarz declares no conflict of interest. Lloyd H. Kasper declares the receipt of grants from Symbiotix Biotherapies, NIH, and NMSS, outside the submitted work. 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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Multiple Sclerosis and Related Disorders (P Villoslada, Section Editor)
The Gut Microbiome in Multiple Sclerosis Daniel W. Mielcarz, PhD* Lloyd H. Kasper, MD Address * Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 1 Medical Center Drive, Lebanon, NH 03756, USA Email: [email protected]
* Springer Science+Business Media New York 2015
This article is part of the Topical Collection on Multiple Sclerosis and Related Disorders Keywords Gut microbiome I Multiple sclerosis I MS I Vitamin D deficiency I Hygiene hypothesis I Immunity I Inflammation
Opinion statement The gut microbiome is made up of a wide range of (chiefly) bacterial species that colonize the small and large intestine. The human gut microbiome contains a subset of thousands of bacterial species, with up to 1014 total bacteria. Studies examining this bacterial content have shown wide variations in which species are present between individuals. The gut microbiome has been shown to have profound effects on the development and maintenance of immune system in both animal models and in humans. A growing body of evidence has implicated the human gut microbiome in a range of disorders, including obesity, inflammatory bowel diseases, and cardiovascular disease. Animal studies present compelling evidence that the gut microbiome plays a significant role in the progression of demyelinating disease, and that modulation of the microbiome can lead to either exacerbation or amelioration of symptoms. Differences in diet, vitamin D insufficiency, smoking, and alcohol use have all been implicated as risk factors in MS, and all have the ability to affect the composition of the gut microbiota. Preliminary clinical trials aimed at modulating the gut microbiota in MS patients are underway and may prove to be a promising and lower-risk treatment option in the future.
Introduction Multiple sclerosis Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination and neurodegeneration [1]. Symptoms of MS arising from demyelinated lesions include fatigue, numbness, loss of coordination, vertigo, vision loss, dizziness, pain, bladder and bowel dysfunction, and
depression. MS presents clinically as a relapsing/ remitting or a progressive disease. Auto-reactive cells attack CNS tissue and cause demyelination and axonal damage; myelin-specific CD4+ cells are thought to be the main pathogenic cells in RRMS [2]. However, recent studies provide strong evidence that B cells play an important role in the pathology of RRMS [3].
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The etiology of MS is not fully appreciated, although strong evidence points to both genetic and environmental factors. The female-to-male ratio of MS has increased over the past decades to a current incidence of 3:1, indicating a potential role for intrinsic factors such as differences in endocrine function and perhaps hormone exposure such as from hormonal birth control [4]. Familial studies have shown a recurrence rate of MS of 20 %. However, MS is not fully genetic, as monozygotic twins only show a 25–30 % lifetime MS risk when the other twin has been diagnosed [5]. Genetic studies have revealed that two HLA alleles, DRB1*1501 and DQ6 are more common in MS patients [6, 7]. These MHC class II genes are involved in the presentation of antigen to CD4+ T cells, and these particular alleles may be more effective at presenting self-antigen in a context that leads to autoimmunity. More recently, genome-wide association studies (GWAS) have demonstrated a role for several other genes involved in the immune response, including MHC class I, supporting the notion that much of the pathology of MS is immune mediated [8•]. Geographical location influences disease incidence as well, with a higher disease incidence at higher latitudes although this distinction has become less apparent in recent years as the world has become progressively more globalized [9]. Hypotheses for the reason behind this difference include differences in sun exposure leading to different levels of Vitamin D and differing regional diets [10, 11]. Vitamin D deficiency irrespective of geography is associated with increased symptoms in MS, and high levels of 25-OH Vitamin D are correlated with reduced MS risk in Caucasians [12]. Vitamin D has been shown to be important for immune function, including promoting T regulatory cell function, which can be important for controlling autoimmunity [13•]. Other dietary factors have also been shown to play a role in MS. Leptin, a hormone produced by adipose tissue, regulates food intake. Leptin deficient mice, while obese and exhibiting immune system alterations, do not develop EAE, and neutralizing leptin during active EAE disease reduces symptoms [14, 15]. In MS, leptin is found in high levels in CNS lesions, sera, and CSF, and shows a correlation with reduced numbers of T regulatory cells, indicating a potential role for this hormone in human disease [16, 17]. Indeed, fasting mice with less circulating leptin show lower EAE disease activity [18]. Another explanation for the geographical differences seen in MS incidence is the hygiene hypothesis. The hygiene hypothesis, first formulated by Strachan in 1989 states that as people experience less microbial
Curr Treat Options Neurol (2015) 17:18 exposure, they are more likely to experience autoimmune disorders such as allergy and MS [19]. In MS studies, much of the research into the relation between the hygiene hypothesis and the geographical variation of MS incidence has focused on helminth infections. Helminth infections typically require a Th2 immune response to control parasite growth, and the hypothesis states that this Th2 response will prevent or counterbalance a self-directed Th1 response. Extensively reviewed in this journal by Jorge Correale, the balance of animal studies have shown significant reductions in EAE clinical scores when mice are infected with one of several different parasites, typically showing a reduction in Th1 response and an increase in Th2 response [20•]. Early clinical trials are underway utilizing helminth infection in MS patients, and while study sizes have been small, treatment appears safe, and improved clinical outcome measures suggest that this approach may prove beneficial. Recently, Pedrini et al. explored the relation of Helicobacter pylori infection and multiple sclerosis, potentially providing further evidence for the hygiene hypothesis [21]. Female MS patients had a significantly lower rate of H. pylori infection than healthy controls, and seropositive MS patients had a lower disability score than seronegative patients. However, in male subjects, there was no relation between infection and MS incidence or disability score, indicating that there may be complex interactions with endocrine or genetic factors.
The gut microbiome and animal models of MS Beyond parasitic infections, differences in gut colonization by normal, non-pathogenic intestinal microflora among individuals and populations may be key mediators in a wide range of altered physiologic conditions including autoimmunity and other degenerative conditions. The gut microbiome is considered to be the entire genome of the host and the extensive genome of the microflora within the gut and consists of an extremely heterogeneous range of microflora that has evolved with the host to be critical for digestion, immune system development, and protection from pathogenic microorganisms [22•, 23]. The gut microbiota represents an important boundary between the environment and the immune system, and a major site for exposure to a wide range of both pathologic and intrinsic antigen. The total number of gut microbiota associated with a given human is 100 trillion cells, 100 times larger than the number of
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Bself^ cells making up the person [24•] and over a million genes compared to the human 28,000. It thus represents a critical and essential organ comprised of non-self genetic material that is involved in our health, well-being, and perhaps susceptibility to a wide range of disease states. Given the complex etiology of MS, with both environmental and genetically influenced immune factors both playing a role, many studies have focused efforts on defining the role of the gut microbiota in animal models of MS (Table 1). The gut-associated lymphoid tissue (GALT) represents the largest immune reservoir in our body containing nearly 80 % of our immune compartment. Gnotobiotic mice (raised in germ-free conditions) show significant defects in both their gut-associated and systemic lymphoid tissue [43••]. Peyer’s patches in these animals
are hypoplasic, and they have greatly reduced numbers of plasma cells that produce IgA and lamina propria resident CD4+ T cells [44]. Systemically, the internal structures of the spleen and lymph nodes are malformed, and the serum is hypergammaglobulinemic [44]. Colonization of germ-free mice with normal flora can reverse these phenotypes. As the gut of a neonatal mouse is sterile, colonization with commensals is a critical part of immune system development [45]. Given the extreme defects in immune system development, it is perhaps not surprising that germ-free mice show resistance to EAE induction, in both spontaneous and inducible models [25, 26], clearly showing an essential role for commensal-mediated immune system development in this disease. Of further note, recent studies demonstrate that gut microbiota influence blood-brain barrier
Table 1. Studies examining the role of the gut microbiome in experimental autoimmune encephalomyelitis Animal model
Intervention
Clinical score
Immune response
Reference
C57Bl6, MOG; SJL MOG-TCR tg
Germ-free housing
Decreased
[25, 26]
C57Bl/6, MOG; SJL, PLP
Broad-spectrum antibiotic treatment
Decrease
C57Bl/6, MOG
Oral administration of wild type B. fragilis
Decrease
C57Bl/6, MOG
Oral administration of PSA B. fragilis Oral treatment with PSA from B. fragilis Oral treatment with S. typhimurium expressing CFA/I fimbriae from E. coli Oral administration of Bifidobacterium animalis Oral administration of Lactobacillus spp. or other lactic acid bacteria Oral administration of Lactobacillus spp. with Bifidobacterium bifidum, and Streptococcus thermophilus Oral administration of Hsp65-tg Lactobacillus lactis
Normal
Reduced Th1 and Th17, increased Treg frequency Increased Treg frequency, decreased inflammatory cytokines Increased Treg frequency, increased Breg frequency Normal Increased CD39+ Treg frequency Increased Treg frequency, decreased Th1 and Th17 (Not examined)
[32, 33]
Decreased Th1 and Th17, increased Treg, IL-10 dependent Decreased Th1 and Th17, increased Treg
[38–40]
Decreased IL-17, increased IL-10, increased Treg and CD4+LAP+ cells
[42]
C57Bl/6, MOG SJL, PLP
Lewis rat, MBP C57Bl/6, MOG; SJL, PLP C57BL/6 MOG; Lewis rat, MBP
C57Bl/6, MOG
Decrease Decrease (both prophylactic and therapeutic) Decreased duration Decreased
Decreased
Decreased
[27••, 28]
[29••, 30, 31]
[29••]
[34–36]
[37]
[41]
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(BBB) permeability in mice. Germ-free mice have disrupted BBB tight junction when compared to pathogen-free adult mice. The permeability is overcome by colonization of the germ free mice with conventional gut flora. This observation suggests that early colonization by microflora may be essential in the development of normal BBB function [46]. While germ-free mice are useful tools for examining the role of commensals in many disease models, the extreme deficits in immune system development create a model that is less relevant to human disease. In our laboratory and others, oral antibiotics have been used to significantly deplete the commensal burden in the gut [27••, 28]. In these depleted animals, EAE severity is significantly reduced, and an increase in FoxP3+ Treg cells was seen in both the mesenteric and cervical lymph nodes, and adoptive transfer of these IL-10 producing Treg cells protected recipient animals [27••]. Only oral administration of the antibiotic mixture was able to confer this protection whereas intraperitoneal administration had no effect. In addition, an increase IL-10producing B cells (B regs) were also observed in antibiotic-depleted animals, a population that has shown protection upon transfer in other EAE studies [30, 47]. Studies elsewhere have demonstrated a role for iNKT cells in the antibiotic-mediated protection from EAE [28]. These studies firmly establish a complex role for gut commensals in the development of EAE beyond simply allowing the immune system to develop normally. Further studies in our laboratory and elsewhere have examined the role for specific commensal species in the pathogenesis of EAE. We have focused on the commensal Bacteroides fragilis, and specifically its capsular polysaccharide A (PSA). B. fragilis PSA is a zwitterionic polysaccharide that has shown to be able to affect the immune response in vitro, and to be sufficient to induce normal GALT development in formally germ-free mice [48–51]. Studies in colitis models show that PSA or colonization with B. fragilis is able to protect from disease, whereas mice colonized with PSA-deficient B. fragilis ( PSA) were susceptible [52, 53]. A reduction in IL-17 and increase in IL-10-producing CD4+ T cells was seen following PSA treatment, and these changes were TLR2 dependent [52, 54]. We have shown PSAdependent reduction in EAE disease when mice are colonized with B. fragilis following antibiotic treatment [29••]. This protection was associated with enhanced numbers of FoxP3+ Treg cells in the cervical lymph nodes, and naïve CD4+ T cells isolated from these mice
Curr Treat Options Neurol (2015) 17:18 more readily converted into IL-10-producing Treg cells in vitro. In contrast, mice reconstituted with PSAdeficient B. fragilis were not protected from EAE, and in fact showed increased susceptibility compared with non-reconstituted antibiotic treated mice. Recent studies in our lab demonstrated that EAE susceptible mice with normal intestinal microflora that were treated with purified PSA from B. fragilis had a profound clinical effect in reducing in EAE clinical score, and that this decrease was both IL-10 and TLR2 dependent [32]. An increased level of CD39+ CD4+ Treg cells was observed in the PSAtreated mice, and these cells are found at higher frequencies in CNS-draining lymph nodes compared to mice treated with a control polysaccharide [32, 33]. Taken together, these results point to B. fragilis PSA as being a potent regulator of EAE disease in mice, and may indicate potential utility in humans. Indeed, preliminary studies in our laboratory show the ability of PSA to induce CD39+ Treg in vitro from naïve CD4+ cells isolated from healthy controls. Studies elsewhere have demonstrated several other gut microbiota-related modification of EAE disease process. Oral treatment of mice with Salmonella typhimurium expressing the CFA/I fimbriae from E. coli leads to a decrease in EAE clinical score in both a prophylactic and therapeutic model of treatment [34–36]. This treatment leads to an increase in Treg frequency and a decrease in Th1 and Th17 cells [34–36]. Adoptive transfer of the Treg cells elicited by treatment was able to confer protection, indicating a key role for this cellular subset [35]. IL-13 and TGF-beta produced by Treg were shown to be important mediators of protection from EAE [34, 55]. This promising vaccination strategy was also shown to reduce disease in a collagen-induced arthritis model, indicating a potential role for changes in the gut microflora for other disseminated autoimmune diseases [56]. Priobiotic species of bacteria have been proposed as modulators of autoimmunity, and oral administration of several species has been tested in the EAE model. Bifidobacterium animalis decreased the duration of EAE symptoms in the Lewis rat model of EAE [37]. In mouse models of EAE, oral administration of Lactobacillus spp. or other lactic acid-producing bacteria have been shown to reduce clinical score [38–40]. Specifically, treatment with these bacteria has been shown to give rise to IL-10producing Treg, and that this IL-10 is responsible for protection [38, 39]. A probiotic mixture of Lactobacillus spp. with Bifidobacterium bifidum and Streptococcus thermophiles administered orally decreased EAE clinical score in both mouse and rat models, along with a decrease in
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Th1 and Th17 cells and an increase in Treg [41]. Lactobacillus lactis modified to express Hsp65 had a profound effect on the EAE immune response, decreasing clinical score, decreasing IL-17, and increasing IL-10, an increasing numbers of Treg and LAP+ CD4 T cells that have produced the anti-inflammatory molecule TGF-β, indicating potential for transgenically modified probiotic species in fighting inflammatory disease [42]. Taken together, these studies provide strong evidence for modulation of the immune response in EAE by probiotic species of bacteria that should inform future human studies.
Examining the gut microbiome in MS While modifications in the gut microbiome have been extensively studied in EAE, studies in MS thus far have been mostly limited to case-control examinations of gut microflora in MS patients versus healthy controls. Recent human studies related to the gut microbiome are presented in Table 2. In a small study, Mowry et al. found that RRMS patients had differing levels of Firmicutes, Bacteroidetes, and Proteobacteria, and patients treated with glatiramer acetate had further differences in Firmicutes [57]. Furthermore, Vitamin D treatment led to increases in Enterobacteria in both healthy controls and RRMS
patients, with changes in Firmicutes, Actinobacteria, and Proteobacteria in RRMS patients. A similar study but larger study was performed by Jhangi et al. in which the gut microbiomes of 53 MS patients (22 untreated, 13 GA treated, and 18 IFN-β treated) were compared with 44 healthy controls [60•]. Treatment effects from GA or IFN-β were absent, but differences between MS patients and healthy controls were observed. A significant increase in Archaea was seen, specifically Methanobrevibacter smithii, which could potentially play a role in inflammation. As in the Mowry study, levels of Firmicutes were reduced in MS patients, along with a reduction in other butyrate producers, Butyricimonas. Butyrate can have anti-inflammatory properties, indicating that the reduction in these species in MS patients may contribute to disease [63]. The gut microbiome of 20 pediatric MS patients was examined in a study by Tremlett et al. [61]. Preliminary results from the study show increases in Shigella, Escherichia, and Clostridium, all species associated with infection and inflammation, and decreases in Eubacterium rectale and Corynebacterium. These observational studies have been somewhat limited in patient number, but show that differences do exist, and that these differences may play a role in the inflammatory processes of MS. A study is underway by the MS Microbiome Consortium which aims to examine much larger numbers of MS patients for microbiome content.
Table 2. Studies examining the role of the gut microbiome in multiple sclerosis patients Study type
Subjects
Details
Reference
Case-control
7 RRMS patients
[57, 58]
Case-control
26 RRMS patients, 4 SPMS patients
Case-control
53 MS patients
Case-control
20 pediatric RRMS
Some differences in Firmicutes, Bacteroidetes, and Proteobacteria. Vitamin D affected levels of Firmicutes, Actinobacteria, and Proteobacteria Clostridium perfringens type B epsilon toxin found in CSF of MS patients/C. perfingens type A was found at lower levels in MS patient stool than in healthy controls Increased Archaea in MS patients compared with healthy controls; decreased Butyricimonas and Lachnospiraceae Increased Shigella, Escherichia, Clostridium; decreased Eubacterium rectale and Corynebacterium PSA from B. fragilis was able to induce suppressive CD39+ Treg in vitro in both MS patients and healthy controls The MS Microbiome Consortium is currently analyzing hundreds of samples from MS patients by 16s ribosomal subunit sequencing
In vitro
Population
Enrolling
[59]
[60•]
[61] [62]
N/A
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In study focused on one specific commensal, prevalence of Clostridium perfringens was examined, and the commensal type A strain found to be less common in the GI tract of MS patients versus healthy controls [59]. Consistent with this, a significant subset of MS patient antibody against C. perfringens epsilon toxin—secreted by type B and D strains—in both serum and CSF, indicating a potential role for this toxin in MS disease process. Given the promising results from the EAE model, our laboratory has begun preliminary studies examining the
effects of B. fragilis PSA on human immune cells. Peripheral blood mononuclear cells were isolated from RRMS patients and healthy controls, and immune subsets sorted and exposed to PSA in vitro. PSA was able to induce the differentiation of T regulatory cells expressing CD39 and HLA-DR [62]. These Treg had increased suppressive capacity and IL-10 production, and were able to suppress monocyte production of TNF-α. This in vitro data—combined with the extensive EAE studies—point to PSA and potentially other microbiome-derived molecules as promising future therapeutics in MS.
Treatment The clinical data on the microbiome in MS is chiefly descriptive thus far, and strategies focused on directly modulating it are not yet supported. However, treatments and lifestyle changes that have been shown to affect MS may be acting through changes in the microbiome. Vitamin D has been shown to correlate with and promote the differentiation of Treg [12, 13•]. Latitudinal differences in MS incidence have been proposed to be due to vitamin D deficiency at higher latitudes [9, 64]. Vitamin D levels have been linked to changes in the gut microbiome, indicating that maintaining healthy Vitamin D levels may be important in maintaining a microbiome/immune system balance [65, 66]. With this evidence, vitamin D supplementation has been tested in several clinical trials for MS treatment. A systemic analysis of five double-blind trials showed one with positive results in a reduction of T1 lesions and four others with no advantage over placebo [67], but given the relative safety of Vitamin D supplementation, it is prudent to recommend screening and supplementation for MS patients, particularly those in higher latitudes. Beyond vitamin D, changes in diet can rapidly alter the composition of the microbiome [68]. Obese individuals have a significantly different gut microbiota than lean individuals [69]. Studies in mice examining the effect of a high-fat Westernized diet have shown alterations in the gut flora, an increase in proinflammatory plasma free fatty acids, and an increase in EAE severity [69–71]. Obesity in humans can be related to reduced diversity of the microbiome and a lower amount of Bacteroidetes [69]. These changes related to obesity and Western diet may have implications for MS pathology, although more study is needed. Experiential information suggests that there are a variety of diets including the Swank and Wahls that have been proposed to be of benefit to those with MS. To date, however, there have been no confirmatory clinical trials performed to demonstrate efficacy in either relapsing or progressive MS. As more information is gathered on the critical role of the gut microbiome in CNS demyelination, it is expected that clinical trials focused on diet with clearly defined outcome measures will be undertaken. Research examining the gut microbiome and EAE/MS has already provided strong evidence for an immune system link between the gut and the CNS. While it is too early to directly proscribe modulation of the gut microbiota for
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treatment of MS, it is clear that said modulation can have profound effects in animal models of MS. We hypothesize that future studies will show the gut microbiome to be an important environmental risk factor in MS, and that treatments targeted at or derived from the microbiome will be safe and effective ways to treat the disease.
Diet and lifestyle & &
No specific diets have been yet been linked to improved outcomes in MS, although a high fat Western diet has been linked to increased severity of EAE in the mouse model. Obesity has been linked to reduced diversity of the gut microbiome and specifically lower prevalence of Bacteroidetes. Given the potential antiinflammatory role of these bacteria, patients should be counseled to maintain a healthy BMI.
Compliance with Ethics Guidelines Conflict of Interest Daniel W. Mielcarz declares no conflict of interest. Lloyd H. Kasper declares the receipt of grants from Symbiotix Biotherapies, NIH, and NMSS, outside the submitted work. 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.
References and Recommended Reading Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1. 2. 3. 4.
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