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REVIEW ARTICLE
Does the buck stop with the bugs?: an overview of microbial
dysbiosis in rheumatoid arthritis
Pulukool SANDHYA,
1
Debashish DANDA,
1
Disha SHARMA
2,3
and Vinod SCARIA
2,3
1
Department of Clinical Immunology and Rheumatology, Christian Medical College, Vellore, Tamil Nadu,
2
GN Ramachandran
Knowledge Center for Genome Informatics, CSIR Institute of Genomics and Integrative Biology (CSIR-IGIB), and
3
Faculty of Life
Sciences, Academy of Scientific and Innovative Research (AcSIR), Delhi, India
Abstract
The human body is an environmental niche which is home to diverse co-habiting microbes collectively referred
as the human microbiome. Recent years have seen the in-depth characterization of the human microbiome and
associations with diseases. Linking of the composition or number of the human microbiota with diseases and
traits date back to the original work of Elie Metchnikoff. Recent advances in genomic technologies have opened
up finer details and dynamics of this new science with higher precision. Microbe-rheumatoid arthritis connec-
tion, largely related to the gut and oral microbiomes, has showed up as a result apart from several other earlier,
well-studied candidate autoimmune diseases. Although evidence favouring roles of specific microbial species,
including Porphyromonas,Prevotella and Leptotricha, has become clearer,mechanistic insights still continue to be
enigmatic. Manipulating the microbes by traditional dietary modifications, probiotics, and antibiotics and by
currently employed disease-modifying agents seems to modulate the disease process and its progression. In the
present review, we appraise the existing information as well as the gaps in knowledge in this challenging field.
We also discuss the future directions for potential clinical applications, including prevention and management
of rheumatoid arthritis using microbial modifications.
Key words: autoimmunity, citrullination, microbiome, periodontitis, Porphyromonas gingivalis, rheumatoid
arthritis.
INTRODUCTION
The human body is populated by a large diversity of
microbial species and recent years have seen extensive
characterization of these co-habitants, popularly called
the human microbiome.
1,2
It is now widely believed
that the host and the microbial population co-exist in
close equilibrium and share a symbiotic relationship.
3,4
In his landmark work The Prolongation of Life: Optimistic
Studies, Elie Metchnikoff postulated the concept of equi-
librium between the host and the microbiome, which
in turn could modulate the transition between healthy
and diseased states. The change in this equilibrium or
dysbiosis has been hypothesized to be associated with
disease pathogenesis. Microbiome also has a role in
development of innate and adaptive immune response
and development of tolerance to certain auto-antigens.
5
It is also now widely recognised that the host-microbe
interaction is essential for recognition and development
of the immune system apart from serving as a barrier
for pathogens.
6,7
The in-depth characterization of the human micro-
biome has been possible with the new sequencing
technologies, popularly known as next-generation
sequencing (NGS) technologies which offer nucleotide
sequencing at high throughputs and at affordable
costs.
8,9
Diversity of the microbiome varies between the
Correspondence: Dr Vinod Scaria, GN Ramachandran Knowl-
edge Center for Genome Informatics, CSIR Institute of Geno-
mics and Integrative Biology (CSIR-IGIB), Mathura Road,
Delhi 110028, India. Email: vinods@igib.in
©2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
International Journal of Rheumatic Diseases 2015
body parts considered and the gut microbiome encom-
passes the largest diversity.
10
It is also now known that
composition of the microbiome is modulated by a
number of factors, including host genetic factors, diet-
ary habits, antibiotics, infectious agents and other envi-
ronmental cues.
1113
Integration of multi-omic profiles
for understanding dysbiosis has added to the repertoire
of methods and approaches to understand disease biol-
ogy.
14
Genetic predispositions to peculiar microbial
populations have been studied in recent years.
15
Re-
ports suggest a possible trans-generational inheritance
of microbiomes.
16,17
In addition, a number of organ-
isms which have the potential to stabilize the architec-
ture and maintain a positive equilibrium in the host
has been studied, and these strains are popularly
known as probiotics.
Dysbiosis of the microbiome has been implicated in
a number of diseases. Nevertheless most of the micro-
biome studies have focused on comparison of the
microbial diversity between diseased individuals and
apparently normal individuals. This precludes the delin-
eation of cause-effect relationships. Currently, human
microbiome studies relate to a spectrum of human ill-
nesses, mostly chronic inflammatory and autoimmune
diseases.
1821
Apart from inflammatory bowel disease,
rheumatoid arthritis (RA) is one of the most extensively
studied major autoimmune disorders with respect to
dysbiosis and deranged microbiome architecture.
2226
RA is a prototype chronic inflammatory process charac-
terized by erosive arthritis, auto-antibody production
and systemic features. Synovial inflammation induces
pannus formation, leading to joint failure unless aggres-
sively treated. The search for a causative agent continues
and remains elusive despite path-breaking information
on the pathogenesis of the disease. Environmental fac-
tors, most likely an infectious agent, act as a trigger for
autoimmunity in a genetically predisposed individual.
Targeted gene scans and genome-wide association stud-
ies (GWAS) have revealed immune-related genes as hot-
spots.
27
However, these could explain only a small
fraction of genetic risk in RA. It has been widely hypoth-
esized and speculated that the microbiome may account
for the missing link in the pathophysiology of RA. The
association of microbes in the pathogenesis of RA has
had its historical roots right from the first report by Max
Sch
uller.
28
It is also worth mentioning the contribution
to this field by Metchnikoff who advocated the wide-
spread and popular practice of dietary modifications for
the amelioration of symptoms of RA in the 1940s.
29
In the present manuscript, we review the current
understanding on the role of human microbiota in the
pathogenesis and disease activity of RA. Apart from pro-
viding insights into the pathophysiological mechanisms
modulating dysbiosis, we also discuss the biological
and clinical relevance of data on this subject from the
published literature. We address the potential of thera-
peutic intervention involving the host-microbe balance.
The final section of the review discusses the gaps in the
understanding of this emerging area, and how this
could be bridged by diverse interest groups.
THE HUMAN MICROBIOME IN RA
The exploration of potential microbial dysbiosis in RA
has been centred around two body sites, namely the
oral cavity and the gut, largely due to historic reasons.
In recent times, studies unravelling the microbiome
composition in RA has gained importance. A number
of disparate clinical observations have been made in
the past, which in the light of the new tools like meta-
genome sequencing provides new connections and
insights. In a nutshell, the potential evidence on the
pathophysiology of RA modulated through the micro-
biome can be put together into discrete models. The
models are schematically represented in Figure 1. The
following sections of the manuscript provide an over-
view of the evidence of oral and gut microbiome
dynamics in RA. A detailed list of human microbiome
associations in RA is summarized in Table 1.
Gut microbiome and RA
The gut microbiome has the maximum diversity and
density of microorganisms in the human body. These
microorganisms shape the development of the intestinal
immune system. Gnotobiotic animals have defective
development of the gut-associated lymphoid tissue
(GALT), Peyer’s patches intestinal epithelial cells,
impaired secretion of antimicrobial peptides and defen-
sins, lower regulatory T (Treg) cells and were also found
to be less efficient in suppressing Th17 response.
3034
The microbiota also determine pro- or anti-inflamma-
tory responses
35,36
which depend on the ability of the
organisms to bind and stimulate Toll-like receptors
(TLRs) and Nod-like receptors (NLRs) present on the
intestinal epithelial cells.
37
Some organisms like Lacto-
bacillus exert anti-inflammatory effects via induction of
Treg cells.
38
Most symbiotic bacteria have anti-inflam-
matory properties that are beneficial to the host and pre-
vent colonization by the pathobionts.
39
However, some
symbionts could also induce a pro-inflammatory state
under certain conditions.
30
Studies have emphasized a
causal role of the microbiome in the pathophysiology of
2International Journal of Rheumatic Diseases 2015
P. Sandhya et al.
RA. For example, introduction of microorganisms was
found to aggravate or alleviate arthritis in experimental
models of RA with diverse genetic background. In addi-
tion, in inflammatory bowel disease (IBD), dysbiosis
was found to tip the balance in favor of a pro-inflamma-
tory state in individuals with genetic susceptibility.
38
In humans, dysbiosis of the fecal flora has been
observed in RA. A number of studies explored the diver-
sity and differences of microbiome in the human gut
and their association with diseases. Organisms like
Clostridium perfringens were found to be more prevalent
in RA.
40
Anaerobic bacteria were also found to vary
between erosive RA and controls in another indepen-
dent study.
25
A recent study comparing patients with
RA and fibromyalgia showed paucity of organisms
like Bifidobacteria and the Bacteroides-Porphyromonas-
Figure 1 Overview of the proposed mechanisms of pathophysiology of rheumatoid arthritis (RA) modulated through microbial
dysbiosis. The oral cavity, lung and gut are believed to be the sites of origin of disease in RA. Microbial dysbiosis at these sites
could act in concert with environmental and host genetic factors to initiate disease. In the oral cavity Porphyromonas gingivalis by
virtue of peptidyl-arginine-deiminase (PAD) and gingipains cause protein citrullination. Translocation of oral pathogens could
also cause a similar phenomenon in the lungs. At these two sites, cigarette smoke could augment the process of citrullination. A
genetically susceptible host mounts an immune response to citrullinated antigens causing T cell and B cell activation and subse-
quent production of anti-citrullinated peptide antibodies. These antibodies are pathogenic in RA. Similarly in the gut, microbial
dysbiosis tilts the balance in favor of pro-inflammatory cytokine production. Infection in gut also breaks down the immune bar-
rier, leading to release of bacterial cell wall components into the circulation. These bacterial cell wall components have been
shown to elicit an immune response in the joints.
International Journal of Rheumatic Diseases 2015 3
Microbial dysbiosis in RA
Prevotella group in RA. A difference was also observed
for the Eubacterium rectale and Clostridium coccoides
group.
26
Recently, a 16S rRNA sequencing study of gut
microbiome on treatment-na
ıve patients with early
onset RA in comparison with chronic treated RA, psori-
atic arthritis and healthy controls showed association of
Prevotella species with new-onset RA. An investigation
of the composition of fecal Lactobacillus communities in
na
ıve early RA patients suggested a higher diversity of
the genera in early disease. The study also revealed an
increased level of Lactobacillus salivarius,Lactobacillus
iners and Lactobacillus ruminis in patients with RA in
comparison with the controls.
23
In addition, Lactobacil-
lus mucosa was found to be uniquely represented in RA
patients. Although lactobacilli are generally known to
be associated with an anti-inflammatory state, it is not
known if the increase in lactobacillus could represent
an anti-inflammatory mechanism in early RA.
Table 1 Summary of selected studies on microbiome profiles in rheumatoid arthritis (RA)
Site Methodology Study samples/description Summary of the findings References
Fecal Estimation of bacterial
counts in fecal culture
25 patients with RA
compared with controls
Higher carriage rate of
Clostridia was observed in RA
patients compared to
controls
Shinebaum et al.
40
Fecal Estimation of bacterial
counts in fecal culture
26 active RA on treatment
with SSZ versus DPA
Clostridium perfringens and
E. coli counts dropped in SSZ
treated group
Neumann et al.
83
Fecal Estimation of bacterial
counts in fecal culture
22 active RA and 26 OA,
both groups on NSAIDs; and
22 OA not on NSAIDs
Study observed a higher
Clostridium perfringens count
in RA and in OA patients
receiving NSAIDs
Dearlove et al.
116
Fecal Estimation of bacterial
counts in fecal culture
31 patients with RA on SSZ Low fecal count of
Clostridium perfringens was
observed after weeks of
treatment
Bradley et al.
117
Fecal Gas-liquid chromatography
of bacterial CFAs
74 treatment-naive early RA
and 91 non-RA controls
Variation in CFA profile of
RA as compared to controls
likely caused by anaerobic
bacteria
Eerola et al.
25
Fecal Comparison of bacterial
cellular fatty acids using gas
liquid chromatography
43 patients divided in two
groups, one with vegan diet
and other control
Vegan diet has an impact on
microbial flora that is
associated with improvement
in RA
Peltonen et al.
79
Fecal 16S rRNA hybridization,
flow cytometry and DNA
staining
51 treatment-naive early RA
versus 50 fibromyalgia
Significantly lower
representation of
Bifidobacteria, Bacteroides-
Porphyromonas-Prevotella and
Eubacterium rectale
Clostridium coccoides groups
in RA
Vaahtovuo et al.
26
Fecal Lactobacillus-specific PCR
amplification of DNA from
stools
15 treatment-naive early RA
versus 15 healthy controls
Study suggested a higher
number and diversity of
Lactobacillus in feces of
patients with RA
Liu et al.
23
Sub-gingival Multiplexed-454 pyro-
sequencing
31 new-onset treatment-
naive RA, 34 chronic RA and
18 healthy controls
P. gingivalis was found to be
associated with PD status but
the microbial diversity was
found to be similar between
RA and controls
Scher et al.
24
CFAs, cellular fatty acids; DPA, D-penicillamine; NSAIDs, non-steroidal anti-inflammatory drugs; OA, osteoarthritis; PD, Periodontitis; SSZ, sul-
phasalazine.
4International Journal of Rheumatic Diseases 2015
P. Sandhya et al.
Development of specific in vitro or in vivo models may
put forward this strategy.
The major challenge in understanding the micro-
biome and its dynamics has been that a vast majority
of the microbes in a niche could not be effectively
cultured and isolated in monoclonal populations.
Simplistic approaches including sorting single cells
followed by genome amplification and sequencing
could provide a new opportunity to sequence and
understand individual bacteria in a mixed population
of cells.
113
Single cell genomics, therefore, help under-
stand genome sequences of individual organisms in a
pool. Complemented by sequencing technologies with
extremely longer reads, it may be theoretically possi-
ble to assemble genomes from single cells or mole-
cules.
114,115
This concept of individual genome
sequence/components in a consortium would cause a
paradigm shift in understanding the metabolic capac-
ity of individual organisms. Such an understanding
could provide the much needed insights into commu-
nity architecture, collaboration and other sociological
aspects of biofilm. Exploitation of this information
may also help engineer specific disease pathways using
synthetic biology.
CONCLUSIONS AND FUTURE
PERSPECTIVE
Involvement of microbes in the pathophysiology of
RA is supported by a number of complementary evi-
dences from independent reports, and many of them
suggest a possible incriminatory role of Porphy-
romonas in the pathogenesis of RA. Research in this
area is fraught with practical difficulties of following
up high-risk individuals over a long period of time
and a non-availability of an in vivo model to emu-
late or replicate the series of pathogenetic events
that follow the trigger. Despite these limitations, a
number of technological advancements have now
made it possible to profile the microbiome and its
dynamics in a culture-independent manner and pro-
vide clues toward the intricate role of microbiome
in disease processes. Understanding of the involve-
ment of microbiome in RA has opened up newer
avenues for improving diagnostic accuracy as well as
a therapeutic window of opportunity to modulate
microbes, including but not limited to diet, antibi-
otics and probiotics. Further research on host-
microbe interaction in health and disease in relation
to host genetics, immune response, dietary factors
and environmental risk factors may hold promise.
KEY MESSAGES
The human microbiome studies strongly suggest an
incriminating role of microbes in the pathophysiology
and progression of RA.
A number of approaches to modulate the micro-
biome like diet, antibiotics and probiotics could have a
potential role in disease management, which require
further in-depth studies to ascertain efficacy and clinical
utility.
Understanding the role of microbes and methods to
measure and modulate them could have immense diag-
nostic and therapeutic applications in RA.
ACKNOWLEDGEMENTS
The authors acknowledge help, discussions and scien-
tific inputs from Dr Sridhar Sivasubbu for preparing
this manuscript.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
FUNDING STATEMENT
This work is funded by Council of Scientific and Indus-
trial Research (CSIR), India through Grant No:
OLP1105 (EMPOWER). The funders had no role in the
content or preparation of the manuscript.
REFERENCES
1 Qin J, Li R, Raes J et al. (2010) A human gut microbial
gene catalogue established by metagenomic sequencing.
Nature 464,5965.
2 Gill SR, Pop M, Deboy RT et al. (2006) Metagenomic
analysis of the human distal gut microbiome. Science
312, 13559.
3 Gilbert SF, Sapp J, Tauber AI (2012) A symbiotic view of life:
we have never been individuals. QRevBiol87,32541.
4 Li M, Wang B, Zhang M et al. (2008) Symbiotic gut
microbes modulate human metabolic phenotypes. Proc
Natl Acad Sci USA 105, 211722.
5 Round JL, O’Connell RM, Mazmanian SK (2010) Coordi-
nation of tolerogenic immune responses by the commen-
sal microbiota. J Autoimmun 34, J2205.
6 Rossi O, van Baarlen P, Wells JM (2013) Host-recogni-
tion of pathogens and commensals in the mammalian
intestine. Curr Top Microbiol Immunol 358, 291321.
7 Chu H, Mazmanian SK (2013) Innate immune recogni-
tion of the microbiota promotes host-microbial symbio-
sis. Nat Immunol 14, 66875.
International Journal of Rheumatic Diseases 2015 9
Microbial dysbiosis in RA
Oral bacterial DNA in joints and direct
damage
There is evidence to suggest direct damage to joints by
oral bacteria. Studies have identified oral bacterial DNA
in serum and synovial fluid of patients with RA and
psoriatic arthritis in comparison to controls; however,
bacterial DNA in periodontal pockets did not differ
between these groups.
65
Bacterial DNA belonging to
P. gingivalis,Eubacterium saburreum,Peptostreptococcus
micros,A. israelii,Selenomonas noxia,Propionibacterium
acnes,Campylobacter showae,Tannerella forsythensis,
C. sputigena,Leptotrichia buccalis and P. intermedia have
been found exclusively in synovial fluid of patients with RA
and psoriatic arthritis.
65
Whileitispossiblethatoralbacte-
rial DNA could translocate to joints, triggering synovial
inflammation, it is more likely that inflamed joints could
entrap these bacterial DNA, leading to augmentation of
inflammation. In addition, P. gingivalis could invade
human chondrocytes, interfere with the cell cycle and cause
apoptosis of these cells, resulting in joint damage.
66
Oral microbes, lung and the pathophysiology
of RA
Apart from the well-studied candidate sites, it is inter-
esting to note that smoking is associated with possible
translocation of the supraglottic bacteria to the lungs,
mostly including species like Prevotella and Porphy-
romonas.
67
This attains great importance in the light of
the fact that both the bacteria along with smoking are
independent risk factors and are closely associated with
the etio-pathogenesis of RA. Evidence also suggests an
increased level of PADs in the lungs of smokers, which
could be another site for citrullination of host pro-
teins.
68
It has also been suggested that the presence of
Porphyromonas,Prevotella and other associated taxa in
the lung, instead of their usual home in the supraglottic
regions, is associated with airway inflammation.
67
Although systematic evaluation and comparison of the
lung microbiome has not been performed in patients
with RA, this would be a lead worth exploring.
EFFECT OF PROBIOTICS AND DIET IN
PATHOPHYSIOLOGY OF RA
Apart from implicating bacteria in the direct causality
and pathophysiology of inflammatory and autoim-
mune diseases, the protective roles of microbiota in dis-
eases also has to be emphasized. The role of microbiota
in modulating the development of innate immunity,
preventing colonization of harmful bacteria, decreasing
gut permeability and modulating inflammation has
been studied. Live microorganisms which confer a
health benefit to the host when administered in ade-
quate amounts are called probiotics.
69
Most commonly
used probiotics belong to Bifidobacterium,Lactobacillus
and Escherichia coli Nissle species. Probiotics act via
modulating both the innate and adaptive immune sys-
tem. In one of the prominent studies, members of the
Clostridium cluster XIVa suppressed inflammation by
inducing colonic Treg cells.
70
Anti-inflammatory effect
of Faecalibacterium prausnitzii in IBD was mediated via
suppression of IL-8 and nuclear factor jB pathway.
71
Bifidobacterium induced IL-10 production and inhib-
ited IL-8 which accounts for its beneficial effect in ulcer-
ative colitis.
72
Lactobacillus casei reduced clinical and
histo-pathological features of active inflammation in
collagen-induced arthritis.
73
These effects were medi-
ated by inhibition of pro-inflammatory cytokine pro-
duction by Th1 cells, cyclo-oxygenase-2 inhibition and
induction of IL-10.
73
In induced arthritis models of
Lewis rats, Lactobacillus GG strain attenuated arthritis.
74
A number of studies have attempted to systematically
understand the clinical outcomes of the use of probi-
otics. To date, four randomized double-blind placebo-
controlled trials of probiotics in RA have been reported.
In the first study, 21 patients with mild RA were ran-
domized to receive Lactobacillus rhamnosus GG or pla-
cebo for 12 months. The study noted no improvement
in disease activity scores or inflammatory markers, but
patients experienced subjective well-being.
75
Similar
findings were reported by Pineda et al.
76
using probi-
otic L. rhamnosus GR-1 and Lactobacillus reuteri RC-14
capsules. Mandel et al. randomized 45 patients with RA
and the treatment arm was administered Bacillus coagu-
lans GBI-30 6086 for a month. The authors observed
improvement in pain scores, patient global assessment
scores and reduction in inflammatory markers.
77
In the
recent trial by Alipour et al.,
78
L. casei 01 was supple-
mented to the treatment arm for 8 weeks. So far, this is
the only study that has reported improvement in dis-
ease activity with reduction in inflammatory markers
and cytokine levels. The variable outcome noted in the
above-mentioned studies could be attributed to differ-
ences in patient profile and characteristics, duration of
supplementation, different species and the dose of pro-
biotics used, possible attenuation of the microbe in the
gut and their interactions with other external factors,
including diet. In summary, although evidence from
experimental animal models suggests beneficial effect
of probiotics in RA, this has not been unequivocally
replicated in clinical settings. Since there is no com-
6International Journal of Rheumatic Diseases 2015
P. Sandhya et al.
pelling and credible evidence at this point of time, rou-
tine use of probiotics in clinical settings cannot be rec-
ommended. Additional studies toward systematic
identification of species providing maximum clinical
benefit, optimum dose and duration of administration
are required.
Several groups have explored the role of diet in
RA.
79,80
Improved disease activity by fasting and vege-
tarian diet was noted by clinical researchers. This effect
could be partly mediated through alteration of the
intestinal microbiome. A significant difference in gut
flora between responders and non-responders in studies
comparing cellular fatty acid profiles supports this
hypothesis. However, a recent systematic review
showed that this and other studies were limited by size
and bias and there were alternate explanations for the
noted improvement.
81,82
Study of diet induced changes
in microbial flora in the metagenomic era is expected to
enhance understanding and provide more scientific evi-
dence to support dietary intervention in management
of RA.
ANTIMICROBIALS AND RA
Antibiotics have been an integral part of the therapeutic
armamentarium against RA. Sulphasalazine is one of the
most common disease-modifying antirheumatic drugs
(DMARDs) used in RA. The sulfapyridine moiety of sul-
phasalazine inhibits non-sporing anaerobes, Clostridia and
Enterobacteria in the gut of patients with RA and inflamma-
tory bowel disease.
83,84
Tetracyclines are the other class of
antimicrobials previously used in the treatment of RA in the
early 1960s, but later lost out to more effective drugs. Both
these drugs are broad-spectrum bacteriostatic antibiotics
with potent anti-inflammatory properties. For example,
tetracyclines inhibit matrix metalloproteinase and nitric
oxide synthase, suppress adaptive immune cells and
increase IL-10, an anti-inflammatory cytokine.
85
Tetracy-
cline derivative minocycline has also been found efficacious
in RA.
86
Antimalarials used in RA too have similar antimi-
crobial and anti-inflammatory properties.
87
Other antibi-
otics used in RA therapy include macrolides, levofloxacin,
dapsone, ceftriaxone and metronidazole.
8891
With the
new-found appreciation of the potential role of micro-
biome in RA, it is all the more plausible that improvement
in the disease with antibiotic drugs could partly be caused
by their antimicrobial properties. In addition, it is also
worthwhile to note that tetracyclines and sulfonamides are
effective against periodontal pathogens.
92,93
That this could
partly account for its benefit in RA is not clearly known. It
only remains to be seen if specific antibacterial therapy for
periodontitis could prevent RA in high-risk individuals. It
should be emphasized that the mechanisms of action, effi-
cacy and toxicity need to be closely evaluated before the
common application of antibiotics become part of the regu-
lar treatment regimen. Apart from the antimicrobials and
anti-inflammatory agents modulating the microbes, there is
an isolated report of the gut microbiome modulating the
metabolism of therapeutic agents in animal models. The
reduction of sulphasalazine to sulfapyridine and 5-amino
acetylsalicylic acid is mediated by the azoreductase activity
of the gut bacteria, particularly the probiotic strains. Probi-
otics increased azoreductase activity and modified sul-
phasalazine metabolism in rats, but this observation could
not be replicated in a preliminary human study.
94,95
DIAGNOSTIC IMPLICATIONS OF THE
UNDERSTANDING OF DYSBIOSIS IN RA
Microbiome studies have been explored with respect to
their diagnostic potential in diseases like type 2 diabetes
mellitus,
9698
obesity
99,100
and IBD.
101,102
Taking clues
from these studies, it appears that microbiome profiling has
the potential to be extensively used for patient stratification
and/or risk prediction in RA. It also has the potential to sup-
plement the present diagnostic modalities, improving on
the combined precision, as it is unlikely that the micro-
biome could act as an independent diagnostic biomarker in
RA. The sum total of the disease susceptibility genes and the
metagenome could potentially be used to create a risk pre-
diction tool for RA. The diagnostic tell-tale features of
microbiome apart from the profiles in various body niches,
could also include their metabolites. Best examples in this
regard include anti-citrullinated peptide antibody (ACPA),
one of the biomarkers used in the diagnosis of RA. ACPA is
a consistent diagnostic as well as prognostic biomarker for
RA.
103
In one of the recent studies, ACPA was also corre-
lated with the onset and severity of the disease.
104
POTENTIAL IMPLICATIONS OF
MICROBIOME IN CLINICAL
MANAGEMENT
Understanding the biological mechanism of dysbiosis
has far-reaching implications in prevention, early
diagnosis and therapeutic intervention. Therapeutic
advances have been made in autoimmune diseases
despite several shortcomings, including lack of clarity
on dynamics of the microbiota and the biological
changes effected by them. Paucity of information
related to microbiome diversity between diseased and
healthy individuals is another limitation. The primary
International Journal of Rheumatic Diseases 2015 7
Microbial dysbiosis in RA
modality to reverse dysbiosis by the use of antibiotics is
limited as most antibiotics have non-specific actions.
Probiotics, or concoctions of bacterial populations
which have a positive effect in maintaining the equilib-
rium have been widely marketed, despite proven bio-
logical and clinical evidence only in a handful of
conditions.
105
Apart from probiotics and antibiotics, a third cate-
gory of molecules called prebiotics has been studied
extensively in the recent years. These classes of mole-
cules promote the binding or attachment of microbes
to the skin or mucosal membrane through specific
interactions and provide a novel opportunity to
specifically promote or inhibit specific classes of
microorganisms in an attempt to normalize the imbal-
ance.
There is also a potential fourth opportunity by mole-
cules which have specific activities promoting or
inhibiting specific recognition and cross-talk, including
those mediated through TLRs and other host-encoded
recognition molecules.
106
The fifth and the most dramatic modality of micro-
bial transplantation has drawn much attention in recent
times, whereby the microbiome from a healthy individ-
ual could be transplanted to an orthologous site.
Notwithstanding ethical questions and recent evidence,
a number of trials in this direction have been initiated
in diseases like IBD.
107
Finally, the sixth and probably the most poorly
explored therapeutic opportunity involving the micro-
bial environment would be the use of bacteriophages.
The extreme selectivity of bacteriophages could be used
to understand the underlying mechanism of homeosta-
sis and dysbiosis on one end, while being effectively
used to modulate the misbalance on the other.
GAPS IN KNOWLEDGE, HYPOTHESES
AND OPPORTUNITIES IN THE FIELD
Human microbiome has two distinct components: the
core components which are static and a more dynamic
variable component which responds to external cues.
The core and variable microbiome components could
possibly vary between geographical regions and/or eth-
nicities and poses one of the major challenges toward
understanding the association of microbiome with
traits or diseases. Core and variable components can,
therefore, be defined specifically in the context of the
population and the environment as well as the genetic
background of the host. This is evident by the wide vari-
ations in microbiome components across different eth-
nicities.
108
Nevertheless, major compositions of the
three enterotypes as defined by multiple gut micro-
biome studies stand true across populations; these asso-
ciations have been reproduced by replication studies.
To address the challenge of characterizing the com-
pendium of microbiome, several studies to catalogue
this diversity from different body sites have been
initiated globally, although not inclusive of all
ethnicities.
109
Apart from environmental influences leading to pop-
ulation scale differences in their microbial composition,
the genetic factors in the host strongly modulate specific
microbial types.
110
Molecular explanations for such
specific genetic controls have not been clearly delin-
eated. Research on genetics of host and microbiome
crosstalk may reveal molecules involved in microbial
identification, attachment, immune response and main-
tenance, which could become eventual targets. It would
also be worthwhile to explore whether specific micro-
biome signatures could affect the prognosis or thera-
peutic outcome of the disease. It should nevertheless be
remembered that drugs and their effects on the micro-
biome are still a poorly understood field. Understand-
ing the effects of drugs on the microbiome profiles
would be important to delineate confounding factors.
The understanding would also contribute to the possi-
bility of preventing and possibly delaying the onset of
disease.
However, the human microbiome will still remain an
associative rather than causative factor unless lengthy
and expensive longitudinal cohort studies in unaffected,
shared epitope-positive first-degree relatives of patients
with RA are carried out. Even if such studies are con-
ducted, the confounding effects exerted by environment
would remain a major gap. These limitations could be
taken care of by in vivo studies in model systems. The
recent studies on HLA transgenic mice and gut micro-
biome offers a new avenue to study the disease mecha-
nism in a model system in vivo.
111
It is formidable that
a number of model systems like mouse and zebrafish
have been reported in recent years.
112
Further character-
ization and modelling of such microbial orthologs
leading to therapeutic strategies are awaited. The devel-
opments of specific antibacterial agents, including
small molecules, peptides and possibly biological
agents like bacteriophages could have immense appli-
cations in modulating and reversing dysbiosis. Such a
strategy could even reverse the disease process through
preventive approaches. Food and nutrition could also
modulate and tilt the balance of the human micro-
biome favorably, leading to prevention of disease.
8International Journal of Rheumatic Diseases 2015
P. Sandhya et al.
Development of specific in vitro or in vivo models may
put forward this strategy.
The major challenge in understanding the micro-
biome and its dynamics has been that a vast majority
of the microbes in a niche could not be effectively
cultured and isolated in monoclonal populations.
Simplistic approaches including sorting single cells
followed by genome amplification and sequencing
could provide a new opportunity to sequence and
understand individual bacteria in a mixed population
of cells.
113
Single cell genomics, therefore, help under-
stand genome sequences of individual organisms in a
pool. Complemented by sequencing technologies with
extremely longer reads, it may be theoretically possi-
ble to assemble genomes from single cells or mole-
cules.
114,115
This concept of individual genome
sequence/components in a consortium would cause a
paradigm shift in understanding the metabolic capac-
ity of individual organisms. Such an understanding
could provide the much needed insights into commu-
nity architecture, collaboration and other sociological
aspects of biofilm. Exploitation of this information
may also help engineer specific disease pathways using
synthetic biology.
CONCLUSIONS AND FUTURE
PERSPECTIVE
Involvement of microbes in the pathophysiology of
RA is supported by a number of complementary evi-
dences from independent reports, and many of them
suggest a possible incriminatory role of Porphy-
romonas in the pathogenesis of RA. Research in this
area is fraught with practical difficulties of following
up high-risk individuals over a long period of time
and a non-availability of an in vivo model to emu-
late or replicate the series of pathogenetic events
that follow the trigger. Despite these limitations, a
number of technological advancements have now
made it possible to profile the microbiome and its
dynamics in a culture-independent manner and pro-
vide clues toward the intricate role of microbiome
in disease processes. Understanding of the involve-
ment of microbiome in RA has opened up newer
avenues for improving diagnostic accuracy as well as
a therapeutic window of opportunity to modulate
microbes, including but not limited to diet, antibi-
otics and probiotics. Further research on host-
microbe interaction in health and disease in relation
to host genetics, immune response, dietary factors
and environmental risk factors may hold promise.
KEY MESSAGES
The human microbiome studies strongly suggest an
incriminating role of microbes in the pathophysiology
and progression of RA.
A number of approaches to modulate the micro-
biome like diet, antibiotics and probiotics could have a
potential role in disease management, which require
further in-depth studies to ascertain efficacy and clinical
utility.
Understanding the role of microbes and methods to
measure and modulate them could have immense diag-
nostic and therapeutic applications in RA.
ACKNOWLEDGEMENTS
The authors acknowledge help, discussions and scien-
tific inputs from Dr Sridhar Sivasubbu for preparing
this manuscript.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
FUNDING STATEMENT
This work is funded by Council of Scientific and Indus-
trial Research (CSIR), India through Grant No:
OLP1105 (EMPOWER). The funders had no role in the
content or preparation of the manuscript.
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