Microbiome in rheumatic disease
Ritasman Baisya

Structure of talk
• Human microbiome- introduction
• Healthy commensal
• Function
• Microbiome & immune system association
• Research in microbiome including animal model
• Microbiome in different rheumatic diseases
• Therapeutic application
• Pharmaco-microbiomics – recent interest
• Take home points

The Human Microbiome
• Collective genomes of the microorganisms (bacteria, bacteriophages, fungi,
protozoa and viruses) that live inside and on the human body ……
• Human gut contains 2 kg bacteria – 3.3 million genes- METAGENOME
• Intestinal tract contains up to 100 trillion microorganisms, majority in the colon
• Healthy human- Firmicutes , Bacteroides , Proteobacteria , Actinobacteria

Evolution of gut microbiome
• At birth, GI tract - sterile.
• Breast milk - crucial role via transmission of the milk microbiota ( Bifidobacterium )
• The activity and composition of microbiota change from infancy to old in response to
the genetic background, diet, immune system , health status of the host
• Dominant bacterial group differs along gut based on - Oxygen availability, presence
of antimicrobial peptides, availability of carbon sources and pH

Site of GI tract Normal commensal microbes
Oral microbiota Firmicutes (Streptococcus salivarius) , Actinobacteria
Stomach Prevotella , Streptococcus, Veillonella, Rothia & Haemophilus
Small intestine Lactobacillaceae & Enterobacteriaceae
Colon Bacteroid , Prevotella, Rikenella, Lachnospira and Ruminococci
Mucosa Mucin degraders ( Bacteroides fragilis & Akkermansia
muciniphila).

Non GI commensal microbes-
• Vagina - Over 200 phylotype -
Firmicutes, Bacteroidetes, Actinobacteria, & Fusobacteria.
• Placenta - non-pathogenic commensal microbiota from the
Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and
Fusobacteria phyla

Functions of the human gut microbiota
1. Digestion and energy harvesting from indigestible food components by increasing
absorption and enzyme release…
2. Supporting and priming the immune system …
3. Providing nutrients to the gut epithelium (such as short-chain fatty acids)…..
4. Resistance against colonization by potential pathogens
5. Influence satiety through the production of SCFA and releasing satiety hormone
peptide YY and glucagon-like peptide
6. Influence state of mind by producing neuroactive compounds that act on the brain

Gut- Immune – Brain axis
Van de Wiele, T., Van Praet, J., Marzorati, M. et al. How the microbiota shapes rheumatic diseases. Nat Rev
Rheumatol 12, 398–411 (2016).

Gut microbiome and immune system

Relationship between gut microbiota and host

Physiologic intestinal barrier
• Thick mucus layer .
• Enteric antimicrobial protein
• Increased sIgA
• Tight adherent cell junction
• Defensin , cathelicidin , C – lectin
• IEL- CD4(+) ,CD8(+) T cell
• Lamina propria – immune armentarium

Immune recognition of gut microorganisms
• Gut-associated lymphoid tissue (GALT) - 70% of the body’s immune system
• 80% of all IgA- producing plasma cells reside in lamina propria
• PRRs involves recognition of microorganism (MAMP) , & engagement of innate
immune system, including antimicrobial peptide
• Antigen-presenting cells - dendritic cells, macrophages and B cells present in lamina
propria

Immune recognition …
• Dendritic cells detect antigens directly in the intestinal lumen, after which they
typically migrate to the mesenteric lymph nodes
• Maturation of B cells into high-affinity IgA- secreting plasma cells occurs within
Peyer’s patches
• Some low affinity IgA- in isolated lymphoid follicule and lamina propria ( T
cell independent )

Brucklacher-Waldert, Verena & Carr, Edward & Linterman, Michelle & Veldhoen, Marc. (2014). Cellular Plasticity of
CD4+ T Cells in the Intestine. Frontiers in immunology. 5. 488

Microenvironments in the gut mucosa
• Mucus layer - inner layer close to the epithelium
& outer layer.
• Inner layer is rigid & devoid of microorganisms
• Outer mucosa – contains micro bacteria
• Mucosal microbiota - fundamentally different
from its luminal or fecal counterparts
• Mucosal microbiome is more abundant in
Firmicutes (especially Clostridium cluster
XIVa) than in Bacteroides
Microbiome

Regulation of intestinal immunity
• In physiologic gut – low effector T cell- Th1,2,17 and more T reg
• Butyrate-producing Clostridia, can modulate Treg cells- lower intestinal
lesion
• Bacteroides fragilis can produce polysaccharide A (PSA) – TH1 & Treg
response and suppresses inflammation ….
• Segmented filamentous bacteria (SFB) direct towards increased production of
TH17 cells – protective response
• NKT cell – prevents commensal microbial colonization by IFN & release of
antimicrobial peptide

Regulation of systemic immunity
• Role of the microbiota in regulating host immune responses is best emphasized in
mice housed under germ-free conditions
• Microbiota contributes to production of antimicrobial molecules REG3γ and
REG3β in mice & impaired production is associated with Listeria and Yersinia
infection ….
• Dysbiosis of microbiota trigger autoimmune attack by autoreactive Th1, Th17
response and increasing proinflammatory cytokine – host –microbiome cross talk

Host and microbiome cross talk in health and autoimmune state
HOCHBERG

Host and microbiome interplay
• Linear or unidirectional model – a primary cause initiates a
one-way directional process - with a particular genetic background
following an environmental trigger
• Multidirectional model- in which the interrelationship between
genetics, the microbiota, environment and immune responses is not
unidirectional and more plastic ….

Example of unidirectional model
Van Praet, J. T. et al. Commensal microbiota influence systemic autoimmune responses. EMBO J. 34, 466–474 (2015) and Knoop, K. A. & Newberry, R. D. Isolated lymphoid follicles
are dynamic reservoirs for the induction of intestinal IgA. Front. Immunol 3, 84 (2012)

Example of multidirectional model

June L. Round, and Noah W. Palm Sci. Immunol. 2018;3:eaao1603

Recent Research method of human microbiome
• 16s rRNA sequencing – unique and highly conserved primer binding site
• Whole genome shotgun sequencing , pyrosequencing
• Metagenomics , meta-tanscriptomics , Metabolomics , proteomics ..
• Enolase by P . gingivalis – sufficient for RA in murine model
• PSA by B fragilis – sufficient to prevent EAE
• TMAO by Prevotella – sufficient for atherosclerosis
• HMP ( by NIH) , MetaHit constorium ( by Europian Commission )

Bikel, Shirley et al . (2015). Combining metagenomics, metatranscriptomics and viromics to explore novel microbial
interactions: Towards a systems-level understanding of human microbiome. Computational and Structural Biotechnology
Journal. 292.

16S based approach & shotgun metagenomic approach
https://journals.plos.org/ploscompbiol/article/figure?id=10.1371/journal.pcbi.1002808.g001

Human microbiome project - The first and second phases
Proctor, L.M., Creasy, H.H., Fettweis, J.M. et al. The Integrative Human Microbiome
Project. Nature 569, 641–648 (2019).

Microbiome in animal model of arthritis
• Gnotobiotic animal –Animals in which the composition of all microorganisms
present is known (Greek words - ‘gnostos’( known ) & ‘bios’(life).
• Germ- free mice- bred & raised under conditions to render them free from all
microorganism
• Humanised mice - faecal microbiota is established in germ- free mice through
the transplantation of fresh or frozen gut microbiota samples
• This models help to identify how local change in intestinal microbial
community can trigger autoimmunity

June L. Round, and Noah W. Palm Sci. Immunol. 2018;3:eaao1603
Demonstrating causality in host-microbe interactions.

Examples of animal model
• Mono-colonization of germ-free IL-1Ra-deficient mice with the gut-residing commensal
bacterium Lactobacillus bifidus - arthritis
• Introduction of SFB in germ-free K/B×N mice – arthritis
• Periodontal pathogens P gingivalis and P. nigrescans markedly aggravate the severity of
arthritis in mice with collagen-induced arthritis (CIA)
• In HLA B27 transgenic rats, presence of the HLA-B27 antigen was associated with an
altered microbial composition of the gut
• Modulation of the intestinal flora influences disease progression in Lupus animal model-

Animal model of microbiome

Examples of microbiome in Rheumatic diseases

Spondyloarthropathy
• Studies from HLA B27 transgenic rats and humans- most evidence
• 2/3 of SpA – subclinical microscopic gut inflammation
• Mucosal inflammation – acute ( like enterocolitis ) or chronic (like Crohn )
• Terminal ileum & colon –most common site
• 20% of patients with chronic intestinal lesions developed clinically overt IBD
over a 5-year period

AS and ERA
• AS, the prototypic form of SpA, occurs in up to 10% of patients with IBD
• HLA-B27 has been suspected of playing a role in shaping the microbiota of AS patients
• Higher prevalence of sulfate-reducing bacteria was found in patients with AS
• Some patients displayed heightened T cell proliferation
• Bacterial families Lachnospira, Ruminococci, Rikenella, Porphyromonus , and
Bacteroid increased significantly, and Veillonella and Prevotella reduced significantly
in terminal ileum of patients with AS (Costello et al.)

AS & ERA
• Lachnospira and Ruminococci occurred together, but Veillonella and Prevotella were
the opposite, indicating possible mutualistic or antagonistic relationships.
• An increase in Prevotella spp. & Bacteroides vulgatus & decrease in Rikenellaceae -
in HLA-B*27 transgenic animals
• In enthesitis-related arthritis , F. prausnitzii decreased in the stools of patients
• Whereas Bacteroides spp. and Akkermansia muciniphila were identified as
disease-associated agents in ERA

IBD –associated arthritis
• Decreased biodiversity (α-diversity) - lower richness and evenness of the intestinal
microbiota
• Lower firmicutes and increased gamma proteobacteria
• Significant increase in enterobacteria
• Acetate ( Ruminococcus ) & butyrate ( Fecalibacterium , Roseburia ) producing
bacteria decreased

Psoriasis & PsA
• Increased risk of Crohn’s disease in US women with psoriasis & an even
higher concomitant PsA
• Lower frequencies of Akkermansia & Ruminococcus ( Scher et al.)
• They have positive correlation with medium-chain fatty acids ( heptanoate
and hexanoate)
• Akkermansia - negative correlation with secretory IgA ( sIgA) and short-
chain fatty acids (acetate and butyrate) in feces.

Skin microbiome of psoriasis
• Psoriatic lesions showed greater diversity in microbial sequences ( Gao et al )
• In skin - Actino , Firmicutes , Bacteroides , Proteobacteria
• Firmicutes - abundant in all samples
• Actionbacteria and proteobacteria lower in the psoriatic lesions than healthy
• Streptococcus/Propionibacterium - over 12-fold higher in disease lesions
compared to healthy controls
• Skin microbiome depends on – moisture , sebaceous content

Decreased

Rheumatoid arthritis

• Periodontal disease occurs more frequently and tends to be more severe in patients
with RA ( risk ratio 1.13, p- 0.0006)
• P. gingivalis ( a red complex bacteria) could contribute to the induction of ACPAs,
a strong predictor of the onset of RA
• DNA of P. gingivalis and P. nigrescens - found in serum and synovial fluid of
patients with RA
• Anaeroglobus – recent discovered , not well known
RA- oral microbiome

Gut in RA
• Dysbiosis in the faecal microbiota of patients with newly diagnosed RA ( NORA)
(Vaahtovuo et al)
• Presence of Prevotella copri & loss of Bacteroides in the fecal microbiome in
untreated NORA ( 16s rRNA sequencing)
• P. copri leads to expansion of the intestinal Th17 cell population – role in RA
pathogenesis

Metagenomics in RA Gut
• A significant shift at the taxonomic and functional level of the RA gut microbiota
• Lower abundance of vitamin metabolism & pentose phosphate pathways.
• Enrichment for lipopolysaccharide biosynthesis , transport, secretion systems,
reductive acetyl-CoA, and acetate to methane conversion
• RA enriched MLGs - correlation with titers of IgA and IgG in serum.

Respiratory in RA
• Relationship between the respiratory mucosa, which harbors its own
characteristic microbiota & RA
• Increased smoking causes relative abundance of Prevotella and
Porphyromonas

Summary of mechanisms by which molecular mimicry of the gut microbiota, and CARD9 genotype can
contribute to systemic inflammation.
RA and the microbiome: do host genetic factors provide the link? Journal of Autoimmunity Volume 99, May 2019, Pages

SLE
• Increase in Bacteroidetes & an over 2-fold reduction in the Firmicutes/Bacteroidetes ratio
( Hevia et al )….
• Bacteroides mediated glycan degradation & liposaccharide synthesis – more in SLE
patients ….
• Germ-free lymphotoxin-deficient animals monocolonized with SFB produced more ANAs
• B. bifidum and B. coccoides can suppress the effects of the SLE gut microbiota & their
abundances can be modified by dietary sources like polyphenols ….
• Synergistetes may also provide avenues for treatment, as they are associated with the
presence of protective IgM proteins in SLE patients….

Sjogren syndrome
• Oral cavity - Increase in mutans streptococci, Lactobacillus & Candida albicans,
and depletion of Fusobacterium nucleatum colony forming units
• Ralstonia, which is ubiquitous and found to colonize the oral mucosa and lungs
of patients on mechanical ventilation - more in pSS patients
• Gut - 1. Increased abundance of Escherichia, Shigella and Streptococcus genera
2. Decreased abundance of Faecalibacterium, Bacteroides, Parabacteroides
& Prevotella

Behcet disease
• Oral cavity - Neisseria and Veillonella - depleted on the oral mucosa ,Rothia
species -colonized the non-ulcer mucosa of BD patients….
• Ulcerated sites of BD patients - overabundance of Streptococcus species
• Salivary microbiota of active oral ulcers - overabundant in Bifidobacterium
dentium, Prevotella histicola, Candida albicans, & Streptococcus…..
• Significant dysbiosis in gut flora - depletion of Roseburia & Subdoligranulum (
Consolandi et al )

Kawasaki disease – gut microbiome
• Rothia & Staphylococcus - increased in abundance during the acute phase
• Ruminococcus, Blautia, Faecalibacterium, and Roseburia - dominant during non-
acute phase
• Streptococcus pneumonia and Streptococcus oralis- over abundant in the acute
phase compared to the non-acute phase and healthy controls.

Therapeutic application

Probiotics
• Probiotics - live microorganisms when administered in adequate amounts confer a
health benefit on the host
• Lactobacillus casei –reduced proinflammatory cytokine levels, increased IL-10,
and improved arthritis scores , HPE change in CIA rat
• In RCT of SpA, no significant benefit over placebo was demonstrated, although
duration of treatment or choice of probiotic may have affected this result
• Small pilot study of RA - probiotics causes improvement in disability scores
(HAQ) but not in ACR20 responses

Future Research—Faecal Microbiota Transplant
• Microbial ecosystem therapeutics & faecal microbiota transplant (FMT)
may be useful in SpA as ileocolonic inflammation in AS may be modulated by the
microbiota
• A potential alternative to FMT is “synthetic stool” therapy , a technique
reported as effective in preliminary studies of CDI

Pharmaco-microbiomics
• Effect of microbiome variation on drug deposition , action , toxicity
• Microorganisms and their enzymatic products can affect the bioavailability , clinical
efficacy & toxicity of drugs through direct and indirect mechanisms
• Microbiome-b​ased precision​ medicine ​approaches ​in​ inflammatory​ arthritis - predict
response to treatment by modulating the microbiome to improve response to
therapy or reduce drug toxicity.
• Human autoimmune diseases have utilized this approach for methotrexate , biologic
therapy based study

Venn diagram of pharmaco-microbiomics interaction

Some examples of rheumatic pharmaco-microbiomics

Pharmacomicrobiomic study in autoimmune & rheumatic disease

COVID 19 and microbiome ….
• Gut– lung crosstalk phenomenon in COVID-19
• No direct clinical evidence that the modulation of gut microbiota plays the
therapeutic role in COVID-19
• severe COVID-19 infection, probiotics may be used to maintain the balance of
intestinal microecology and prevent secondary bacterial infection
( China's National Health Commission and National Administration of Traditional Chinese Medicine
recommended )
1. Gao, Q.Y., Chen, Y.X. and Fang, J.Y. (2020), 2019 Novel coronavirus infection and gastrointestinal tract. J Dig Dis, 21:
125-126.

1. Openshaw, P. Crossing barriers: infections of the lung and the gut. Mucosal Immunol 2, 100–102 (2009)
Viral effect on gut microbiome

Take Home Message
• Human microbiome – 2 Kg of body weight , 100 trillion organism
• Normal commensal differs along length of gut
• Its function of wide range- immune shaping is very important of them
• Microbione-host immune homeostasis is mediated by multiple factors
• Dysbiosis can trigger autoimmunity including arthritis
• Gene sequencing and animal model are helpful for microbiome study
• SpA – very strong association , HLAB27 plays important role
• Oral-gut-lung crosstalk in RA
• SLE - ,Sjogren, Behchet , Vasculitis- microbiome association
• Probiotic , FMT- therapeutic application
• Pharmacomicrobics – recent advance

References
1. Van de Wiele, T., Van Praet, J., Marzorati, M. et al. How the microbiota shapes rheumatic diseases. Nat Rev
Rheumatol 12, 398–411 (2016).
2. Scher, J.U., Nayak, R.R., Ubeda, C. et al. Pharmacomicrobiomics in inflammatory arthritis: gut microbiome
as modulator of therapeutic response. Nat Rev Rheumatol (2020)
3. Patrick Coit, Amr H. Sawalha, The human microbiome in rheumatic autoimmune diseases: A comprehensive
review, Clinical Immunology (2016)
4. Yeoh, Nigel & Burton, Jeremy & Suppiah, Praema & Reid, Gregor & Stebbings, Simon. (2013). The Role of
the Microbiome in Rheumatic Diseases. Current rheumatology reports. 15. 314
5. Moiseev S, Rameev V, Karovaikina E, et al . Gut microbiome in rheumatic diseases. Annals of the
Rheumatic Diseases Published Online First: 14 November 2019

6. A.M. Fricker et al. What is new and relevant for sequencing-based microbiome research? A mini-
review. Journal of Advanced Research 19 (2019) 105–112
7. Gao, Q.Y., Chen, Y.X. and Fang, J.Y. (2020), 2019 Novel coronavirus infection and
gastrointestinal tract. J Dig Dis, 21: 125-126.
8. Marc Hochberg ,Ellen Gravallese . Rheumatology . 7th Edition
References