Host-commensal interactions have increasingly been shown to play a role in the induction of autoimmunity both in experimental animals and human diseases including inflammatory bowel disease, rheumatoid arthritis, type 1 diabetes and experimental autoimmune encephalomyelitis8,27,28. The origin of the autoimmune process in multiple sclerosis is still poorly understood and whether the inciting factors that trigger inflammation primarily occur in the central nervous system or in the periphery is unknown. Given that disease concordance in MS is 25% in monozygotic twins, both genetic and environmental factors likely contribute to the development of disease29, and the gut microbiota might be one such environmental factor.

We undertook studies to define the community structure of the faecal microbiome in MS patients using high-throughput 16S rRNA gene sequencing. We found alterations at the phylum level with increases in Euryarchaeota and Verrucomicrobia. At the genus level, more specific shifts were observed, including increases of Methanobrevibacter and Akkermansia and reduction in Butyricimonas. Given that discrepancies may occur due to variability in primer selection and sequencing reads, we utilized two separate sequencing methodologies and observed that the majority of taxonomic shifts were consistent in both platforms.

Several of the organisms identified in this study as being altered in MS have been demonstrated to drive inflammation or have been associated with autoimmunity. The archaeon Methanobrevibacter has been implicated in inflammation by its capacity to recruit inflammatory cells and activate human dendritic cells17 and its role in inflammatory diseases, including periodontitis, asthma and inflammatory bowel disease24,30,31. In addition, archaeosomes derived from Methanobrevibacter have potent adjuvant properties secondary to their unique lipid structure and have been used as adjuvants for vaccines32. Methanobrevibacter is distributed throughout the small bowel and colon and is tightly adherent to the mucosa via regulated expression of adhesin-like proteins, placing it in close proximity to the gut-associated lymphoid tissue33. Consequently, when mucosa-associated (rather than luminal) microbiota were studied in inflammatory bowel disease, Methanobrevibacter smithii was increased more than threefold in mucosal samples from patients with both Crohn’s disease and ulcerative colitis compared to controls30. Methanobrevibacter smithii is also recovered more frequently in children with obesity, a known risk factor for the development of MS in adult life34. Furthermore, in a pilot study of pediatric multiple sclerosis, children colonized with Methanobrevibacter had a shorter time to relapse35.

We also found that the phylum Verrucomicrobia was increased in MS patients and was driven by the genus Akkermansia, which was also reported in a pilot study of 7 MS patients36. In contrast to our findings in MS, Akkermansia species have been reported to be decreased in other autoimmune diseases including psoriatic arthritis37. Akkermansia has been reported to have both regulatory and inflammatory properties, and is a mucin-degrader that converts mucin to short-chain fatty acids that may mediate the immunoregulatory effects38. Alternatively, Akkermansia has been correlated to proinflammatory pathways including upregulation of genes involved in antigen-presentation, B- and T-cell receptor signalling, and activation of complement and coagulation cascades39. These proinflammatory features may be related to its ability to degrade mucus, leading to breakdown of the gut barrier and increased exposure of resident immune cells to microbial antigens40.

We found lower abundances of Butyricimonas, a butyrate-producing genus, in MS patients. Butyrate is a short-chain fatty acid produced by microbes that induce colonic regulatory T cells41. Reductions in colonic butyrate can disrupt barrier function and promote inflammation. Similar to our findings, reductions in butyrate producers have been noted in numerous autoimmune and inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis and type 1 diabetes5,42,43.

We investigated untreated MS patients, to examine the microbiota independent of MS disease-modifying therapy. We found that the genera that were altered in the entire MS cohort (Methanobrevibacter, Akkermansia and Butyricimonas) were also altered in the untreated population, suggesting that these effects are not specifically correlated with therapy. Furthermore, within the untreated MS subset, we observed reductions in genera belonging to the family Coriobacteriaceae, including Collinsella and Slackia. Reductions in Coriobacteriaceae have been reported in relatives of patients with inflammatory bowel disease44.

Patients on disease-modifying therapy had increased abundances of the genera Prevotella compared with untreated patients. Although Prevotella has been reported to be increased in rheumatoid arthritis and inflammatory bowel disease5, Prevotella has been previously correlated to the intake of high-fibre diets, whose primary substrate, fibre, can drive the generation of the immunoregulatory metabolite butyrate45. We found that Prevotella was low in untreated MS, and that treatment with disease-modifying therapy was associated with increased relative abundance of Prevotella. In a smaller cohort of 20 MS patients compared with 40 controls in Japan, the authors detected a decrease in Prevotella11 in MS. Given this consistent finding, future studies investigating the role of Prevotella in MS are warranted.

We also observed increases in the genus Sutterella and decreases in Sarcina in MS patients on therapy. Sutterella was found to be increased in healthy controls compared to patients with new-onset Crohn’s disease46. Sarcina species are reported to be increased in the gut microbiota of autistic patients47. Since immunomodulatory treatment in MS was associated with increases in relative abundances of Prevotella and Sutterella and decreases in Sarcina, it is conceivable that treatment may act to normalize a proinflammatory microbiota.

Our finding that some of MS patients have elevated exhaled methane, a surrogate for levels of Methanobrevibacter in the gut, is consistent with our 16S rRNA sequencing results; it would be interesting to use this rapid, in vivo test to investigate Methanobrevibacter in larger populations of MS patients. While we did not measure stool abundance of Methanobrevibacter in this second cohort, previous studies have shown that the amount of breath methane strongly correlates with the quantity of M. smithii in the stool26. In other anatomical sites, the presence of particular microbial species can be identified based on their metabolic activity, as is routinely done for diagnosing Helicobacter pylori presence with a positive gastric urease test. Future studies employing simultaneous collection of breath, faecal and blood samples in MS patients will address the potential role of breath methane as a biomarker in MS.

The gut microbiota are known to modulate host immune gene expression either by direct contact with cell wall components, or by secretion of factors that can signal through host receptors or through epigenetic modifications that may alter methylation or acetylation of transcriptional promoters48. Microbial colonization by a single organism or by groups of organisms into the gut of germ-free mice can modulate the expression of innate and adaptive immune genes as early as 4 days after microbial inoculation in varying cellular compartments48. In a study of inflammatory bowel disease in humans, associations between microbes and host gene expression were found in innate and adaptive immune pathways49.

We examined relationships between microbial abundance and immune genes implicated in MS pathogenesis. Consistent with the inflammatory properties of Methanobrevibacter and Akkermansia32,33,34,36,45,46, we found positive correlations with these organisms and gene expression in T cells and monocytes involved in key pathways previously implicated in MS pathogenesis, including increased expression of the MAPK family in monocytes (MAPK1 and MAPK14), genes directly involved in both the initiation phase of innate immunity and activating adaptive immunity50. In T cells, Methanobrevibacter and Akkermansia positively correlated with TRAF5, a known regulator of T-cell activation and known to be overexpressed in MS, as well as STAT5B, whose expression is indispensible for the encephalitogenicity of autoreactive CD4+ T cells in EAE50,51. Methanobrevibacter or Akkermansia negatively correlated with TNFAIP3, previously shown to have reduced expression in studies of the MS transcriptome21,50. In the case of Akkermansia, these relationships were even stronger among untreated MS patients alone. Butyricimonas had negative correlations with genes known to be increased in MS among T cells and monocytes, suggesting that reduction in Butyricimonas is associated with increased proinflammatory gene expression, however, directionality cannot be determined from this study. These correlations were observed among all subjects and in untreated MS patients, but not in controls alone, suggesting that the relationship between microbial abundance and gene expression was MS-specific, but not solely driven by differences between controls and MS patients. Although we could detect correlation, we cannot determine the causal direction or conclude whether the microbes drive immunological changes, or whether the disease or altered immunity drives changes in the microbiota.

While our investigation of the gut microbiota in MS provides initial insights into understanding the potential role for the microbiome in this disease, our study has certain limitations. First, we cannot assign a direct cause to the associations we describe in the gut microbiome and the MS immunophenotype. The altered microbes may play a role in disease-related immunological changes, or MS-related changes in physiology may drive microbial alterations. Methanobrevibacter, for example, is recovered more frequently from individuals with constipation-variant irritable bowel disease52; while we excluded patients with irritable bowel disease from our study, it is possible, for example, that subtle changes in gut motility or in the enteric nervous system in MS patients or co-existing constipation may produce conditions favourable for the growth of this microbe. Second, our findings could be influenced by cohort-specific confounders. To minimize the effects of confounders, we used strict exclusion criteria eliminating the potential influence of antibiotics, pregnancy or other autoimmune or gastrointestinal conditions. Medications taken before the window of our study—such as prior courses of steroids—may also influence gut microbial populations. Furthermore, MS patients often consume alternative diets that might favour the growth of particular microbial niches53. Although we did not find any large differences in dietary intake in our cohort, more sensitive assays may reveal dietary variation that may be responsible for the observed changes in the gut microbiome in our disease population. Although the methodology used to collect stool samples was relatively uniform, variability in time of sample collection and interval of previous dietary intake may have also contributed to changes in microbes recovered. While age, gender and BMI have been previously shown to drive changes in the microbiome, our multi-factorial model suggests that these factors did not confound the observed microbial differences between the groups. Although we did not find a relationship between changes in the gut microbiome and clinical parameters such as disease duration or disability, most of our patients had low levels of disability (average EDSS 1.2). Future studies with larger cohorts and longitudinal collection of samples will be required to investigate these clinical associations, including subjects with progressive forms of the disease. Third, we analysed the microbiota using primers targeting the 16S rRNA gene. While this is useful for providing taxonomic information, shotgun metagenomic, metatranscriptomic and metabolomic profiling of faecal samples may reveal changes in the abundance of microbial genes, their expression or the presence of microbial metabolites that would complement phylogenic and taxonomic data. Finally, our study is limited by the fact that we collected samples after disease onset. It is possible that critical changes in the gut microbiome in MS may occur in early or preclinical stages of disease. For example, in type 1 diabetes, changes in the gut microbiome were apparent before the onset of illness in high-risk individuals with the concomitant appearance of anti-islet antibodies54. Investigation of the microbiota in paediatric or early-onset MS may provide further evidence of associations between the composition of the gut microbiome and MS pathogenesis.

In summary, we have found alterations of the human gut microbiome in MS that correlate with changes in the immune transcriptome and treatment. It is possible that treatment strategies of MS in the future may include therapeutic interventions designed to affect the microbiome such as probiotics, faecal transplantation and delivery of constituents of organisms isolated from the microbiome10, although more work is required. In addition, characterization of the gut microbiome in MS may provide biomarkers for assessing disease activity and could theoretically be an avenue to prevent MS in young at-risk populations.