Clinical Immunology 215 (2020) 108415

Contents lists available at ScienceDirect

Clinical Immunology
journal homepage: www.elsevier.com/locate/yclim

Review Article

Gut microbiota in chronic inflammatory disorders: A focus on pediatric T

inflammatory bowel diseases and juvenile idiopathic arthritis
⁎
Amanda Ricciutoa,b, , Philip M. Shermana,b, Ronald M. Laxerb,c
a
Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada
b
Department of Paediatrics, Faculty of Medicine, University of Toronto, Canada
c
Division of Rheumatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: The gut microbiota is integral to human health, including maintaining the delicate balance between tolerance
Dysbiosis and protection against potentially harmful pathogens. A growing body of evidence implicates the intestinal
Fecal microbial transplantation microbiome in immune-mediated inflammatory disorders; these data span the spectrum from genetic and en-
Inflammatory bowel disease vironmental disease risk factors, to animal studies (particularly germ-free and gnotobiotic models) and human
Juvenile idiopathic arthritis
studies, including evidence of dysbiosis in diseased individuals compared to healthy populations. In this review,
Microbiota
we summarize both animal and human data supporting a link between the gut microbiota and inflammatory
bowel diseases (IBD) and systemic inflammatory arthritis, as models for chronic inflammatory disorders, while
offering a pediatric focus (pediatric IBD and juvenile idiopathic arthritis). We discuss relevant mechanisms
related to the crosstalk between the gut microbiota and the innate and adaptive immune system. We close with a
brief discussion of emerging microbe-altering interventions, including fecal microbial transplantation and its
immunologic effects.

1. Introduction tremendous interest of late. Numerous studies document differences in

Immune-mediated inflammatory disorders (IMIDs), including
chronic inflammatory bowel diseases (IBD) and juvenile inflammatory
arthritides (JIA), are highly prevalent in industrialized countries. It is
projected, for example, that by the year 2030, the prevalence of IBD
will approach 1% in Canada [1]. More alarming still is that young
children represent the fastest growing group [2]. While overall in-
cidence rates are stabilizing in the developed world, they are now in-
creasing in developing countries, posing a growing global burden [3].
IMIDs are currently thought to result from a complex interplay be-
tween host genetics and various environmental factors. A fascinating
epidemiologic phenomenon is that the risk of certain IMIDs in im-
migrants to high prevalence regions approximates that in host re-
sidents, such as the risk of ulcerative colitis (UC), an IBD subtype, in
South Asians [4]. Observations like these, coupled with rapid fluxes in
disease rates over relatively short periods of time, indicate that genetic
factors cannot be solely to blame. Rather, these findings point strongly
to the role of environmental risk factors.
Among environmental factors, the gut microbiota has garnered
the microbial community structure (i.e., dysbiosis) of individuals af-
fected by various diseases (including IMIDs) compared to healthy
controls (HCs). Moreover, animal studies demonstrate the transfer-
ability of disease phenotype upon transfer of fecal material from dis-
eased humans or animals to germ-free (GF) mice, offering particularly
compelling support for a causal link between gut microbes and disease.
The reciprocal, multi-faceted interactions between the gut microbiota
and human immune system add biological plausibility to the hypothesis
that gut microbes are causally implicated in immune-mediated dis-
orders. This has exciting implications for the development of potential
therapies with microbe-altering properties, such as nutritional inter-
ventions, pro- and prebiotics, antibiotics and fecal microbial trans-
plantation (FMT).
In what follows, we review the role of the gut microbiota in both
IBD and inflammatory arthropathies, as models for chronic in-
flammatory disorders, while offering a pediatric focus. We begin by
summarizing methods used to characterize the microbiota, the influ-
ence of the gut microbiota on the immune system and evidence sup-
porting an association between the microbiome and both IBD and

Abbreviations: IBD, inflammatory bowel diseases; JIA, juvenile idiopathic arthritis

⁎
Corresponding author at: Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8,
Canada.
E-mail addresses: amanda.ricciuto@sickkids.ca (A. Ricciuto), philip.sherman@sickkids.ca (P.M. Sherman), ronald.laxer@sickkids.ca (R.M. Laxer).

https://doi.org/10.1016/j.clim.2020.108415
Received 13 February 2020; Received in revised form 6 April 2020; Accepted 6 April 2020
Available online 09 April 2020
1521-6616/ © 2020 Elsevier Inc. All rights reserved.
A. Ricciuto, et al.
arthritis. We then review microbial changes reported in both condi-
tions, while highlighting pediatric findings. We close with a discussion
of selected future novel therapies, including FMT.
2. Microbiota characterization methods
The human body includes a complex mix of mammalian and pro-
karyotic cells, present in roughly equal proportions [5]. The microbial
flora harbored by humans is termed the “microbiota.” It includes
symbiotic and commensal microorganisms, as well as pathobionts
(bacteria with the potential to be pathogenic). The term “microbiome”
denotes the collective genomes of the microbiota.
Standard culturing techniques are unable to identify at least 80% of
the human microbiota [6]. The flurry of microbiome-focused research
in recent years was spurred by the advent of technologies that enable
more comprehensive microbial characterization, particularly high
throughput DNA sequencing. This method uses the 16S small ribosomal
subunit (16S rRNA), a gene unique to bacteria, to identify bacteria and
Archaea [7]. The 16S rRNA gene includes elements that are conserved
between most bacteria and hypervariable elements that enable the
differentiation of species. An analogous approach can be applied to
fungi, which uses the internal transcribed spacer (ITS), 18S rRNA or 26S
rRNA regions. While these methods have proven extremely useful, they
have certain limitations. These include a limited ability to differentiate
commensal from pathogenic strains, especially in the setting of hor-
izontal gene transfer (the exchange of virulence factors between species
in the same environment) [8], and to identify non-bacterial strains.
Metagenomics, the study of the collective genomes (bacterial,
fungal and viral) present in an environmental sample, allows even more
detailed compositional profiling of a microbial community. Shotgun
metagenomics refers to a method by which DNA extracted from an
environment is sheared into small fragments, which are then se-
quenced. Importantly, whole genome sequencing can be used to deduce
microbial function, including enzymatic capacity. Metagenomics is
complemented by the methods of metatranscriptomics, metaproteomics
and metabolomics, which respectively examine gene expression, and
protein and metabolite production by a microbial community.
Limitations of whole genome sequencing currently relate to the
2
Clinical Immunology 215 (2020) 108415
Fig. 1. Functions of the gut microbiota in human
health.
Short chain fatty acids are derived from indigestible
complex carbohydrates; they serve as an energy
source for colonocytes and have important im-
munoregulatory functions. “Colonization resistance”
refers to mechanisms by which gut microbes protect
themselves and the host against incursion by poten-
tially harmful microorganisms.
relatively high cost compared with 16S rRNA profiling and the issue of
reproducibility of findings both within samples and between labora-
tories.
3. The microbiome in human health
Most intestinal bacteria belong to 4 phyla: Firmicutes,
Bacteroidetes, Proteobacteria and Actinobacteria, with the first two
predominating in health. Microbial diversity increases along the gas-
trointestinal tract, from stomach to colon and, at the terminal ileum,
prevalent species transition from aerobes to facultative and strict
anaerobes. Microbial populations differ substantially between the gut
lumen and mucosal surfaces; the latter are physically closer to the in-
testinal epithelium and, consequently, may exert a greater effect on the
host, including the innate and adaptive arms of the immune system. For
example, in trinitrobenzenesulfonic acid (TNBS)-induced colitis in
mice, the microbiomes of feces, cecum and mucus are distinct, and the
mucus microbiome correlates most closely with disease severity [9].
Moreover, in a human study of functional gastrointestinal disorders,
mucosa-associated Faecalibacterium and Ruminococcus were inversely
correlated with Toll-like receptor (TLR)4 expression while, in luminal
samples, there was no correlation with TLR4 at any taxonomic level
[10]. It is pertinent, therefore, to consider the source of microbes (fecal
or mucosal) in interpreting findings reported in various studies of the
gut microbiota.
Over a person's lifetime, the diversity and composition of the mi-
crobiota fluctuate with age and extrinsic factors, such as diet, lifestyle,
antibiotics and other environmental exposures. Somewhat surprisingly,
the functional capacity of the healthy microbiome is relatively stable
irrespective of its specific composition [11]. The gut microbiota per-
forms numerous functions that are integral to human health. These are
outlined in Fig. 1 and can be grouped into 3 main categories: nutrition
and metabolism; protection against pathogens (or “colonization re-
sistance”); and immune system development. The latter is further de-
tailed below.
A. Ricciuto, et al.
4. The effects of the gut microbiota on the human immune system
4.1. The innate and adaptive immune system: a refresher
The gut faces the remarkable challenge of maintaining an intricate
balance between tolerance to the 500 to 1000 commensal bacteria
present in the gut and protection against potentially harmful pathogens.
The predominantly tolerogenic immunologic response to intestinal lu-
minal antigens (microbial and dietary) is enabled by the recognition of
antigen patterns by innate immune cells, which in turn direct T cell
reactivity. Microbial sensing by mononuclear phagocytes (MNPs) of the
innate system (macrophages, dendritic cells (DCs)) and intestinal epi-
thelial cells (ECs) is mediated by pattern recognition receptors (PRRs),
such as TLRs and nucleotide-binding oligomerization domain (NOD)-
like receptors (NLRs). TLRs and NLRs recognize pathogen-associated
molecular patterns (PAMPs), such as lipopolysaccharide (LPS). TLRs
also stimulate the production of antimicrobial peptides (AMPs), such as
regenerating family protein III (Reg III β/γ). Innate lymphoid cells
(ILCs) also belong to the innate arm of the immune system. They do not
bear antigen-specific T or B cell receptors but share certain functional
characteristics with CD4+ T helper (Th) cells. There are several distinct
types; ILC3s constitute the majority of intestinal ILCs. They secrete in-
terleukin (IL)-17 and IL-22 and are generally considered the innate
counterpart of Th17 cells.
Within the adaptive arm of the immune system, T helper cells are
classically categorized into Th1 and Th2 subsets; Th1 considered the
primary mediator of responses to extracellular pathogens (through
production of interferon (IFN)-γ, which recruits macrophages), and Th2
considered the primary mediator of response to parasites (through se-
cretion of IL-4, IL-5 and IL-13). IBD includes two main subtypes, UC and
Crohn's disease (CD). Based on this framework, UC was traditionally
considered to be predominantly Th2-mediated and CD to be Th1-
mediated. However, this paradigm was expanded and refined following
the discovery of complementary Th subsets, including proinflammatory
Th17 cells (CD4+ IL-17-secreting T cells) and anti-inflammatory T
regulatory (Treg) cells, which express the nuclear transcription factor
Forkhead box P3 (FoxP3).
Th17 cells play an important role in mucosal defense, while Tregs
dampen the proliferation and functions of other T cells. Although Th17
and Treg cells herald from a common CD4+ naïve T cell, their differ-
entiation is contingent on specific inputs, specifically transforming
growth factor (TGF)-β in the presence of retinoic acid for Tregs, and
TGF-β, IL-6 and IL-21 (in humans) for Th17. The proinflammatory/anti-
inflammatory balance between Th17 and Treg cells is now recognized
to be fundamental to the etiopathogenesis of T-cell mediated IMIDs like
IBD and JIA [12,13]. The complexity of the Th framework continues to
grow, with more recent data demonstrating the existence of additional
Th subsets, such as regulatory type 1 (Tr1) cells (CD4+ T cells that
possess immunoregulatory properties and produce IL-10, but are
FoxP3-negative) as well as follicular Th (Tfh) cells (which promote
Th1/Th17 polarization), Th9 cells and Th22 cells.
4.2. The reciprocal interactions between the microbiota and the immune
system
The paramount importance of the gut microbiota in shaping a
healthy immune system is highlighted by the immunologic defects that
arise in mice raised in germ free (GF) environments. These animals
display lymphoid hypoplasia in the spleen, lymph nodes, thymus and at
mucosal surfaces; they have fewer peripheral CD4+ T cells (with a Th2
bias); and have lower immunoglobulin (Ig) levels [14,15].
The influence of the gut microbiota on the immune system is ap-
parent within both the innate and adaptive arms. Communication be-
tween gut microbes and innate MNPs is key to maintaining intestinal
homeostasis. TLR activation by commensal bacteria can induce, but
also dampen and regulate, intestinal inflammation. For example,
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Clinical Immunology 215 (2020) 108415
repeated TLR activation by commensal microbes suppresses proin-
flammatory NF-ΚΒ signaling, downstream chemokine (IL-8) production
and recruitment of neutrophils. Moreover, PRR triggering activates and
induces antigen-presenting cells (APCs), which in turn promote Treg
differentiation [16]. In keeping with this, feeding LPS to GF mice
challenged with dextran sulfate sodium (DSS) to induce colitis is pro-
tective, and associated with an increase in CD103+ DCs and induction
of Tregs in the distal intestine [17]. Several microbial metabolites also
affect innate immune responses. SCFAs (acetate, propionate and buty-
rate) are produced from microbial fermentation of host-indigestible
complex carbohydrates, largely by Firmicutes and Bacteroidetes. SCFAs
influence innate cells via G protein-coupled receptors; they suppress the
production of proinflammatory cytokines, such as TNF-α, IL-6 and ni-
tric oxide (NO) [18]. Bacterial-derived tryptophan metabolites have the
capacity to directly modulate ILC3 functionality via the aryl hydro-
carbon receptor (AhR) [19].
Within the adaptive arm of the host immune system, the microbiota
is critical for the development of the Th17 compartment in the gut la-
mina propria [20,21]. This is illustrated by the absence of Th17 cells in
mice reared under GF conditions [21]. Among commensal bacteria in
mice, segmented filamentous bacteria (SFB) have a particularly potent
ability to induce Th17 cells; the addition of SFB alone to GF mice is
sufficient for Th17 cells to accumulate in the small bowel [22,23].
Based on a more recent study, adhesion of microbes to intestinal ECs is
an important cue for Th17 induction; this was observed with SFB, as
well as the murine-specific enteropathogen Citrobacter rodentium, the
human enteric pathogen Escherichia coli serotype O157:H7 and a mix-
ture of 20 bacterial strains derived from feces of adult UC patients [24].
There are also ample data to support the role of gut bacteria and
their metabolites in promoting Tregs. SCFAs can stimulate colonic Tregs
directly, through physical inhibition of histone deacetylases (HDACs)
involved in the post-translational regulation of the transcription factor
FoxP3, or indirectly, via G protein-coupled receptors. SCFAs also pro-
mote the differentiation of CD4+ naïve T cells into Tregs. Mouse and in
vitro studies support the ability of SCFAs and SCFA-producing bacteria
to induce Tregs and demonstrate their association with FoxP3 expres-
sion and higher IL-10 levels [25,26]. Colonic Treg abundance correlates
with butyrate concentrations and butyrate is protective against the
development of colitis in an adoptive T cell transfer model of re-
combination activating gene (RAG)−/− mice (lacking both T and B
cells) [27]. Retinoic acid and tryptophan products are additional mi-
crobial metabolites with Treg-stimulating effects. Retinoic acid is gen-
erated by DCs in a process that requires commensal support. This re-
tinoic acid then leads to increased gut-homing IL-10-producing Tregs
and enhanced Treg differentiation [28]. Microbial tryptophan meta-
bolites (kynurenines), produced by bacteria like Bifidobacterium longum,
B. thetaiotaomicron and Faecalibacterium prausnitzii, drive intraepithelial
CD4+ Th cells toward an immunoregulatory phenotype, an effect
mediated by AhR [29–31].
In addition to microbial metabolites, several specific bacteria, in-
cluding Clostridia, Bacteroides fragilis and Faecalibacterium prausnitzii,
are directly implicated in Treg induction. In seminal work by Atarashi
et al. [32,33], the transfer of specific Clostridia strains from human
stool into GF mice triggered a significant increase in Tregs, which were
low at baseline. This occurred in conjunction with production of anti-
inflammatory molecules, such as IL-10, TGF-β1, cytotoxic T lymphocyte
antigen-4 (CTLA-4) and inducible T cell co-stimulator (ICOS)), as well
as SCFAs. The polysaccharide A (PSA) of the capsule of B. fragilis sti-
mulates differentiation of naïve CD4+ T cells into IL-10-secreting Tregs
or Tr1 cells via plasmacytoid DC stimulation [34,35]. In the absence of
APCs, PSA can directly activate TLR2 on FoxP3+ T cells to trigger the
production of immunoregulatory molecules like IL-10 [36]. As dis-
cussed in 5.1.3 below, F. prausnitzii has garnered significant attention in
IBD. This bacterium displays several anti-inflammatory effects, in part
mediated by its product, microbial anti-inflammatory molecule (MAM),
which blocks NF-ΚΒ activation and IL-8 production [37–39]. F.
A. Ricciuto, et al.
prausnitzii is associated with a tolerogenic serum cytokine profile,
characterized by low levels of IL-12 and IFN-γ, and high IL-10 [40].
Thus far, we have described the role of the gut microbiota in
shaping local immune responses. Only recently was it shown that in-
testinal microbes influence early lymphocyte development in the
thymus as well, as supported by impaired thymic lymphocyte devel-
opment observed in GF mice. Congruent with the above discussion, B.
fragilis monocolonization restored this defect but not when a PSA-de-
ficient strain was used [41].
5. Evidence supporting the role of the gut microbiome in IBD and
inflammatory arthropathies
5.1. Evidence relating to disease risk factors
Both genetic and environmental risk factors for IBD and in-
flammatory arthritis point to the possible role of the gut microbiome in
disease pathogenesis. Many of the susceptibility loci for IBD relate to
interactions between the immune system and the microbiota.
Illustrative genes include NOD2, autophagy-related 16-like 1
(ATG16L1), caspase recruitment domain-containing protein 9 (CARD9)
and C-type lectin domain family 7 member (CLEC7A). NOD2 encodes
an intracellular PRR. Mice deficient in NOD2 have intestinal dysbiosis
and a heightened susceptibility to colitis. Furthermore, in the setting of
NOD2 deficiency, commensal microbes, like Bacteroides vulgatus, trigger
mucosal barrier disruption and inflammatory gene expression [42]. In
humans, NOD2 mutations are associated with lower IL-10, increased
mucosa-associated bacteria [43], as well as depletion of Faecalibacteria
and enrichment of Escherichia [44–47]. Many environmental factors
that have been identified as risk factors for IBD (and other IMIDs) also
invoke change in the gut microbiota. Briefly, these include exposure to
bouts of acute gastroenteritis, breastfeeding, early antibiotic use,
changes in diet (particularly high fat content), cigarette smoking and
the hygiene hypothesis more broadly [42].
5.2. Evidence relating to IBD – animal studies
Observations of attenuated colitis in GF animals and animals treated
with antibiotics offer compelling support for the involvement of the gut
microbiome in the pathogenesis of intestinal inflammation [48–50]. For
example, IL-10−/− mice (a common colitis model) do not develop co-
litis when GF. Intriguingly, the reintroduction of specific bacteria may
be beneficial (Lactobacillus plantarum 299 V) or harmful (Enterobacter
faecalis) in this model [50–52]. Seminal work from the 1990s showed
that the adoptive transfer of naïve CD4+ T cells fails to induce colitis in
RAG1−/− mice raised under GF conditions [53,54]. However, upon
activation by gut microbial antigens, transfer of CD4+ T cells into se-
vere combined immunodeficiency (SCID) mice effectively induces co-
litis [55]. In a more recent study, colonization of GF mice using fecal
samples from IBD patients led to an increased abundance of Th17 and
Th2 cells and decreased Tregs, compared to fecal samples from healthy
controls [56]. Moreover, the transfer of IBD microbial samples wor-
sened disease in a RAG1−/− T cell transfer model.
Animal models of colitis reveal enrichment of specific bacteria, such
as Bacteroides, Enterobacteriaceae, Porphyromonas, Akkermansia mu-
ciniphila and Clostridium ramosum, which are associated with increased
mucosal inflammation [48]. A relatively novel method, termed IgA-seq,
can be used to identify bacteria coated with IgA. The assumption is that
these IgA-coated bacteria have a greater capacity to stimulate in-
flammatory responses and, therefore, disease. Using this method, 35
bacterial species uniquely coated with IgA were identified in the stool
samples of IBD patients. In mice, these bacteria exacerbated DSS colitis
[57]. Interestingly, compared to CD patients without arthritis, those
with peripheral spondyloarthritis (SpA), are enriched in IgA-coated E.
coli resembling adherent and invasive E. coli (AIEC) [58]. Colonization
of GF mice with CD-SpA-derived E. coli triggers Th17 expansion.
4
Clinical Immunology 215 (2020) 108415
Transfer of these bacteria into IL-10-deficient mice and K/BxN mice (an
arthritis model) exacerbates colitis and arthritis, respectively, lending
support to the pathogenicity of these bacteria. F. prausnitzii, on the
other hand, displays a protective effect against colitis in animals; the
oral administration of F. prausnitzii (or its supernatant) mitigates TNBS-
induced colitis in mice [37].
5.3. Evidence relating to IBD – human studies
A few general observations lend support to the role of the gut mi-
crobiota in IBD pathogenesis. These include the fact that IBD pre-
dominates in gut regions of high bacterial load (terminal ileum and
colon); fecal diversion is associated with improvement of CD actvity
[59,60]; and antibiotic therapy is helpful for selected types of IBD, such
as perianal CD and pouchitis in UC [42].
Numerous studies demonstrate a state of dysbiosis in adult- and
pediatric-onset IBD. While these reports are consistent in showing dif-
ferences between IBD and control cohorts, they are otherwise hugely
heterogeneous, in term of both the methods employed and the specific
findings arising, which limits interpretability. Several such studies are
discussed below, particularly within the framework of a recent meta-
analysis of the microbiome in IBD [61]. Table 1 provides a summary of
microbiota studies in pediatric IBD (discussed below), further high-
lighting the heterogeneity of findings between studies. Contributors to
heterogeneity include sample source (fecal vs mucosal), specific
methods used to characterize the microbiota, medication exposure, as
well as patient age and geographic location of residence.
Despite these limitations, a few generalizations can be made. The
first is that IBD is associated with an overall reduction of microbial
diversity (i.e., the total number of bacterial species). Data from the
MetaHIT consortium suggest that, on average, IBD patients have 25%
fewer microbial-derived genes than are present in healthy controls
[62]. Temporal instability is another characteristic of the IBD micro-
biota. This was demonstrated in a longitudinal study, in which 128 IBD
patients were serially sampled. Microbial fluctuations were particularly
marked in individuals with ileal CD post surgical resection [63].
Secondly, IBD is associated with a loss of commensal bacteria and
enrichment of pathobionts. At a phylum level, this typically translates
into a relative loss of Firmicutes and enrichment of Proteobacteria.
Among commensal microorganisms, SCFA producers are often de-
pleted, including multiple Firmicutes, such as Clostridium cluster IV
(which includes Ruminococcaceae family members such as F. praus-
nitzii), Clostridium cluster XIVa (which includes Ruminococcus and
Lachnospiraceae family members such as Roseburia and Blautia),
Leuconostocaceae, Phascolarctobacterium, as well as certain
Bacteroidetes, such as Odoribacter splanchnius [64–66]. Both F. praus-
nitzii and Roseburia are butyrate producers and have been found to
correlate inversely with disease activity in UC [65]. Decreased abun-
dance of F. prausnitzii is also a risk factor for post-operative CD recur-
rence [37]. In addition to these bacteria, certain studies show reduc-
tions of other Firmicutes (such as Lactobacillus), Actinobacteria (such
as Bifidobacterium), Bacteroidetes (such as Bacteroides) and selected
Proteobacteria (such as Sutterella) in IBD populations [66–68], al-
though agreement is imperfect between studies. In light of the anti-
inflammatory effects of SCFAs and organisms like F. prausnitzii and
Bacteroides, elaborated above, it is enticing to hypothesize that their
loss is causally implicated in disease processes; however, it is exceed-
ingly difficult to disentangle whether dysbiotic changes represent cause
or consequence in the setting of chronic intestinal inflammation.
Pathobionts belonging to the Proteobacteria phylum are frequently
enriched in IBD, including members of the Enterobacteriaceae family,
such as E. coli, Shigella, Proteus mirabilis and Klebsiella pneumoniae. AIEC
strains have been isolated from the ileal tissue of CD patients [69].
Other Proteobacteria reported to be increased in IBD (though with some
inconsistency) include Pasteurellaceae, Veillonellaceae and Desulfovi-
brio. Desulfovibrio can reduce sulfate, generating hydrogen sulfide
 Table 1
Pediatric studies published since 2010 comparing the gut microbiota in IBD to a control group.
Author Region Patients Sample Method Depleted Enriched Diversity
Year
A. Ricciuto, et al.

CD
Aomatsu 2012 Japan 10 CD Stool 16S rRNA T-RFLP Clostridium IV (including F. prausnitzii), Clostridium Clostridium XIVa (different OTU), Lactobacillales ↓ vs HC/UC
[129] 27 HC XIVa, Bacteroides
Assa 2016 [75] Canada 10 CD TI or ICV bx V6 hypervariable region of F. prausnitzii, Oscillospira, unclassified No difference vs HC
15 HC 16S rRNA Ruminococcaceae
Ijaz 2016 [130] UK 13 + 6 CD Stool 16S rRNA V4 region and Ruminococcaceae, Lachnospiraceae, Enterobacteriaceae, Pasteurellaceae, Veillonella, Dorea, ↓ vs HC
14 HC shotgun metagenomics Phascolarctobacterium, Parabacteroides, Akkermansia, Anaerostipes, Enterococcus, Clostridium_XVIII,
Methanobrevibacter Clostridium XIVa
Kaakoush 2015 Australia 5 CD Stool V1–3 16S rRNA Number of OTUs decreased dramatically upon starting ↓ vs HC
[131] 5 HC pyrosequencing, shotgun exclusive enteral nutrition
metagenomics, Ion Proton
system
Knoll 2017 Germany 5 + 1 CD Stool Whole genome shotgun Nil significant in CD subgroup Nil significant in CD subgroup
[132] 12 HC metagenomics
Makonnen Finland 10 CD Stool 16S rRNA RT-PCR, DGGE of Bifidobacteria, Lachnospiraceae and Coriobacteriaceae; ↑ in patients with
2015 8 HC V6–8 region and culture Bacteroides and Bifidobacteria diversity lower vs HC; medium-high
[133] Ruminococcaceae diversity lower vs HC/UC activity vs HC
Quince 2015 UK 23 CD Stool 16S rRNA sequencing of V4 Coprococcus, Pseudobutyrivibrio, Ruminococcus, Unknown Ruminococcaceae, Parvimonas, Actinomyces, ↓ – decreased further
[115] 8 HC region, shotgun metagenomics Subdoligranulum, Faecalibacterium, Lachnospira, Enterococcus, Peptostreptococcus, Lactococcus, during exclusive
Clostridium, Alistipes, Parabacteroides, Anaerotruncus, Escherichia-Shigella, Eggerthella, Megasphera, enteral nutrition
Roseburia, Turicibacter, Anaerostipes, Catabacter, Prevotella, Gemella, Mogibacterium, Peptoniphilus,
Bifidobacterium, Bilophila, Slackia, unknown Abiotrophia, Streptococcus, Lachnospiraceae
Coriobacteriaceae genus, Dialister Clostridium clostridioformes, S. anginosus, Enterococcus
Lachnospiraceae – specifically E. rectale and Roseburia faecalis, Ruminococcus gnavus

5
inulinivorans
B. adolescentis, Ruminococcus bromii, Eubacterium spp.,
F. prausnitzii, Coprococcus eutactus, Subdoligranulum
variabile
Wang China 11 CD Stool 16S rRNA V3–4 region Anaerostipes, Blautia, Coprococcus, Faecalibacterium, Enterococcus ↓
2018 16 HC Lachnospira, Odoribacter, Roseburia, Ruminococcus,
[134] Sutterella
Sila 2020 Croatia 19 IBD Stool 16S rRNA gene TRFLP Clostridium genus, Firmicutes Streptococcus, Lactococcus, Enterococcus ↓ vs healthy siblings
[135] 20 HC (sib) Proteobacteria
19 HCs
Kowalska 2019 Poland 64 CD Stool 16S rRNA V3–4 region Bifidobacterium adolescentis, Roseburia (R. faecis), F. Enterococcus ↓ vs HC
[136] 18 HC prausnitzii, Gemmiger formicilis, Ruminococcus bromiii,
Veillonellaceae (Dialister), Adlercreutzia, Clostridium
elatum, Coprococcus
Kolho 2016 Finland 36 CD Stool Phylogenetic microarray Clostridium clusters IV and XIVa, F. prausnitzii Bacteroides fragilis, Clostridium ramosum, Eubacterium
[77] 8 HCs (HITChip) cylindroides

UC Depleted Enriched
Aomatsu 2012 Japan 14 UC Stool 16S rRNA TRFLP Clostridium IV, Bacteroides Lactobacillales No difference vs HC
[129] 27 HC
Knoll 2017 Germany 3 + 3 UC Stool Shotgun metagenomics Eubacterium rectale, F. prausnitzii, Bilophila wadsworthia E. coli, Clostridium ramosum, Ruminococcus gnavus, ↓
[132] 12 HC (trend) Fusobacterium nucleatum
Maukonen Finland 12 UC Stool RT-PCR, DGGE of 16S rRNA Bifidobacteria, Lachnospiraceae, Coriobacteriaceae, Bacteroides ↑ in patients with
2015 8 HC gene V6-V8 region, culture Lactobacillus medium-high
Bacteroides and Bifidobacteria diversity lower vs HC, activity vs HC
Ruminococcaceae diversity lower vs HC
(continued on next page)
Clinical Immunology 215 (2020) 108415
 Table 1 (continued)

Author Region Patients Sample Method Depleted Enriched Diversity

Year
A. Ricciuto, et al.

Michail 2012 Canada, USA, 27 ASUC Stool 16S rRNA microarray Phylum: Firmicutes (5% decrease), Lentisphaerae, Phylum: Proteobacteria (3.6% increase), Fusobacteria, ↓, especially steroid
[137] Australia, Israel 26 HC Verrucomicrobia Spirochaetes non-responders
Class: Clostridia, Lentisphaerae, Coriobacteridae, Class: Actinobacteridae, Bacilli, Erysipelotrichi,
Verrucomicrobiae Fusobacteria, Alphaproteobacteria, Betaproteobacteria,
Deltaproteobacteria, Epsilonproteobacteria,
Gammaproteobacteria, Spirochetes
Species: E. coli
Shah 2016 [76] USA 10 UC Colonic bx 16S rRNA V4-V6 region Roseburia, Akkermansia Pasteurellaceae, RF32, Phenylobacterium, Similar UC vs HC
13 HC Haemophilus, F. prausnitzii (numeric only)
Sila 2020 Croatia 19 IBD Stool 16S rRNA gene T-RFLP Clostridium genus, Firmicutes Streptococcus, Lactococcus, Enterococcus ↓ vs healthy siblings
[138] 20 HC (sib) Proteobacteria
19 HCs
Turner 2019 Israel, Canada, 28 UC Stool 16S rRNA V4 Illumina MiSeq Gammaproteobacteria ↓ vs remission
[139] USA, Europe (vs remission)
Kolho 2016 Finland 26 UC Stool Phylogenetic microarray Sutterella wadsworthensis, C. difficile
[77] 8 HCs (HITChip)

Bx – biopsy; CD – Crohn's disease; DGGE – denaturing gradient gel electrophoresis; HC – healthy control; ICV – ileocecal valve; OTU – operational taxonomic unit; RT-PCR – reverse transcription polymerase chain
reaction; T- RFLP – terminal-restriction fragment length polymorphism; TI – terminal ileum; UC – ulcerative colitis.

6
Clinical Immunology 215 (2020) 108415
A. Ricciuto, et al. Clinical Immunology 215 (2020) 108415

(H2S), which is of relevance because H2S impairs the utilization of Table 2

butyrate (a source of energy) by colonocytes. Certain Firmicutes, in- Microbiota changes reported in chronic inflammatory bowel diseases.
cluding Ruminococcus gnavus and C. difficile, Bacteroidetes, including Depleted Enriched
Prevotellaceae, and Fusobacteria are also reported to be increased in
IBD [67,68,70]. Bacteria • Firmicutes phylum • Proteobacteria
The above findings are supported by a large pediatric study con- • Clostridium cluster IV phylum

ducted by Gevers et al. which examined fecal and mucosal samples

(Ruminococcaceae like
F. prausnitzii)
• Enterobacteriaceae (E.
coli, including adherent
from 468 treatment-naïve children newly diagnosed with CD and 229
healthy controls [71]. The CD group displayed increased abundances of
• Clostridium
XIVa
cluster and invasive E. coli
(AIEC), Shigella, P.
Pasteurellaceae, Veillonellaceae, Neisseriaceae, Fusobacteriaceae and (Ruminococcaceae and mirabilis, K.
Lachnospiraceae like pneumoniae)
E. coli, and decreased abundances of Bacteroides, Clostridiales, Faeca-
Roseburia and Blautia) • R. gnavus
libacterium, Roseburia, Blautia, Ruminococcus and Lachnospiraceae. It
• Christensenellaceae • Veillonellaceae
is interesting to note that microbial alterations were more marked in • Coriobacteriaceae • Pasteurellaceae
mucosal than fecal samples, and that sample composition was more • Bacteroides • Neisseriaceae
• Bifidobacterium • Actinomyces
• •
similar between ileum and rectum than between feces and mucosa.
Sutterella Fusobacteria
A systematic review of adult and pediatric case-control studies
• Akkermansia • Desulfovibrio
published since 2010 comparing the gut microbiota between IBD and • E. rectale
control cohorts was recently conducted [61]. The most striking finding Fungi [73] • S. cerevisiae • C.C. albicans
is the lack of consistency across studies: among 34 CD and 29 UC stu- • Ascomycota diversity • M. parapsilosis
• Basidiomycota
restricta
dies, no microbial changes were seen in more than three studies.
• Caudovirales diversity
Moreover, only four bacterial groups were consistently altered in three
studies. Small sample sizes and the sources of heterogeneity outlined
Viruses/Bacteriophages •
above were common limitations. Despite this lack of reproducibility,
the authors drew a few conclusions based on more consistent ob- rectale, uncultured Clostridiales and Vibrio species, and a lower abun-
servations. In CD, all three studies examining Christensenellaceae and dance of S. mitis were associated with a greater likelihood of response to
Coriobacteriaceae and six of 11 studies examining F. prausnitzii found a treatment with an anti-tumor necrosis factor-α (TNF-α) monoclonal
decrease, while two studies each of Actinomyces, Veillonella and E. coli antibody [77]. In a more recent study, disruptions of an association
found an increase in these microorganisms. In UC, Eubacterium rectale network including Lachnospiraceae and Ruminococcaceae (SCFA pro-
and Akkermansia were reduced in all three studies, and E. coli pro- ducers) were associated with frequently relapsing disease, a poor clin-
portions were increased in four of nine studies. Diversity was either ical response to anti-TNF-α and a higher risk of CD recurrence after
lower or not different in IBD patients compared to controls. Chris- surgical resection [78].
tensenellaceae and Eubacterium are Firmicutes and butyrate producers. While the focus to date has been on taxonomic classification, a far
Coriobacteriaceae, which belong to the Actinobacteria phylum, include more salient question to pose than “who is there” is “what are they
species such as Collinsella, Eggerthella, Slackia and Atopobium, of doing?” In other words, the functional repertoire of a microbial com-
which some have the ability to transform bile acids. This is pertinent as munity bears more relevance to host health than its composition. Multi-
bile acid metabolites influence both Th17 and Treg cell differentiation omics methods (i.e., metagenomics, metatranscriptomics, metabo-
[72]. lomics, metaproteomics) represent powerful tools to interrogate mi-
It is important to recall that bacteria are not the sole non-mam- crobial function. Using a multi-omics approach, Lloyd-Price and col-
malian inhabitants of the human gut. Fungi (the “mycobiome”) and leagues examined 132 pediatric and adult IBD patients serially over one
viruses (the “virome”) are present as well. Exploration of these elements year [79]. Molecular disruptions of transcription, among Clostridia for
of the human gut microbiota is still in its infancy, but data supporting example, and metabolite pools (SCFAs, bile acids and acylcarnitines)
dysbiosis at these levels are emerging. Studies support overgrowth of C. were apparent. Although the IBD group generally displayed decreased
albicans and C. parapsilosis, and decreased abundance of S. cerevisiae in microbiome stability over time, the relative abundances of Prevotella
IBD [73]. There is also enrichment of Caudovirales bacteriophages and copri remained stable in IBD patients. This is of interest given the link
loss of viral-bacterial correlations in the mucosa of UC patients [74]. between P. copri and inflammatory arthritis, particularly rheumatoid
Bringing together the above findings, Table 2 summarizes some of arthritis (RA) (considered below).
the more consistent dysbiotic changes observed in IBD. Fig. 2 illustrates
the disruption of microbial composition and related aberrant immune
responses in the setting of IBD. 5.4. Evidence relating to inflammatory arthropathies – animal studies
The discussion thus far has collapsed adult and pediatric studies, but
the pediatric population presents several important advantages, in- As is the case for colitis, support for the role of the microbiota in the
cluding temporal proximity to disease origin and less frequent co- pathogenesis of inflammatory arthritis derives from multiple observa-
morbidities, all of which minimize confounding. Table 1 presents a tions of absent arthritis in GF animal models. Examples include the IL-1
summary of the pediatric studies in the systematic review discussed, as receptor antagonist knockout mouse (IL1rn−/−), K/BxN T cell receptor
well as selected subsequent pediatric publications. Although most transgenic mouse, ANKylosing ENThesopathy (ANKENT) mouse, HLA-
findings are aligned with those presented above, it is interesting to note B27 transgenic rat and SKG mouse (point mutation in ZAP-70, a T cell
that the only two studies to demonstrate a numeric increase in F. transduction molecule), all of which develop arthritis under normal
prausnitzii abundance (rather than a decrease) were performed in chil- circumstances, but fail to do so when raised under GF conditions. In all
dren (though one was not statistically significant) [75,76]. Similarly, cases, inflammatory arthritis ensues upon reintroduction of specific
although most studies support a decrease in Bacteroides, two pediatric microbes [80–83]. The IL1rn−/− GF mouse displays dysbiosis char-
studies showed a decrease. Such findings raise the possibility of age- acterized by decreased richness and diversity, and depletion of Rumi-
dependent changes in the gut microbiota. nococcus and Prevotella. K/BxN T cell receptor transgenic mice develop
The potential utility of the gut microbiota as a prognostic and/or inflammatory arthritis due to T cell recognition of a self-antigen. Re-
predictive biomarker of disease course and treatment response is of colonization of the GF K/BxN T cell receptor transgenic mouse with SFB
great interest. In a study including 68 children with IBD, higher base- results in arthritis and reinstatement of Th17 cells in the gut lamina
line abundances of Bifidobacterium, Clostridium colinum, Eubacterium propria. The latter 3 models develop arthritis resembling SpA. In

7
A. Ricciuto, et al. Clinical Immunology 215 (2020) 108415

Fig. 2. Genetic, environmental and immune-mediated microbiome interactions in the pathogenesis of chronic inflammatory bowel diseases (IBD).
In health, the gut microbiota promotes homeostasis and anti-inflammatory processes. In the setting of appropriate genetic and environmental risk factors, dysbiotic
changes occur in IBD and contribute to aberrant immune responses by the innate and adaptive immune system. Reprinted from J Allergy Clin Immunol, 145(1), Glassner
et al., The microbiome and inflammatory bowel disease, 16–27, Copyright (2020), with permission from Elsevier.

humans, SpAs predominantly include ankylosing spondylitis (AS) and
psoriatic arthritis (PsA). SpAs are characterized by axial and peripheral
asymmetric joint involvement, enthesitis and dactylitis.
Interestingly, gut inflammation occurs frequently with SpA, with
roughly 10–15% of SpA patients developing IBD. Conversely, up to 30%
of IBD patients manifest signs of inflammatory arthritis [84]. Even in
the absence of gastrointestinal symptoms, approximately half of SpA
patients have microscopic intestinal inflammation [85]. HLA-B27
transgenic rats exhibit a higher abundance of Paraprevotella and lower
Rikenellaceae [86]. The dysbiosis appears to be age-dependent, with a
decrease in Firmicutes and increase in Proteobacteria and Akkermansia
muciniphila, as well as IgA-coated bacteria, with age. These microbial
changes are associated with increased expression of Th1 and Th17
proinflammatory cytokines, colonic Th17 expansion, dysregulated AMP
production and upregulation of bacterial-specific IgA [87]. Propionate
administration improves the arthritis in this model [88]. SKG mice re-
main arthritis-free when treated with antifungals and develop arthritis
upon exposure to fungal elements, findings that highlight the im-
portance of the mycobiome [89]. Transferability of phenotype with
microbial transplantation was demonstrated in this model: instillation
of feces from RA patients (characterized by increased P. copri) into
BALB/c ZAP-70 mutant mice provokes severe arthritis in a Th17-
dependent manner [90].
Chronic recurrent multifocal osteomyelitis (CRMO) is an autoin-
flammatory disorder characterized by bone lesions and fever. CRMO is
associated with pediatric IBD [91]. Mice homozygous for a mutation in
proline-serine-threonine phosphatase interacting protein 2 (Pstpip2),
termed Pstpip2cmo mice, develop similar manifestations [92]. Experi-
ments in these mice highlight the potential role of the microbiota in
CRMO. The critical cytokine for promoting disease in Pstpip2cmo mice is
IL-1β. PSTPIP2 appears to negatively regulate IL-1β secretion in a ca-
pase-1- and NLRP3 inflammasome-independent fashion and neu-
trophils, rather than macrophages, are the key innate immune cells
implicated [93]. Interestingly, Pstpip2cmo mice fed a high fat diet (HFD)
are completely protected from osteomyelitis; this protection occurs in
conjunction with a microbial shift toward a greater ratio of Lactoba-
cillus to Prevotella. Moreover, fecal transplantation from protected HFD
mice to pre-disease, normal chow Pstpip2cmo mice is associated with
decreased Prevotella and IL-1β production, and is protective against
clinical disease. Further support for the impact of the microbiota on IL-
1β derives from the finding that CD45+ cells from the colons of GF
mice express significantly lower levels of Il1b transcript than specific
pathogen-free mice [94]. Collectively, these observations offer the
possibility that microbe-altering interventions could well have

8
A. Ricciuto, et al.
therapeutic implications for autoinflammatory disorders, like CRMO
[95].5.1.5 Evidence relating to inflammatory arthritis – Human studies.
It is challenging to reconcile microbiota findings in systemic in-
flammatory arthropathies as this umbrella captures several distinct
entities. Nevertheless, the following includes a few notable observations
from adult studies. Firstly, compared to controls, Prevotella copri is
present at higher abundance in new-onset RA, with a concomitant de-
crease in Bacteroides [96]. More recently, Zhou et al. also demonstrated
increased P. copri in an AS cohort [97]. In this latter study, P. copri
abundance fell with treatment and more significantly so in patients
receiving anti-TNF-α or disease-modifying anti-rheumatic drugs, rather
than non-steroidal anti-inflammatory agents only. In a study of RA and
SpA, the most striking finding was a 2- to 3-fold increase in the abun-
dance of R. gnavus (a mucin degrader) in SpA compared to both healthy
controls and RA patients [98]. Furthermore, R. gnavus correlated with
SpA activity in patients with a history of IBD. A recent study by
Klingberg et al. also demonstrated increased R. gnavus in AS patients
[99]. In this study, patients with elevated fecal calprotectin, a bio-
marker of intestinal inflammation, had a lower abundance of bacteria
with anti-inflammatory effects, like F. prausnitzii and Clostridium, but
higher levels of Streptococcus.
Proteobacteria are often enriched in patients with inflammatory
arthropathies, including AS (E. coli and Shigella spp. increased) [99]
and RA populations [98]. Akkermansia muciniphila, another mucin-de-
grading bacterium, is frequently decreased; it was almost absent in a
PsA cohort [100], and present at lower abundances than controls in the
AS study by Zhou [97]. It should be noted, however, that not all studies
have consistently supported this finding [99]. Reports have also been
conflicting about Bacteroides, with many showing depletion
[96,99,101,102], but others enrichment [97,103]. In fact, in one study,
Bacteroides coprophilus was the AS-enriched bacterium with the stron-
gest ability to identify AS (from controls) [97]. Zhou et al. used shotgun
metagenomics to investigate microbial function in AS; investigators
identified three major pathways in the AS group, including oxidative
phosphorylation, LPS biosynthesis and glycosaminoglycan degradation.
Notably, the metabolism pathway of butanoate, a SCFA, was decreased
in the AS group and there was a trend toward a decrease in propionate
metabolism as well.
The potential roles of many of these microorganisms in perpetuating
or regulating inflammation are considered in the IBD section. In that
vein, it is interesting to note several similarities between the dysbiotic
changes described in IBD and arthritis populations, such as decreased F.
prausnitzii, and increased Proteobacteria and R. gnavus. There are also
data suggesting that P. copri strains can contribute to human disease by
inciting chronic inflammation. Prevotella strains primarily act via TLR2
to stimulate APCs to produce Th17-polarizing cytokines. In addition,
Prevotella strains stimulate ECs to produce IL-8, IL-6 and the chemo-
kine CCL20, which can trigger Th17 immune responses and neutrophil
recruitment [104]. Moreover, two RA-specific autoantigens that share
sequence homology with P. copri are highly expressed in inflamed RA
synovium [105].
Specific consideration of pediatric studies is worthwhile here as
well. JIA includes 7 subtypes: 1) systemic-onset; 2) persistent or ex-
tended oligoarthritis (≤4 joints); 3) Rheumatoid Factor (RF)-negative
polyarthritis (≥5 joints); 4) RF-positive polyarthritis; 5) psoriatic; 6)
enthesitis-related arthritis (ERA); and 7) undifferentiated. ERA re-
sembles adult SpA and RF-positive polyarthritis resembles RA. A very
recent review indicates that, to date, there have been 7 studies on the
gut microbiota in JIA: 3 focus on children with oligo- or polyarticular
JIA; 3 focus on ERA; and 1 includes both oligo−/polyarticular JIA and
ERA [106]. Table 3 summarizes the findings of these studies.
As with IBD, despite substantial heterogeneity between JIA studies,
a few general themes have emerged. Firstly, a few microorganisms are
fairly consistently altered in JIA. These include a decrease in F. praus-
nitzii, in line with reduced microbial genetic potential for butyrate
production [107]; an increase in Ruminococcaceae, which may
9
Clinical Immunology 215 (2020) 108415
correspond to R. gnavus, as reported in IBD and adult inflammatory
arthritis; and an increase in Bacteroides. The second theme is that ob-
servations in adults cannot necessarily be extrapolated to children. In
other words, there may be an age-dependent effect for certain micro-
organisms, as already raised in 5.1.5. The surprisingly consistent in-
crease in Bacteroides in the JIA studies contrasts with the majority of
adult arthritis studies, which favour depletion, although, as already
acknowledged, this has not been a universal finding across all studies.
Interestingly, one JIA study reported an increase in F. prausnitzii, rather
than a decrease, invoking the two pediatric IBD studies cited above that
similarly documented numerically higher F. prausnitzii abundances in
IBD patients than healthy controls. The authors of the JIA review also
highlight the finding of increased Akkermansia in the arthritis cohort of
the 2014 study by Stoll et al., which again departs from the pre-
ponderance of the evidence in adult cohorts. In this case, however,
pediatric IBD studies are typically aligned with adult arthritis (and
adult IBD) studies in suggesting depletion of Akkermansia. It is unclear
whether this reduction in IBD is cause or consequence, the latter po-
tentially due to IBD-related depletion of goblet cell-derived mucins,
which are the energy substrate for Akkermansia. Interestingly, there are
animal data to support that Akkermansia muciniphila may be deleterious
in arthritis models, but beneficial in colitis models; this microorganism
was recently shown to be permissive to arthritis in the K/BxN mouse
[108],and to augment bacterial invasiveness [109], while ameliorating
DSS colitis in mice [110].
6. Microbe-altering therapies
6.1. Nutritional interventions
Numerous potential therapies exist to modulate the gut microbiota.
These include nutritional interventions, pre- and probiotics, oral anti-
biotics, as well as fecal microbial transplantation (FMT). Exclusive
enteral nutrition (EEN), the elimination of table food and substitution
with a polymeric or elemental formula for a several-week period, is an
effective induction treatment for pediatric CD [111]. Notably, there is a
case report and a case series suggesting a similar beneficial effect of
EEN in JIA [112,113]. Seemingly paradoxically, fecal microbial di-
versity appears to decline further with EEN, including a decrease in
organisms already depleted at baseline and thought to have anti-in-
flammatory properties, such as Faecalibacterium [114,115]. Such per-
plexing findings underscore our incomplete understanding of the
complex microbial processes at play in these chronic inflammatory
disorders.
6.2. FMT – animal studies
FMT, defined as the restoration of physiologic microbial community
by means of transplanting microbiota via feces, is an exciting emerging
therapy. FMT has demonstrated efficacy for recurrent C. difficile infec-
tion [116]. There are 4 randomized controlled trials (RCTs) and nu-
merous case series in IBD, which support transient benefit [117–120].
There are no studies of FMT for inflammatory arthritis.
Animal experiments offer important insight into the immunologic
mechanisms mediating the effects of FMT for treating colitis. In one
study, the gavage of mucus and feces from healthy mice into mice with
DSS colitis improved the colitis and was associated with an increase in
IL-10 levels, the number of IL-10-producing CD4+ T cells and invariant
NK (iNK) T cells, and a decrease in macrophages, neutrophils, ILC2s,
ILC3s and MHC-II-expressing APCs in the lamina propria. There was
also a non-significant increase in FoxP3+ Tregs and upregulation of
AMPs. The increased IL-10 levels normalized when the inflammation
resolved and IL-10 receptor blockade abrogated the effects, suggesting
that the benefit of FMT is IL-10-dependent [121]. Lamina propria
mononuclear cells (LPMCs) exposed to FMT-derived microbiota gen-
erated less TNF-α, IL-1β and IFN-γ in this study, and FMT was
A. Ricciuto, et al. Clinical Immunology 215 (2020) 108415

Table 3
Studies of the microbiota in juvenile idiopathic arthritis; adapted from [106].
Author Region Patients Treatment Method Depleted Enriched
Year

Stoll USA 25 ERA DMARDs Amp of V4 rRNA Faecalibacterium prausnitzii Bacteroides

2014 [140] 13 HC Illumina HiSeq, Lachnospiraceae Akkermansia
HiSeq
Large and statistically significant decreased abundance of Faecalibacterium prausnitzii (3.8% vs 10%), smaller decrease in unspecified Lachnospiraceae (7% vs 12%); 2 distinct clusters
on PCoA, one driven by Akkermansia muciniphila and the other by Bacteroides
Tejesvi Finland 13 oJIA, 15 RF- pJIA, 1 RF+, Treatment-naïve Amp of V4-V5 rRNA Firmicutes Bacteroides
2016 [141] pJIA, 1 ERA Ion Torrent
27 HC
At phylum level, relative depletion of Firmicutes and enrichment of Bacteroidetes; at genus level, increased Bacteroides (44% vs 33%)
Aggarwal India 33 ERA DMARD-naïve x Amp of V3 rRNA Prevotellaceae (trend) Bacteroides
2017 [142] 14 HC 6 weeks Illumina HiScan SQ Klebsiella
Strong depletion of Prevotellaceae family (Bacteroidetes phylum) (9% vs 66%); increased abundance of Bacteroides genus (3% vs 0.06%) and Klebsiella genus (82% vs 43%)
Di Paola Italy 10 pJIA, 19 ERA DMARDs V5-V6 Peptostreptococcaceae (ERA) Ruminococcaceae (both)
2016 [143] 29 HC Roche 454 Faecalibacterium (non-ERA) Veillonellaceae (ERA)
Decreased Peptostreptococcaceae (0.3% vs 1.1%); small, non-significant decrease in Faecalibacterium genus (0.2% vs 0.35%); increase in Veillonellaceae (1.4% vs 0.4%); both ERA and
non-ERA JIA showed increased Ruminococcaceae (22% ERA, 27% non-ERA vs 12%)
Muller Netherlands 8 RF- pJIA Treatment -naïve Phyla-specific Alistipes finegoldii Bacteroides fragilis
2017 [144] 22 HC primers; Sanger Prevotella multisacharivorax
PCoA revealed substantial differences in the composition of the Bacteroidetes phyla; at genus level, JIA patients had unspecified increased fecal B. fragilis and decreased Alistipes
finegoldii and Prevotella multisacharivorax
Stoll USA 24 ERA Treatment -naïve Amp of V4 rRNA; F. prausnitzii A1–165 Bacteroides fragilis
2018 [107] 19 HC WGS
Illumina MiSeq
Overall no difference in F. prausnitzii but regulatory strain decreased in ERA at expense of increased non-regulator L2/6 strain; increased B. fragilis (2% vs 0.45%); whole genome
sequencing showed decreased carriage of a pathway resulting in butyrate production
Dijkhuizen Italy, Netherlands Italy: Treatment -naïve V1-V3 Allobaculum Erysipelotrichaceae
2019 [145] 47 oJIA, 24 RF- pJIA, 2 RF+ Roche 454 Gemellaceae F. prausnitzii
pJIA, 3 psJIA, 2 U-JIA Propionibacterium acne Parabacteroides
79 HC Turicibacter Enterococcus
Netherlands: Ruminococcaceae
14 oJIA, 4 RF- pJIA, 2 ERA, 1
psJIA
28 HC

Substantial difference in age between patients and controls in Italian cohort (8 vs 3.9 y); random forest methods in Italian group separated patients and HC with near
100% accuracy (bacteria driving this were Erysipelotrichaceae, F. prausnitzii, Parabacteroides, Enterococcus and Ruminococcaceae (increased in JIA) and
Allobaculum, Gemellaceae, Propionibacterium acnes and Turicibacter (increased in HC); in Dutch cohort, random forest also successfully separated groups but no
single organism differed significantly between groups.
Amp – amplification; ATB – antibiotics; DMARD – disease-modifying anti-rheumatic drug; ERA – enthesitis-related arthritis; HC – healthy control; JIA – juvenile
idiopathic arthritis; o – oligoarticular; p – polyarticular; PCoA – principal coordinate analysis; ps – psoriatic; RF – rheumatoid factor.

associated with an increase in bacterial commensals, including SCFA-
producing taxa, such as Ruminococcaceae and Erysipelotrichaceae. Si-
milarly, in another DSS mouse colitis study, FMT led to improvement of
mucosal inflammation, along with recovery of Bifidobacterium and
Lactobacillus, as well as an increase in tryptophan [122]. There was
also an increase in the expression of IL-10, TGF-β and the ARh receptor,
for which tryptophan metabolites are ligands, as discussed in 4.1.2.
microorganisms (such as those belonging to Clostridium clusters IV and
XIVa), which induce immunoregulatory Tregs and IL-10 [119,127,128].
In keeping with this finding, in the RCT by Moayyedi et al., the stool of
the donor associated with the highest response rate was significantly
enriched in Lachnospiraceae and Ruminococcaceae [120].
7. Conclusions

6.3. FMT – human studies
Only limited human data exploring the immunologic consequences
of FMT are available. In a randomized controlled trial, peripheral blood
mononuclear cells (PBMCs) showed a slight increase in gut-homing
CD4+ T cells following FMT [117]. Two smaller studies support an
increase in colonic mucosal Tregs after a single FMT infusion, and a
decrease in Th1 cells [123,124]. In a pivotal study of 20 UC patients
treated with a single FMT by colonoscopic infusion, a positive corre-
lation was seen between CD4+ T cell production of IFN-γ from rectal
biopsies and the relative abundance of total gut virome [125]. Speci-
fically, the bacteriophage Caudovirales, known to be increased in IBD
compared to healthy controls, was significantly enriched in FMT non-
responders.
Generally, successful FMT is associated with increased alpha di-
versity or richness and a shift in the recipient microbiota toward that of
the donor [126]. The gut microbiota in FMT responders also con-
sistently demonstrates a shift toward butyrate-producing
The gut microbiota is intimately tied to human health and is central
to maintaining immunologic homeostasis. Evidence supporting an as-
sociation between perturbations in the gut microbiome and various
human diseases, including IBD and JIA, continues to rapidly accrue.
Despite substantial progress in this field, the specific underlying me-
chanisms at play (and, at an even more fundamental level, whether
perturbations represent cause or consequence) continue to elude in-
vestigators.
Numerous unknowns remain to be elucidated. Whether local ma-
nipulation of the gut microbiota has the ability to affect and treat sys-
temic autoimmune disorders (e.g., the use of enteral nutrition or FMT
for systemic diseases like JIA) remains to be established. Whether there
is a “window of opportunity” relative to disease onset beyond which
such interventions are futile is also unclear. The focus of investigations
thus far has been on bacterial inhabitants of the gut; future work must
delineate whether other microbiota constituents (fungi, viruses) are
important mediators of gut health and determine their role(s) in various
human diseases. Carefully conducted clinical trials with large enough

10
A. Ricciuto, et al.
sample sizes to discern statistical power are needed to more accurately
determine the efficacy of microbe-altering therapeutic interventions.
Such studies must include longer follow-up duration to carefully assess
for potential adverse effects of microbial manipulation, particularly at
early life stages. As yet, the long-term safety implications of interven-
tions like FMT are unknown. Heterogeneity between studies has been a
significant impediment to progress; accordingly, meticulous attention
and great effort should now be dedicated to standardizing methods.
Finally, the focus must shift from identification to functional character-
ization; this will move the field from a cataloguing of which microbes
are present in the gut, to a careful dissection of what functions various
constituents of the intestinal microbiota are playing across the age
continuum, in both health and various chronic diseases, including IBD
and arthropathies. Only the latter approach will allow us to unravel the
intricate ties between the gut microbiota and human disease.
Funding sources
This review did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.
Acknowledgments
PMS is the recipient of a Canada Research Chair in Gastrointestinal
Disease.
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