Following upon the discoveries that specific bacteria in the gut may modulate the onset of experimental autoimmune encephalomyelitis, an animal model for MS, the human gut microbiome has been investigated in the human disease. Data in MS have been mostly generated from small to moderate size studies using stool samples from patients with relatively long disease duration and often receiving disease-modifying therapies that can affect microbial communities. A consistent finding across these studies is that the alpha and beta diversity of gut microbial communities appears overall similar in MS patients and controls, i.e. the overall bacterial composition is not dramatically different. In contrast, increased or decreased abundance of specific microbes has been reported in cases compared to controls, with some current research emphasis on bacterial cluster differences. While a few of these differences are consistent across studies, many are not, possibly due to variability in geographical region of origin, diet, comorbidities, use of MS drugs and age of the patients. Immune changes in relation to gut microbial alterations have been reported in MS compared to controls. Very few publications have addressed so far the association between the gut microbiome characteristics and the risk of relapse or disability progression. A critical focus of current research is to unravel the changes in metabolic function resulting from alterations in microbial communities (i.e. the dysregulation of specific metabolic pathways due to variations in the presence or abundance of specific bacteria). Ongoing pilot trials of probiotic supplementation or microbiome transplant will determine the feasibility of these interventions and allow the design of future studies to attempt disease modification.
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) and the most frequent neurological disorder in young adults that leads to irreversible deficits and premature retirement. The etiology of MS is still unclear, although genetic predisposition, viral infections, vitamin D deficiency and dietary components along with microbiome dysbalance are discussed as potential risk factors.
The presentation as outlined here will provide novel insights into current paradigm shifts regarding our understanding of the disease. In particular, it will be discussed whether MS pathology is restricted to the CNS or whether also peripheral organs have to be considered as disease starting points, in particular the gut. Over 60% of patients complain of gastrointestinal dysfunction and over 30% of patients report such symptoms even before the onset of MS.
Experimental autoimmune encephalomyelitis (EAE), which is the common mouse model of MS, was used to study enteric nervous system (ENS) pathology by histology, immunohistochemistry and electron microscopy of paraffin and ultra-thin sections as well as whole mounts of both the submucosal and myenteric plexus. Functional analyses comprised measurements of the gastrointestinal transit time and local gut wall motility. Results were transferred to the human disease by performing assessment of colon resectates from MS patients.
Our results show that pathology of the ENS occured prior to CNS degeneration, in particular in the myenteric plexus. This pathology was antibody-mediated and involved autoantigenic targets that are shared between ENS and CNS such as myelin basic protein (MBP) and proteolipid protein (PLP). Pathology was accompanied by significantly reduced gut motility. Likewise, analysis of colon resectates from MS patients revealed nerve fiber degeneration and enterogliosis of the myenteric plexus.
Further research is needed to corroborate these findings in a larger patient cohort and to unravel the etiology of ENS degeneration. Interestingly, ENS involvement is increasingly discussed also in Parkinson’s and Alzheimer’s disease as well as in amyotrophic lateral sclerosis so that there might be a common denominator for several neurodegenerative disorders.
Metagenomic sequencing reveals the functional potential of the gut microbiome, and may explain how the gut microbiome influences pediatric-onset multiple sclerosis (MS) risk.
To examine the gut microbiome functional potential by metagenomic analysis of stool samples from pediatric MS cases and controls using a case-control design.
Persons ≤21 years old enrolled in the Canadian Pediatric Demyelinating Disease Network who provided a stool sample and were not exposed to antibiotics or corticosteroids 30 days prior were included for study. All MS cases met McDonald criteria, had symptom onset <18 years of age and had either no prior disease-modifying drug (DMD) exposure or were exposed to beta-interferon or glatiramer acetate only. Twenty MS cases were matched to 20 non-affected controls by sex, age (± 3 years), stool consistency (Bristol Stool Scale, BSS) and, when possible, by race. Shotgun metagenomic reads were generated using the Illumina NextSeq platform and assembled using MEGAHIT. Metabolic pathway analysis was used to compare the gut microbiome between cases and controls, as well as cases by DMD status (DMD naïve vs DMD exposed MS cases vs controls). Gene ontology classifications were used to assess α-diversity and differential abundance analyses (based on the negative binomial distribution) reported as age-adjusted log-fold change (LFC) in relative abundance, 95% confidence intervals (CI), and false discovery rate adjusted p-values.
The MS cases were aged 13.6 mean years at symptom onset. On average, MS cases and controls were 16.1 and 15.4 years old at the time of stool collection and 80% of each group were girls. MS cases and controls were similar for body mass index (median: 22.8 and 21.0, respectively), stool consistency (BSS types 1-2: n=4, types 3-5: n=16, for both MS and controls) and race (Caucasian: 11 and 9, respectively). Eight MS cases were DMD naïve. Richness of gene ontology classifications did not differ by disease status or DMD status (all p>0.4). However, differential analysis of metabolic pathways indicated that the relative abundance of tryptophan degradation (via the kynurenine pathway; LFC 13; 95%CI: 8–19; p<0.0005) and cresol degradation (LFC 19; 95%CI: 13–25; p<0.0001) pathways were enriched for MS cases vs controls. Differences by DMD status were also observed, e.g., choline biosynthesis was enriched in DMD exposed vs naïve MS cases (LFC 21; 95%CI: 12–29; p<0.0001).
We observed differences in the functional potential of the gut microbiome of young individuals with MS relative to controls at various metabolic pathways, including enrichment of pathways related to tryptophan and metabolism of industrial chemicals. DMD exposure affected findings, with enrichment of pathways involved in promoting CNS remyelination (e.g., choline).
Two studies have reported increased intestinal permeability in multiple sclerosis (MS) patients. Other studies have observed increased intestinal permeability during experimental autoimmune encephalomyelitis (EAE). However, the mechanisms for increased intestinal permeability during EAE/MS remain poorly understood.
Since trypsin regulates gut permeability via activation of proteinase activated receptor 2 (PAR2), the goal of this study is to investigate stool trypsin activity during EAE.
Stool were collected from adult C57BL/6J mice prior to EAE induction as well as at several time points post EAE induction. Stool trypsin activity was measured at time points of interest. Terminal ileum was collected from naïve and EAE mice to examine PAR2 expression. To investigate the role of the gut microbiota on gut trypsin activity during EAE, we induced EAE in mice receiving water or vancomycin. Next, germ-free (GF) mice were colonized with either control mice microbiota or microbiota from vancomycin treated mice and EAE was induced. Feces were collected from the colonized GF mice on the day of EAE induction.
We did not observe change in stool trypsin activity during EAE in untreated mice. We found a 1.5 fold increased in PAR2 expression in the ileum of untreated EAE mice. We found that vancomycin treatment ameliorates EAE. GF mice colonized with microbiota from vancomycin treated mice developed less severe disease than GF mice colonized with feces from control mice. Unlike control mice, vancomycin treated mice had an intact intestinal permeability during EAE. In addition, stool trypsin activity was decreased in vancomycin treated mice during EAE. GF mice colonized with microbiota from vancomycin treated mice had lower stool protease activity compare to GF mice colonized with control microbiota.
Upregulation of intestinal PAR2 drives increased gut permeability during EAE. Vancomycin treatment preserves intestinal permeability via inhibition of stool trypsin activity. Vancomycin effect on stool trypsin activity is mediated via the microbiota. Identification of communities of gut derived bacteria that modulate stool trypsin activity could lead to the development of a novel class of drugs for the prevention and treatment of MS.
Neuromyelitis optica (NMO) is a severe demyelinating disease of the central nervous system (CNS) causing irreversible neurological damage. Initial analyses of gut microbiota in NMO, multiple sclerosis and healthy controls (HHC) revealed dysbiosis in the NMO group, suggesting that the gut microbiome may regulate inflammatory responses
We hypothesized that gut microbiota from NMO patients may participate and promote inflammatory responses in NMO pathogenesis.
Wild-type (WT) C57BL/6 germ-free mice were colonized with fecal samples from one untreated NMO patient (n = 10), one household HC (HHC) (n = 9) or vehicle (n = 13) for five weeks and then examined for susceptibility to MOG p35-55-induced experimental autoimmune encephalomyelitis (EAE) for 30 days post immunization. Upon termination of the study, lymphocytes from spleen, lamina propria of small (LP-SI) and large (LP-LI) intestine, mesenteric lymph nodes (MLN), Peyer’s patches (PP), brain, and spinal cord were examined for the expression of IL-17, IFN-γ, Foxp3, CD25, RORγt and Helios.
In comparison to the mean EAE score of the vehicle group (1.9 ± 0.3), severity was greater (p ≤ 0.01) in mice colonized with fecal microbiota from NMO (3.1 ± 0.8) and HHC (2.7 ± 0.7). The mean clinical score of mice colonized with NMO gut microbiota was significantly greater than mice colonized with gut microbiota from HHC or vehicle (p ≤ 0.001). The frequency of CD4+Foxp3+CD25+ cells was decreased in LP-SI, LP-LI, PP and MLN compartments in NMO and HHC compared with vehicle group (p ≤ 0.01). CD4+Foxp3+Helios+ (another regulatory T cell subpopulation) was significantly decreased in MLN and LP-SI of NMO and HHC compared to vehicle group (P ≤ 0.01).
Our data suggest that NMO fecal microbiota increases EAE susceptibility. Reduction in frequency of Tregs in the gut of mice colonized with NMO fecal material may contribute to EAE exacerbation. Further analysis of microbiota and lymphocyte populations in mice colonized with fecal material from NMO and HHC samples are needed. Results from our ongoing study should provide valuable insight regarding the potential role of gut microbiota in NMO.