This review focusses on the role of role of gut microbiota in health and disease, specifically multiple sclerosis. It looks at the interaction of gut microbiota, enteric nervous system, central nervous system, neuroendocrine system in the pathogenesis of multiple sclerosis

1. Gut Microbiome and
Multiple Sclerosis
Dr. Pramod Krishnan, M.D, D.M
Consultant Neurologist
HOD Neurology
Manipal Hospital, Bengaluru
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2. Introduction: Gut microbiome in health and disease
• Gut microbiota is currently a research “hot spot”
• Using next-generation sequencing (NGS) approaches, largescale studies such
as the Human Microbiome Project (HMP) and the Metagenomics of the
Human Intestinal Tract (MetaHIT) project have provided essential references
regarding the microbiota composition in humans.
• Gut microbiota are related to a variety of diseases:
1. Metabolic disorders
2. Inflammatory bowel diseases (IBD)
3. Cancers
4. Autoimmune diseases
5. Disorders of the central nervous system.

3. Introduction: Gut microbiota and nervous system
• The intestinal microbiota constitutes a complex ecosystem in constant
reciprocal interactions with the immune, neuroendocrine, and neural
systems of the host.
• The CNS and the gut microbiota are connected reciprocally through the
hypothalamic-pituitary-adrenal axis, the immune system, and the enteric
nervous system.
• These interactions have critical impacts on immune homeostasis, intestinal
barrier permeability and can regulate CNS inflammation and disease.
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4. SFB: Segmented filamentous
bacterium;
PSA: Polysaccharide A
The CNS alters the intestinal
microenvironment by
regulating gut motility and
secretion as well as mucosal
immunity via the neuronal-
glial-epithelial axis and
visceral nerves.
Bacteria react to these
changes by producing NTMs
or neuromodulators in the
intestine to impact the host
CNS. These include choline,
tryptophan, short-chain fatty
acids (SCFAs), intestinally
released hormones such as
ghrelin or leptin.

5. 5
Dietary metabolism and roles of the gut microbiota

6. Dysregulation of the gut microbiota in brain disorders
Multiple Sclerosis

7. Gut barrier stabilizers or enhancers investigated in chronic inflammation
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8. Physiological role of gut microbiome
The normal microbiome has many functions:
• Maintenance of the motility and permeability of the gut.
• Synthesis and secretion of essential vitamins, such as vitamin B12, folate,
vitamin K, nicotinic acid, biotin, riboflavin, pyridoxine, panthotenic acid,
and thiamine.
• Maintenance of intestinal epithelial functions, such as absorption and
secretion.
• Local stimulation of the development of innate and adaptive immune
systems via GALT secreting immune cells, cytokines, and IgAs.

9. Mucus secreted by
goblet cells separate
the microbiota from
the upper epithelium
Tight junction (TJ)
made up of proteins
occludes and claudins
regulate paracellular
permeability Immune cells include M
cells, intraepithelial
lymphocytes, long
processes of dendritic
cells, Paneth cells that
secrete antibacterial
peptides.

10. The ANS, along with the
HPA axis and neuroendocrine
signaling, can induce CNS-
modulated changes in the gut.
Bacterial molecules and
neural-immune-endocrine
pathways impact the
development of the CNS,
giving rise to a dynamic
communication network in
the MGB axis.
The development and function of the ENS are controlled by gut
microbiota, through direct or indirect mechanisms. Bacterial
molecules can pass through the intestinal epithelial barrier to directly
interact with enteric plexuses or act on non-neuronal intermediary
cells (e.g., enteric immune cells, ICCs, EGCs, EGCs, and enteric L
cells), whose products can be detected by enteric neurons.
MGB axis, microbiota-gut-brain axis; AD, Alzheimer’s disease; PD,
Parkinson’s disease, ALS, amyotrophic lateral sclerosis; ASD, autism spectrum
disorder; MS, multiple sclerosis; GI, gastrointestinal; ENS, enteric nervous
system; ICCs, interstitial cells of Cajal; EGCs, enteric glial cells; ECCs,
enterochromaffin cells; EA, esophageal achalasia; HSCR, Hirschsprung
disease; FGIDs, functional gastrointestinal disorders; VZV, varicella-zoster
virus; H. pylori, Helicobacter pylori; UC, ulcerative colitis; CD, Crohn’s
disease.
Effects of GI microbiota
on the ENS and MGB axis

11. Gut microbiome in MS

12. Gut microbiota in Multiple Sclerosis
Probable microbiome signatures in MS:
• Higher Firmicutes/Bacteroidetes ratio in MS.
• Increased Streptococcus and decreased Prevotella strains in active MS.
• Increased Akkermansia muciniphila and Acinetobacter calcoaceticus, and
reduced Parabacteroides distasonis in patients compared to controls.
• MS-derived microbiota was capable of inducing or worsening experimental
models of disease, while Prevotella histicola, a human gut-derived
commensal bacteria, could suppress Experimental autoimmune
Encephalitis (EAE).

13. MS pathogenic loop centred
on the gut barrier disruption
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14. Role of gut microbiota in health and Multiple Sclerosis

15. Technical approaches in gut microbiome studies
• Current technologies do not allow the cultivation of all gut bacteria.
• Two widely adopted culture free approaches to study the microbiome:
1. Targeted sequencing/ marker gene sequencing: This includes 16S ribosomal
RNA (rRNA), internal transcribed spacer (ITS), and 18S rRNA sequencing.
16S rRNA sequencing achieves only ~ 80% accuracy in the genus level and is
not able to fully resolve taxonomic profiles at the species level or strain level.
2. Metagenomic sequencing: Shotgun sequencing is used for the
comprehensive profiling of the DNA from microbiota.
• Other technologies including metatranscriptomics, metaproteomics, and
metabolomics to study RNAs, proteins, and metabolites.

17. • Microbial DNA was extracted from frozen faecal samples and
16s rDNA sequencing was performed.
• Gene expression profiling was performed on circulating
monocytes and T cells.
• Peripheral blood mononuclear cells were collected to conduct
proliferation and cytokine assays in response to specific
microbial stimulation.
• Sera was collected for ELISA-based techniques to capture
serologic activity directed against specific microbes.
• Breath samples were collected from a second subject cohort
to determine breath methane concentrations.

18. (a) Relative occurrence of Euryarchaeota and Verrucomicrobia in the faecal microbiota of healthy controls (N= 43), all
MS patients (N= 60), untreated MS (N= 28) and treated MS patient (N= 32) subgroups as analysed by two
independent sequencing technologies, 454 or MiSeq (b) Relative occurrence of prevalent microbiota.

19. Relative abundances of genera in the faecal microbiota that are significantly altered between healthy controls
(N= 43) and MS patients (N= 60; MS-effect) or between untreated (N= 28) and treated MS patients (N= 32)
(disease effect) as analysed by two independent sequencing technologies. Significance was determined by P
<0.05. Bars represent average, and error bars depict s.e.

20. Correlations between microbiota abundances and immune gene
expression. Gene expression was measured from circulating T cells and
monocytes by the Nanostring Immunology panel in MS patients (N= 18) and
healthy controls (N= 18). (a) canonical pathways significantly altered in MS
patients and healthy subjects with an activation z-score >|1.5|in both T cells
(black bars) and monocytes (grey bars) (Ingenuity Pathway Analysis).
(b) Altered gut microbiota abundances correlate with immune gene
expression in MS patients. Spearman’s correlations (σ) between the relative
abundance of significantly altered microbes and the relative expression of
genes significantly altered between healthy controls and untreated MS
patients. Color and slope of ellipse indicate magnitude of correlation, with σ
value superimposed on ellipse. Subject groups MS patients and controls
together (All), untreated MS patients (MS-U) or healthy controls (HC).

21. Summary
Increased in MS
Phylum
• Euryarchaeota
• Verrucomicrobia
Genus
• Methanobrevibacter
• Akkermansia
Decreased in MS
Genus
• Butyricimonas
• Prevotella
Treated MS
Increase in
• Prevotella
• Sutterella
Decrease in
• Sarcina
Methanobrevibacter and Akkermansia show positive
correlations with gene expression in T cells and monocytes
involved in key pathways previously implicated in MS
pathogenesis.
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
Some MS patients had
elevated exhaled methane,
a surrogate for levels of
Methanobrevibacter.

22. Applications in the management of Multiple
Sclerosis
• Therapy in MS focusses on symptom improvement after a disease attack,
preventing new attacks and decreasing the rate of CNS neurodegeneration.
• Existing therapies include immunosuppressants, and immunomodulating
drugs, which are long term and have potential side effects.
• Gut microbiota plays an important role in the development of MS.
Alterations in the gut microbiota in MS can potentially influence the
clinical symptoms and inflammatory factors, which could help us find a
new strategy or target to treat MS.

23. 1. Dietary modifications
• Obese individuals have reduced diversity in their microbiome especially
at a lower level of Bacteroidetes.
• Westernized diet with high fat on mice have shown changes in the gut
flora, with an increasing in proinflammatory plasma free fatty acids and
increased severity in EAE.
• A restricted calorie diet can improve the EAE symptoms, whereas a
high-salt diet causes disease exacerbation in EAE by promoting the
expansion of macrophages and proinflammatory T cells, and Th17
differentiation, and also causing restraint in remyelination

24. 2. Drugs
• Broad-spectrum antibiotics alter the population of T cells in the GALT and in
peripheral lymphoid tissues to reduce the susceptibility to EAE.
• Minocycline was shown to reduce disease severity in EAE both prophylactically
and therapeutically. Antibiotic therapy may be beneficial in the treatment of MS.
• Fingolimod, Teriflunomide, and DMF have been shown to inhibit C. perfringens
growth; therefore, the inhibition of C. perfringens may contribute to the clinical
efficacy of these disease-modifying drugs.

25. 3. Probiotics treatment
• Probiotics can influence systemic immune responses by:
1. Maintaining the function of the gastrointestinal–epithelial barrier
2. Increasing antimicrobial peptide production
3. Helping the activation of the host immune system in response to pathogens.
• Probiotics could be used as adjuvant therapy in MS.
• Long bifidobacterium (b. Longum), Breve bifidobacterium, Bifidobacterium
infantis, Lactobacillus helveticus, Rhamnose lactobacillus, plant
Lactobacillus, and Lactobacillus casei have been shown to improve behaviour,
such as anxiety and depression, in animal models.

26. 4. Fecal Microbial Transplantation (FMT)
• FMT can restore intestinal microecological balance, which may be
efficacious with less adverse reactions. It has been studied in intestinal
disorders, metabolic, autoimmune and allergic diseases, and cancer.
• FMT can improve the walking ability in MS, alleviate autistic behaviour,
improve the neurological symptoms of PD.
• Borody et al. reported three patients with MS with severe constipation
treated with FMT, which reduced the neurological symptoms and
normalized walking.

27. 5. Other therapies
• Parasites, especially helminths, have an effect on Th2 cell induction to
produce anti-inflammatory cytokines, like IL-4, IL-10, IL-13, and TGF-b.
• Helminths have provided therapeutic effects in patients suffering from MS
and ulcerative colitis.
• In addition, patients with MS naturally infected with helminths had fewer
relapses than uninfected patients, and elimination of the parasites
worsened their condition.
• Based on these findings, helminths may be a new method of MS treatment.

28. The Gut-Brain Axis and Effects of
Dimethyl Fumarate (DMF) on the
Gut Microbiome
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29. Immune signaling
including:
1. Inflammasome
2. Type 1 interferon
3. NF-κB
CNS
disorders
Cellular components:
Neuron
Astrocyte
Microglia
Treg
Th1 Th17
Microbiota
Toxic metabolites,
SCFAs
Dysbiosis and dysregulation of the microbiome in patients with MS may contribute to MS disease
• NF-κB=nuclear factor kappa B; SCFA=short-chain fatty acid; Treg=regulatory T cell.
• 1. Tierney BT et al. Cell Host & Microbe 2019;26:283-295; 2. Ma Q et al. J Neuroinflamm. 2019;16:53.
• The human microbiome is the collection of
all the microorganisms living in association
with the human body and consists of
>45,000,000 non-redundant genes1
• Microorganisms in the gastrointestinal tract
of humans impact physiological
and pathological activities1,2
• Disruption of the gut barrier may allow
contents of the gut to “leak” into
bloodstream, which may lead to a
proinflammatory state and trafficking
of immune cells across BBB2
• Disruption of gut homeostasis could
therefore lead to changes in immunological
signaling and influence CNS disorders such
as MS2
The Gut-Brain Axis: Microbiota Can Influence CNS
Components and Pathology

30. Hypothesized DMF Effects on the Microbiome
Normalizes Microbiome Composition
• Analyses of stool samples have shown that dysregulated microbial populations in patients with MS may
be normalized after treatment
Immunoregulation
• DMF treatment leads to an anti-inflammatory lymphocyte phenotype. Some aspects
of this shift may originate from changes in the gut
Neuroprotection via Gut-Brain Axis
• DMF may decrease gut permeability, reducing the ability of toxic metabolites and chemokines to affect
the CNS
• Normalized bacterial populations may lead to decreased toxic metabolic signaling and increased
neuroprotective signaling
• 1. Tierney BT et al. Cell Host & Microbe 2019;26:283-295; 2. Ma Q et al. J Neuroinflamm. 2019;16:53.

31. DMF Effects on the Microbiome
BEFORE DMF Treatment
Microbial populations in people
with MS may be dysregulated
compared to healthy controls
People with MS may have an
altered gut morphology
leading to leakage of bacteria-
originating neurotoxic material5,8,9
People with MS have elevated
inflammatory immuno-signaling
and an inflammatory
lymphocyte population
• IFN-γ=interferon gamma; SCFA=short-chain fatty acid. 1.Tremlett H et al. J Neurol Sci. 2016;363:153-157 2.Takewaki D et al. Proc Natl Acad Sci U S A. 2020;117(36):22402-
22412;3. Storm-Larsen C et al. Mult Scler J Exp Transl Clin. 2019:1-13; 4. Achiron. Unpublished. ISR-BGT-17-11113 5. Jangi S et al. Nat Commun. 2016;7:12015 6. Katz Sand I
et al. Neurol Neuroimmunol Neuroinflamm. 2018;6(1):e517; 7. Rumah KR et al. Front Cell Infect Microbiol. 2017;7:1 8. Ntranos A et al. Brain. 2021; Escribano BM et al.
Neurotherapeutics. 2017;14:199-211; 10. Huan J et al. J Neurosci Res. 2005;81:45-52 11. Kallaur AP et al. Mol Med Rep. 2013;7:1010-1020 12.
Macrophage
Microglia
Microglia/
macrophages
FOXP3
Signaling10
Th1712
Neurotoxic metabolites
Neuroprotective metabolites
SCFAs
Bacteroidetes1 Roseburia2 Actinobacteria3,4 Akkermansia2,5 Clostridia6,7
IFN-γ11

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44. Conclusion
• Dysbiosis in the gut microbiome may be one of the causes of the numerous
diseases, including MS.
• Gut therapies including dietary modification, drug treatment, probiotics and FMT
may be used in MS in the future.
• DMF may affect gut microbiome as part of its therapeutic action.
• However, there is still a long way to go, as more rigorous scientific evidence with
larger sample sizes are required for clinical application.
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45. THANK YOU
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