Review

The Cross-Talk between Microbiome and Metabolome in

Rheumatoid Arthritis
Lidia La Barbera 1,† , Chiara Rizzo 1,† , Giulia Grasso 1 , Federica Macaluso 2 , Federica Camarda 1 ,
Francesco Ciccia 3 and Giuliana Guggino 1, *

1 Rheumatology Section, Department of Health Promotion, Mother and Child Care, Internal Medicine and
Medical Specialties, University of Palermo, Piazza delle Cliniche 2, 90110 Palermo, Italy
2 Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia,
AUSL-IRCCS, Via Giovanni Amendola, 2, 42122 Reggio Emilia, Italy
3 Division of Rheumatology, Department of Precision Medicine, University of Campania Luigi Vanvitelli, S.
Andrea delle Dame—Via L. De Crecchio 7, 0138 Naples, Italy
* Correspondence: giuliana.guggino@unipa.it; Tel.: +39-091-655-2148
† These authors contributed equally to this work.

Abstract: Modern “omics” sciences, including metabolomics and microbiomics, are currently being
applied to inflammatory autoimmune diseases, such as rheumatoid arthritis (RA), to investigate the
interplay between microbiota, metabolic function, and the immune system. In recent decades, robust
evidence has suggested that disruption of the normal composition of the microbiome, known as
dysbiosis, in the gut and mouth of RA patients contributes to immune dysregulation and alterations
in the metabolic pathways, shaping the pathogenesis of the disease and playing a central role in
the risk and progression of RA. Metabolic pathways can be influenced by various agents such as
the surrounding environment, lifestyle, and exposure to microbiota imbalance. In turn, the body’s
metabolic homeostasis influences the immune response, making metabolomics helpful not only to
understand pathogenesis pathways, but also to improve early disease detection and therapeutic
chances. Combined gut microbiome and metabolome studies set out to unravel the interactions
between these two entities, providing insights to discover new treatment targets and potential
biomarkers to prevent joint damage. The purpose of this review is to summarize the main recent
Citation: La Barbera, L.; Rizzo, C.; findings that suggest promising new research directions for the pathogenesis of RA.
Grasso, G.; Macaluso, F.; Camarda, F.;
Ciccia, F.; Guggino, G. The Cross-Talk
Keywords: microbiome; dysbiosis; inflammation; metabolome; rheumatoid arthritis
between Microbiome and
Metabolome in Rheumatoid Arthritis.
BioChem 2023, 3, 47–60. https://
doi.org/10.3390/biochem3010004
1. Introduction
Academic Editors: Buyong Ma and
Rheumatoid arthritis (RA) is a systemic autoimmune disease, clinically characterized
Yehia Mechref
by poly-articular involvement with chronic synovial inflammation culminating in bone
Received: 31 January 2023 erosions and disability [1].
Revised: 26 February 2023 RA is considered a multistep disease [2]. In the preclinical phase of the disease,
Accepted: 6 March 2023 genetic predisposition and environmental triggers increase susceptibility to the onset
Published: 13 March 2023 of RA, resulting in the activation of the immune system outside the joint compartment,
mainly in the mucosal surfaces. Despite the typical joint involvement in established
RA, the pathogenesis of the disease likely begins far from joint structures, in the lungs,
periodontium, or intestine [3]. In a second phase, the immune response is amplified
Copyright: © 2023 by the authors.
systemically, with an expansion of the autoantibody repertoire. Finally, the synovial
Licensee MDPI, Basel, Switzerland.
compartment becomes the target of a local inflammatory response that culminates in
This article is an open access article
clinically evident synovitis [4].
distributed under the terms and
conditions of the Creative Commons
Growing evidence has proposed gut microbiome dysbiosis as a trigger environmental
Attribution (CC BY) license (https://
factor for the dysregulation of innate and adaptive immune responses and the onset of RA.
creativecommons.org/licenses/by/
The suggested mechanism for the gut–joint inflammatory axis is related to intestinal
4.0/). leakage, which can expose the immune system to microorganisms and their products,

BioChem 2023, 3, 47–60. https://doi.org/10.3390/biochem3010004 https://www.mdpi.com/journal/biochem

BioChem 2023, 3 48

triggering a systemic immune response resulting in a local inflammatory process within

the joints.
In the RA scenario, the loss of immunological tolerance and disease development
are related events. The immunological mechanisms that lead to cell differentiation and
expansion both depend on antigen recognition and activation of metabolic pathways, which
provide cellular energy supply [5].
Early stages of the disease involve innate immune cells, such as monocytes and
dendritic cells (DCs), that orchestrate the inflammatory cascade, which subsequently leads
to the activation of lymphocytes and autoantibodies’ production [6]. Their activation
is stimulated by Toll-like receptors (TLR) that bind antigens, driving and supporting
pathogenetic mechanisms through their role as antigen-presenting cells, phagocytes, and
chemokine producers [7].
In particular, macrophages are abundant in RA synovium and represent the major
source of lytic enzymes, chemokines, and cytokines, leading to joint damage and neoangio-
genesis. In addition, they exhibit a long-lasting immune memory capable of remodulating
when stimulated [8]. Naive T lymphocytes in the RA inflammatory milieu age quickly, and
drive cellular metabolic reprogramming [5] and macrophage activation.
Similarly, chronic inflammation in RA appears to be related to metabolic alterations, as
confirmed by the finding of some metabolites linked to rapid disease progression, although
they may precede the onset of symptoms by many years.
Combined studies on immune cells, gut microbiome, and metabolome aim to investi-
gate the interactions between these entities, providing new insights for the improvement of
therapeutic strategies and for the identification of potential biomarkers in order to provide
new tools to interfere with the disruption of the immunological homeostasis that drives
RA, in order to prevent disability and joint damage.
Nowadays, the identification of a specific biomarker or a panel of molecules to profile
RA patients, especially in the initial stage of disease or in the case of seronegative disease,
is a need that has still not been met in rheumatology. The lag time between symptom onset
and diagnosis is up to 9–17 months [9], meaning that patients may experience the risk
of losing the so-called ‘window of opportunity’ for treatment initiation that lasts about
3 months after first symptoms and has been identified as the optimum time to achieve
remission [10].
In this regard, the development of sequencing technologies and multi “omics” method-
ologies may allow a better characterization of RA to help risk prediction, early diagnosis,
prognosis, and proper monitoring.
Over the last decade, thanks to metabolomics, a deeper description of the metabolic
profile, at both serum and tissue level, of RA has emerged. The metabolic fingerprinting
in such a disease is a valuable tool to address specific questions regarding the link be-
tween dysbiosis, bacterial metabolites, and disease development in order to acquire more
knowledge on RA pathogenesis and management [11].
Therefore, the purpose of the present review is to dissect the current literature regard-
ing the interaction between the microbiome, and specifically gut dysbiosis, and RA through
the information derived from metabolomic studies, which stand out as one of the most
promising high-precision techniques in rheumatology.

Research Methodology
A literature search was performed with the aim of achieving a comprehensive and
structured analysis of studies. We searched the major databases (MEDLINE, EMBASE,
ScienceDirect, PubMed, Scopus) to identify relevant articles for inclusion. Two reviewers
independently screened the literature search results and abstracted data from included
studies. A systematic search was performed using keywords related to the diseases of
interest (RA) and the main topics (microbiome, dysbiosis, and metabolome). The reference
lists of all included articles and recent reviews were evaluated for their relevance to disease
pathogenesis and their impact on outcome prediction and disease management.
BioChem 2023, 3 49

2. Metabolome in RA
Metabolomics is an emerging science that is part of the omics group (e.g., proteomic,
transcriptomics, etc.). It allows the identification of small molecules, known as metabolites,
in a biologic system, catching the alteration of the metabolic status of different tissues and
fluids that mirror the cellular perturbation occurring during disease.
Metabolic pathways can be influenced by various agents such as the surrounding
environment, lifestyle, and exposure to microbiota imbalance. In this regard, links between
diet-related metabolites and changes in the microbiome have been extensively investi-
gated [12]. In turn, the body’s metabolic homeostasis influences the immune response [13],
making metabolomics helpful not only to understand pathogenesis pathways, but also to
improve early disease detection and therapeutic chances [13].
Nowadays, in the “omics era”, numerous metabolomics assays of peripheral blood,
tissues, and urine in patients with RA have been conducted, and many techniques have
been employed to characterize metabolomics. Metabolomics is based on two main high-
throughput technologies: nuclear magnetic resonance (NMR) and mass spectrometry (MS).
After the acquisition of experimental data, multivariate statistical analysis is performed in
order to identify the patient’s metabolic profile. MS is more sensitive, but requires sample
pre-treatment through separation of individual constituents using liquid chromatography
or gas chromatography; this results in greater variability and lower reproducibility. In
contrast, NMR does not require sample pre-treatment, yielding more reproducible NMR
despite lower sensitivity. In both cases, a plot is obtained that identifies the different
metabolites representing the metabolome of a specific sample [14].
Metabolomics has been applied in various fields of medicine, as it has enabled the
identification of new possible biomarkers through new methods. This justifies the growing
interest in its application in the study of rheumatic diseases.
A recent study demonstrated a correlation between inflammation in the early stages of
immune diseases, as detected using C reactive protein (CRP) levels, and the serum/urinary
metabolome. The most abundant metabolites present in the samples included glucose,
amino acids, lactate, and citrate [15]. In 2013, Young et al. showed a relationship between
the early inflammatory stages of RA and the levels of specific metabolites such as low-
density lipoproteins, lipids, lactate, glucose, methylguanidine, amino acids, and their
derivatives [16]. Nevertheless, inflammation has been related to metabolites derived from
oxidative stress processes, the urea cycle, and catabolism protein [17], raising the profile of
the urinary metabolome, which, so far, has been studied mainly to evaluate and predict
pharmacological response [18] and to accelerate the diagnostic process [19].
However, no differences in metabolites were found between RA patients in the early
stage and advanced stage, or between the seropositive and seronegative forms.
Interestingly, a negative correlation was identified between the levels of CRP and
citrate, which can be explained by the metabolic reprogramming process resulting from the
activation of the immune response [20]. Once innate immune cells such as macrophages
and DCs are activated, the glycolysis and pentose phosphate pathways are upregulated,
while the citrate pathway, oxidative phosphorylation, and fat acid oxidation are reduced.
However, the increase in glycolytic flux can lead to an accumulation of citrate and succinate
in the mitochondria, which, once transported to the cytosol, is broken down to form
substances necessary for the activation of macrophages and DCs [21].
Conversely, a positive correlation between CRP levels and succinate was highlighted.
This finding is consistent with the ability of succinate to stimulate IL-1ß production during
the inflammatory process and, furthermore, post-translational processes such as succinyla-
tion are able to sustain inflammation [22].
Despite the positive association with glucose and lactate, serum CRP levels correlated
negatively with pyruvate levels. Several mechanisms could justify elevated glucose levels
during an inflammatory response in order to satisfy the increased cellular demand [23].
Mainly, the activation of glycolysis and the reduction of pyruvate to lactate are both
pathways required for the perpetuation of the immune response [24].
BioChem 2023, 3 50

Since the establishment of the inflammatory response requires adequate bioavailability

of metabolites to meet the cellular demand necessary for clonal expansion and cytokine
production, some metabolites are, therefore, considered potential biomarkers that can
predict the onset of the disease and, simultaneously, potential therapeutic targets [5].
However, it should be considered that the major cell lines adapt to different metabolic
conditions: T cells reduce glycolysis and prefer anabolic reactions, while macrophages, on
the other hand, store glucose in favor of cytokine production. The reason why major cell
lines rely on different pathways could be related to the reprogramming of immune cells,
influenced by the inflammatory environment.
The different metabolic adaptations mirror the main roles of the involved cell lines.
T cells mainly perform a regulatory and memory function. Macrophages predominantly
act as effector cells [25]. Anabolic reactions seem to be the exclusive prerogative of T
lymphocytes, in particular of naive T lymphocytes and memory cells, while macrophages
have developed an adaptation in a hypermetabolic sense and this imprinting is already
present in undifferentiated monocytes [5].
Metabolic signature can predict arthritis onset in its early stages and can influence
autoantibodies’ production, also according to genetic susceptibility.
Studies of genome-wide association (GWAS) highlighted the presence of many loci
associated with metabolite levels, which are involved in various metabolic pathways,
indicating the genetic influence on the metabolome. These pathways include amino acids,
lipid metabolism, carnitines, fatty acid metabolism, intermediates of purine and pyrimidine
metabolism, glucose homeostasis, vitamin, and cofactor levels; the genetic polymorphisms
of genes linked to these metabolic pathways in RA have been described [26,27].
Specifically, GWAS studies have identified approximately 100 genetic loci implicated
in the etiopathogenesis of RA [28].

3. Microbiome in RA
Beyond the genetic susceptibility, which is also strongly related to the presence of
human leukocyte antigen (HLA)-DRB1 shared epitope (HLA-SE) alleles, great interest has
developed around other environmental factors that may trigger the autoimmune process
in RA (Figure 1) [29]. The underlying inflammatory and autoimmune mechanisms are
triggered approximately one decade before RA clinical onset, resulting in the detection of
anti-citrullinated protein antibodies (ACPA) at different sites during the pre-clinical phase
of the disease [30].
Numerous studies on microbiota composition and on the different expression of its
metabolites have been conducted, highlighting a correlation between RA etiopathogenesis
and microbiome imbalance at multiple levels [31–33]. Disease development could be
prompted by a regulatory mechanism induced by the microbiota and its metabolites, which
are able to drive the immune response and the inflammatory process [34]. Specifically, the
process of protein citrullination mediated by Porphiromonas gingivalis (P. gingivalis) and the
activation of T helper (Th) 17 pathways by Prevotella copri (P. copri) are recognized among the
mechanisms underlying the aberrant immune responses occurring during disease [35,36].
Several studies have reported the increased risk of developing RA in patients affected
by periodontitis. The involvement of the bacteria coming from the oral cavity has an
important role and influence on the immunity response in RA. The well-established link
between periodontitis and RA is defined by the colonization of the oral cavity by the
Gram-negative bacterium P. gingivalis [37]. P. gingivalis is involved in the production of
citrullinated proteins through its enzyme P. gingivalis peptidyl-arginine deiminase (PPAD),
which is able to convert arginine residues to citrulline [38]. Mechanistic studies in animal
models have revealed that this periodontal bacterium significantly worsens the severity of
collagen-induced arthritis (CIA) by the activation of the Th17-related pathway [39].
m 2023, 3, FOR PEER REVIEW 5
BioChem 2023, 3 51

Figure 1. Dysbiosis and altered metabolic homeostasis in rheumatoid arthritis. Genetics, infections,
Figure 1.and other environmental factors are involved in dysbiosis, triggering the immunological mecha-
Dysbiosis and altered metabolic homeostasis in rheumatoid arthritis. Genetics, infections
nisms underlying RA development. Gut dysbiosis and altered metabolic pathways contribute to
and other environmental factors are involved in dysbiosis, triggering the immunological mecha-
RA onset and progression. RA: rheumatoid arthritis; PAD: peptidyl-arginine deiminases; ACPA:
nisms underlying RA development. Gut dysbiosis and altered metabolic pathways contribute to RA
anti-citrullinated protein antibodies; SCFA: short-chain fatty acids; LCA: lithocholic acid; DCA:
onset and progression. RA: rheumatoid arthritis; PAD: peptidyl-arginine deiminases; ACPA: anti-
deoxycholic acid; isoLCA: isolithocholic acid; 3-oxoLCA: 3-oxolithocholic acid.
citrullinated protein antibodies; SCFA: short-chain fatty acids; LCA: lithocholic acid; DCA: deoxy-
cholic acid; isoLCA:
Recently,isolithocholic
an alternative acid; 3-oxoLCA:
periodontal 3-oxolithocholic
pathogen, acid.
Aggregatibacter actinomycetemcomitans,
has been identified as trigger of autoimmunity in RA [40]. Its virulence factor, leukotoxin A
(LtxA),
Numerous may cause hypercitrullination
studies of neutrophils and
on microbiota composition in RAon and,
thethus, allow the
different bacteria of its
expression
to evade host immune defenses [41]. Looh et al. [42] hypothesized that LtxA-mediated
metabolites have been conducted,
hypercitrullination may involve the highlighting a correlation
abnormal activation of PAD between RA etiopathogene-
enzymes, which leads to
sis and the
microbiome imbalance at multiple levels [31–33]. Disease
citrullination of proteins and autoantigens recognized by ACPA. These mechanisms development could be
prompted by a regulatory
can further accelerate themechanism
development of induced by the microbiota
RA in susceptible individuals. and its metabolites
RA susceptibility is not only influenced by
which are able to drive the immune response and the inflammatory oral microbiota, but also by gut dysbiosis,
process [34]. Specifi-
as demonstrated in multiple animal and human studies (Table 1). Xiaofei Liu et al., through
cally, the process of protein citrullination mediated by Porphiromonas gingivalis (P. gingi-
16S rRNA sequencing, characterized the intestinal microbiome in DBA1 mice that did or did
valis) and
notthe activation
develop CIA [43].ofInTCIA-susceptible
helper (Th) 17 pathways
mice, by Prevotella
they observed an enrichmentcopri
of (P. copri) are rec-
operational
ognizedtaxonomic
among unitsthe mechanisms
(OTUs) affiliatedunderlying
with the genus the aberrantbefore
Lactobacillus immune responses
the arthritis occurring
onset and
an increase
during disease [35,36].in OTUs affiliated with the families Bacteroidaceae, Lachnospiraceae, and S24-7
after the disease development.
Several studies have reported the increased risk of developing RA in patients affected
Strikingly, the authors described a higher frequency of arthritis induction in germ-free
by periodontitis. The involvement
(GF) mice conventionalized withofthethe bacteria
microbiota coming
from from themice
CIA-susceptible oralcompared
cavity has to an im-
portantthose
role and influence on
conventionalized thethe
with immunity
microbiotaresponse in RA. The
from CIA-resistant well-established
mice. Finally, they alsolink be-
tween periodontitis
reported higherand RAconcentration
serum is defined by the colonization
of interleukin (IL)-17 and of increased
the oral cavity by the
proportions of Gram-
CD8+ T cells and Th17 lymphocytes in the spleen of CIA-susceptible mice
negative bacterium P. gingivalis [37]. P. gingivalis is involved in the production of citrulli- [43].
In an elegant study, Chen Y. et al. analyzed the composition of the gut microbiome in
nated proteins through its enzyme P. gingivalis peptidyl-arginine deiminase (PPAD)
RA patients using 16S rRNA gene sequencing, and identified different fecal metabolites,
which issuggesting
able to convert arginine
that alterations residues
in fecal to citrulline
microbiota may result [38]. Mechanistic
in alterations in fecal studies in animal
metabolites.
modelsThe have revealed
results that this
of this analysis periodontal
showed that the RA bacterium
microbiome, significantly worsens
as well as metabolites, the severity
differed
substantially from those of healthy controls (HCs). Firmicutes, Bacteroidetes,
of collagen-induced arthritis (CIA) by the activation of the Th17-related pathway [39]. Proteobacteria,
and Actinobacteria were the principal components in the gut microbiota in both groups, but
Recently, an alternative periodontal pathogen, Aggregatibacter actinomycetemcomitans
they differed mainly in abundance and not in composition [44].
has been identified as trigger of autoimmunity in RA [40]. Its virulence factor, leukotoxin
A (LtxA), may cause hypercitrullination of neutrophils in RA and, thus, allow the bacteria
to evade host immune defenses [41]. Looh et al. [42] hypothesized that LtxA-mediated
hypercitrullination may involve the abnormal activation of PAD enzymes, which leads to
the citrullination of proteins and autoantigens recognized by ACPA. These mechanisms
BioChem 2023, 3 52

The causes of altered gut flora include the reduction in probiotics, such as Bifidobac-
terium, and the increase in dangerous species. Such disruption would affect different
metabolic pathways, leading to the onset of disease. However, some controversial results
have been described.
For instance, although the presence of Bifidobacterium was constantly reduced, Lacto-
bacillus was more abundant in HCs in one study, while it was overexpressed in the RA
group in another report [37]. This suggests that the probiotic imbalance could follow the
different stages of RA [45].
In addition, some peptides (Val Thr Ile, Ile Gly Gly Ile, Val Asn Ile, etc.), amino acids
(lysine, methionine), and nucleotides (thymidine, deoxyuridine, deoxyinosine, deoxyguano-
sine, etc.) were overexpressed in the RA group, whereas some lipids were decreased. Two
fatty acids metabolites were identified as potential biomarkers; specifically, long-chain fatty
acids (9,12-octadecadiynoic acid and 10Z-nonadecenoic acid) were increased in the RA
group and were related to the presence of Verrucomicrobia and Akkermansia, suggesting that
these two metabolites can influence the disease development [33].
These results confirm what has already been demonstrated in other studies in which
fatty acids were increased in RA patients and their presence was related to the inflammatory
process [46]. Consistently, some metabolic pathways, including the biosynthesis process
of fatty acids and the degradation of glycosaminoglycans, were increased in subjects with
RA compared to HCs [47]. Some long-chain fatty acids have shown a protective role, im-
proving symptoms of RA, while short-chain fatty acids (SCFAs), the products of microbial
metabolism, have a pivotal role in the gut–joint axis in animal models [48–50]. Several
factors contribute to the maintenance of intestinal homeostasis, mainly SCFAs, followed
by IgA secreted by plasma cells in the lamina propria, α-defensins derived from Paneth
cells, polysaccharide A from the commensal Bacteroides fragilis (B. fragilis), and RegIIIγ, an
antibacterial lectin expressed by epithelial cells [50]. Disruption of this delicate balance
can result in the development of chronic inflammatory diseases, including RA. SCFAs,
mainly butyrate, propionate, and acetate, are derived from the bacterial fermentation
of non-digestible carbohydrates such as fiber; they act as histone deacetylase (HDAC)
inhibitors and G- protein-coupled receptor (GPCR) ligands modulating the function of
immune cells in the gut and other tissues [51]. Furthermore, SCFAs, as active microbial
metabolites, regulate intestinal homeostasis by facilitating the assembly of tight junctions
and the production of antimicrobial peptides. SCFAs can also affect drug response, as
demonstrated by the role of butyrate in the beneficial effect of B. fragilis on the efficacy of
therapy with methotrexate (MTX). When added to drinking water during MTX treatment,
butyrate restored the therapeutic effect of MTX in gut microbiota-deficient CIA mice to a
similar level as that achieved with B. fragilis gavage [52]. These findings further support a
potential microbial intervention strategy to improve the management of RA.
Another study showed the development of adjuvant-induced arthritis (AIA) only in
GF rats, whereas the animals colonized with Gram-negative bacteria, such as Escherichia
coli or Bacteroides, showed only a mild phenotype of arthritis [53]. The same group has
also described a protective role of some Gram-negative Enterobacteria (Escherichia coli and
Bacteroides species) when introduced into otherwise GF arthritis-susceptible rats [54], sup-
porting the theory of bacterial modulation in disease onset and development [55].
In humans, although it has been observed that a lower risk of developing RA is associ-
ated with recent gastrointestinal and/or urogenital infections [56], there is a substantial
body of evidence supporting the association between RA and intestinal dysbiosis.
Several studies have reported alterations of the intestinal epithelial barrier and in-
creased gut permeability in RA patients, especially linked with the intake of non-steroid
anti-inflammatory drugs (NSAIDs) and the severity of disease activity [57–61].
Additionally, it has been proven that a reduction in gut microbial diversity occurs in
RA patients compared to HCs, characterized by an abundance of some rare taxa, such as
Actinobacteria. Moreover, an association between the increase in Collinsella, Eggerthella, and
Faecalibacterium genera and the production of IL-17A has been described [32].
BioChem 2023, 3 53

Zhang et al. investigated the oral and gut microbial community of RA patients
and controls, detecting a dysbiosis at both sites in RA patients, partially attenuated after
treatment with disease-modifying antirheumatic drugs (DMARDs) [62].
In particular, they described a rise in Gram-positive bacteria and a depletion of Gram-
negative bacteria, such as Proteobacteria and Firmicutes, in the gut of RA patients. Moreover,
at all sites, in individuals with RA, they observed a reduction in Haemophilus spp., negatively
correlated with titers of the RA-specific autoantibodies, and an increase in Lactobacillus
salivarius, associated with high disease activity.
In an interesting study, Scher et al. characterized and compared intestinal microbiota
between new-onset DMARDs/steroids naive RA patients (NORA), chronic treated RA
patients, psoriatic arthritis (PsA) patients, and HCs [63]. They performed sequencing of the
16S gene on stool samples, identifying higher levels of P. copri associated with decreased
commensal beneficial Bacteriodes in NORA patients. Additionally, they highlighted the
capacity of P. copri to regulate the gut microbiota and to induce colitis in an inflammatory
bowel disease (IBD) mice model.
A recent study has also shown a marked sequence homology between two autoanti-
gens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), that are highly ex-
pressed in the synovial tissue of RA patients, and epitopes from Prevotella sp. This result
further supports the molecular mimicry theory in RA and the association between mucosal
and joint inflammation [64].

4. Metabolomics Applied to Treatment and Interference with Microbiome Pathological

Dysfunction in RA
The metabolic profile of RA patients changes importantly during disease course, as
reviewed above, but treatments could even contribute to altering metabolite levels, for
example increasing anti-inflammatory molecules. In this regard, metabolomics could help
in identifying new biomarkers to assess response to treatment with the final goal of profiling
responders and non-responders, and of creating a more personalized approach to prevent
treatment failure [65,66].
Currently, a large proportion of patients still shows a poor response to both conven-
tional (c)DMARDs and biologic (b)DMARDs [67].
Regarding cDMARDs, the metabolome of RA patients revealed that the levels of
tryptophan are decreased under treatment with MTX or leflunomide [68]. Tryptophan is
involved in the metabolic pathway that drives TLR activation, danger molecule recogni-
tion, and IL-6 production, so its reduction could mirror the efficacy of MTX [69,70]. In
an early RA cohort of 82 patients treated with MTX, a recent study demonstrated that
the baseline concentrations of homocysteine, taurine, adenosine triphosphate, guanosine
diphosphate, and uric acid were significantly lower in the plasma of non-responders versus
responders, while glycolytic intermediates 1,3-/2,3-diphosphoglyceric acid, glycerol-3-
phosphate, and phosphoenolpyruvate were significantly higher in non-responders [71]. In
addition, in a mouse model of RA, low serum levels of N-methylisoleucine were revealed
as a potential biomarker of response to MTX [72]. The effects of MTX even include the
normalization of taurine, aspartate, alanine, lactic acid, adenosine, and guanine, whose
levels are reestablished after treatment [73].
In relation to gut microbiome, Artacho et al. reported a possible association between
gut bacteria and response to MTX in new-onset naive RA. In non-responders, gut bacteria
showed an increased metabolism of MTX, possibly accounting for a decrease in drug
availability for patients, leading to a worsened clinical response [74].
Among biologic agents, most evidence linking metabolomics to treatment focused on
TNF-inhibitors (TNFi), as TNF is a strong proinflammatory cytokine renowned to influence
glucose and lipid metabolism [75]. In particular, an increase in hippuric acid, citrate, and
lactic acid was described after infliximab use, while an increase in choline, phenylacetic
acid, urea, creatine, and methylamine was observed after treatment with etanercept [76].
BioChem 2023, 3 54

A specific metabolite signature was even outlined between responders and non-
responders to TNFi both at the blood and urine level. After TNFi therapy, two different
metabolic profiles differentiated good responders from non-responders, and carbohydrate
derivates, such as D-glucose, D-fructose, sucrose, and maltose, emerged as determinants
of the therapeutic response [77]. Additionally, five metabolites (glycerol 3-phosphate, be-
tonicine, N-acetylalanine, hexanoic acid, and taurine) were identified as potential predictors
of good response to TNFi.
In urine samples, histamine, glutamine, phenylacetic acid, xanthine, xanthurenic acid,
and creatinine were found to be upregulated in patients responding to TNF antagonist, while
ethan-olamine, hydroxyphenylpyruvic acid, and phosphocreatine were downregulated. Three
metabolites, namely 3-aminobutyric acid, citric acid, and quinic acid, were shown to
positively predict response to abatacept [78].
Changes in arachidonic acid metabolism with the consequent modulation of the
inflammatory pathway related to IL-6 signaling were depicted in a study focused on
tocilizumab efficacy [79]. Moreover, tocilizumab responders present higher levels of 3-
hydroxybutyrate and phenylalanine, which can improve the ability to predict tocilizumab
responders [80].
Rituximab administration determines a downregulation in phosphatidylethanolamines,
phosphatidyserines, and phosphatidylglycerols levels, once again stressing the im-portance
of amino acids and lipid perturbations in RA [81,82].
Coming to new treatments, specifically JAK-inhibitors, higher levels of omega-3 fatty
acids and docosahexaenoic acid in treated patients were demonstrated and correlated with
an important effect on pain reduction [83].
To improve the identification of possible responders to treatments, the combination
of metabolites and clinical data is crucial [84], and new players, such as gut bacteria and
alternative therapies targeting their functions, should carefully be taken into account.
On the basis of substantial evidence regarding the correlation between gut dysbiosis
and RA, the effect of probiotics in RA patients has been recently investigated, with some
encouraging results. Probiotics are live bacteria that, when administered to the host in
appropriate doses, provide significant health benefits. Specifically, exogenous bacteria can
improve gut microbiota homeostasis, modulate the immune response, and support gut
barrier integrity, treating dysbiosis and related pathological conditions. Several studies
have investigated the role of probiotic bacteria in systemic immune responses. In these
studies, the supplementation with probiotics correlated with a decrease in disease activity
score (DAS28), CRP, and systemic inflammatory cytokines, such as tumor necrosis factor-α
(TNFα), IL-12, and IL-6, and at the same time, with an increase in IL-10 [45,85]. To identify
potential probiotic gut microbes that can ameliorate the development of RA, microbiota
profiling in RA patients was studied using 16S rDNA bacterial gene sequencing and shot-
gun metagenomics. Interestingly, the relative abundance of Parabacteroides distasonis (P.
distasonis) in RA patients was decreased, and this decrease was negatively correlated with
DAS28. These findings were explained by the effects of microbial metabolites derived from
P. distasonis, lithocholic acid (LCA), deoxycholic acid (DCA), isolithocholic acid (isoLCA),
and 3-oxolithocholic acid (3-oxoLCA), on CD4+ T-cell differentiation and macrophage polar-
ization. In particular, 3-oxoLCA and isoLCA promoted the M2 polarization of macrophages
and, simultaneously, inhibited Th17 cell differentiation. Consistently, oral treatment of
arthritic mice with live P. distasonis significantly mitigated RA development, pointing to
P. distasonis as a potential probiotic agent in the treatment of RA [86]. Another potential
probiotic agent suggested for RA is Bifidobacterium pseudocatenulatum, which is also involved
in bile acid (BA) metabolism. B. pseudocatenulatum prevented joint damage in a CIA mouse
model by elevating the enzymatic activity of bile salt hydrolase and increasing the levels
of unconjugated secondary BA to suppress aberrant Th 1/17-type immune responses [87].
Probiotics have been proposed as an adjuvant treatment in the therapeutic strategy of
immune-mediated disorders. MTX, one of the most widely used DMARDs in inflamma-
tory arthritis, alters the composition of the gut microbiota, helping to restore the altered
BioChem 2023, 3 55

microbial balance [62]. Further introduction of a probiotic has demonstrated a synergistic

effect that can effectively control the inflammatory status. In this regard, the positive influ-
ence of the Lactobacillaceae family on the microbiota in RA has been optimized in patients
taking DMARDs, as demonstrated by the ability of Lactobacillus casei (L. casei) to reduce
RA symptoms and inhibit proinflammatory cytokines [88]. In their study, Alipour et al.
showed that L. casei supplementations decreased serum CRP levels, and reduced tender
and swollen joint counts; furthermore, serum levels of interleukins (IL-10, IL-12) and TNF-α
were improved in the probiotic group [88]. The impact of lactobacilli on joint inflammation
has also been explored in the CIA model. The addition of L. casei or L. acidophilus in a rat
model of CIA proved to be as effective as standard treatment with indomethacin [89]; the
anti-inflammatory properties of L. casei in the CIA rat model were confirmed by its ability to
inhibit the COX-2 enzyme and downregulate proinflammatory cytokines [90]. Recently, the
effect of Lactoplantibacillus plantarum LS/07 (LB) was investigated in combination therapy
with MTX in adjuvant arthritis (AA) in rats. LB administered in combination with MTX
helped relieve hind leg swelling and body weight loss for the whole duration of the experi-
ment. The combination therapy was more effective in reducing the inflammation than MTX
alone. Regarding the positive anti-inflammatory effect of Lactiplantibacillus plantarum LS/07,
administration of LB to AA rats reduced the concentrations of IL-17A, metalloproteinase-9
(MMP-9), and monocyte chemotactic protein-1 (MCP-1), with the latter two involved in
macrophage recruitment and joint destruction during RA [91].

Table 1. The table reports the main studies on predisposing/protective gut bacteria involved in
rheumatoid arthritis.

Metabolites References
Collinsella, Eggerthella, and
Predisposing Bacteria [32]
Faecalibacterium genera
9,12-octadecadiynoic acid and
Verrucomicrobiaand Akkermansia [33] (animal study)
10Z-nonadecenoic acid
Lactobacillus salivarius [62]
P. copri [63]
Prevotella sp. [64]
Protective Bacteria B. fragilis Butyrate [52] (animal study)
[53] (animal study), [54]
Escherichia coli and Bacteroides species
(animal study)
Haemophilus spp. [62]
Lactiplantibacillus plantarumLS/07 [82] (animal study)
LCA, DCA, isoLCA and
P. distasonis [86] (animal study)
3-oxoLCA
Bifidobacterium pseudocatenulatum unconjugated secondary BA [87] (animal study)
[88,89] (animal study), [90]
L. casei
(animal study)
L. acidophilus [89] (animal study)
LCA: lithocholic acid; DCA: deoxycholic acid; isoLCA: isolithocholic acid; 3-oxoLCA: 3-oxolithocholic acid; BA:
bile acid.

5. Conclusions and Future Perspectives

The association between the gut microbiome and metabolites has not been fully
evaluated yet in RA. Nevertheless, combined gut microbiome and metabolome studies have
suggested promising new research directions to deepen our knowledge of RA pathogenesis.
The microbiome is critical for the balance of the host immune system, and its changes
are essential for modulating inflammation and promoting the loss of immune tolerance, as
BioChem 2023, 3 56

demonstrated by the correlation between gut microbiota dysbiosis and the development of
arthritis in several mouse models. The early stages of the pathological immune response
associated with autoimmune diseases occur in mucosal tissues, such as the intestinal or
oral mucosa, and are strongly associated with an abundance of specific bacterial species.
Several studies, including animal studies, have pointed out an interaction between the
gut microbiome, local/systemic immunity, and activation of joint inflammation. The
link between these domains could be intimately connected to the activation of specific
metabolic pathways. Metabolomics is an approach that has not been fully explored, but
certainly holds promise for identifying biomarkers in different fields of medicine, including
rheumatic diseases. Despite the undisputed potential, some limitations emerge, such as
the lack of clear standardization of the technique used (NMR or MS) and sample collection
methods, as well as the challenging availability of these methods and the specific laboratory
skills required.
This review summarizes recent studies that have shown the correlation between the
development of RA and the cross-link between the microbiome and metabolome.
Further evidence is needed to confirm what has been highlighted so far regarding
the possible role of the microbiome and its influence on the metabolome in shaping the
pathogenesis of RA. Identifying the prevalent metabolites and studying the gut–joint axis
and intestinal dysbiosis could represent the fundamental steps needed to broaden the ther-
apeutic strategies scenario in the era of personalized medicine to achieve a comprehensive
“treat-to-target” approach. Moreover, the identification of these markers can be a valuable
tool for predicting the course of the disease and preventing its progression.

Author Contributions: L.L.B. and C.R.: writing—original draft preparation, writing—review and
editing, conceptualization; G.G. (Giulia Grasso) and F.M.: visualization, formal analysis, methodology,
resources; F.C. (Federica Camarda): visualization, resources; F.C. (Francesco Ciccia): supervision,
project administration; G.G. (Giuliana Guggino): supervision, project administration, conceptualiza-
tion. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. McInnes, I.B.; Schett, G. The Pathogenesis of Rheumatoid Arthritis. N. Engl. J. Med. 2011, 365, 2205–2219. [CrossRef] [PubMed]
2. McInnes, I.B.; Buckley, C.D.; Isaacs, J.D. Cytokines in rheumatoid arthritis—Shaping the immunological landscape. Nat. Rev.
Rheumatol. 2016, 12, 63–68. [CrossRef] [PubMed]
3. Petrovská, N.; Prajzlerová, K.; Vencovský, J.; Šenolt, L.; Filková, M. The pre-clinical phase of rheumatoid arthritis: From risk
factors to prevention of arthritis. Autoimmun. Rev. 2021, 20, 102797. [CrossRef] [PubMed]
4. Romão, V.C.; Fonseca, J.E. Disease mechanisms in preclinical rheumatoid arthritis: A narrative review. Front. Med. 2022, 9, 689711.
[CrossRef]
5. Weyand, C.M.; Goronzy, J.J. Immunometabolism in the development of rheumatoid arthritis. Immunol. Rev. 2020, 294, 177–187.
[CrossRef] [PubMed]
6. Edilova, M.I.; Akram, A.; Abdul-Sater, A.A. Innate immunity drives pathogenesis of rheumatoid arthritis. Biomed. J. 2021, 44,
172–182. [CrossRef] [PubMed]
7. Saferding, V.; Blüml, S. Innate immunity as the trigger of systemic autoimmune diseases. J. Autoimmun. 2020, 110, 102382.
[CrossRef]
8. Siouti, E.; Andreakos, E. The many facets of macrophages in rheumatoid arthritis. Biochem. Pharmacol. 2019, 165, 152–169.
[CrossRef]
9. Chan, K.-W.A.; Felson, D.T.; Yood, R.A.; Walker, A.M. The lag time between onset of symptoms and diagnosis of rheumatoid
arthritis. Arthritis Rheum. 1994, 37, 814–820. [CrossRef]
10. O’Dell, J.R. Treating rheumatoid arthritis early: A window of opportunity? Arthritis Rheum. 2002, 46, 283–285. [CrossRef]
BioChem 2023, 3 57

11. Xu, L.; Chang, C.; Jiang, P.; Wei, K.; Zhang, R.; Jin, Y.; Zhao, J.; Xu, L.; Shi, Y.; Guo, S.; et al. Metabolomics in rheumatoid arthritis:
Advances and review. Front. Immunol. 2022, 13, 961708. [CrossRef] [PubMed]
12. Coras, R.; Murillo-Saich, J.D.; Guma, M. Circulating Pro- and Anti-Inflammatory Metabolites and Its Potential Role in Rheumatoid
Arthritis Pathogenesis. Cells 2020, 9, 827. [CrossRef] [PubMed]
13. Li, C.; Chen, B.; Fang, Z.; Leng, Y.-F.; Wang, D.-W.; Chen, F.-Q.; Xu, X.; Sun, Z.-L. Metabolomics in the development and
progression of rheumatoid arthritis: A systematic review. Jt. Bone Spine 2020, 87, 425–430. [CrossRef] [PubMed]
14. Rizzo, C.; Camarda, F.; Donzella, D.; La Barbera, L.; Guggino, G. Metabolomics: An Emerging Approach to Understand
Pathogenesis and to Assess Diagnosis and Response to Treatment in Spondyloarthritis. Cells 2022, 11, 549. [CrossRef]
15. Jutley, G.S.; Sahota, K.; Sahbudin, I.; Filer, A.; Arayssi, T.; Young, S.P.; Raza, K. Relationship Between Inflammation and Metabolism
in Patients With Newly Presenting Rheumatoid Arthritis. Front. Immunol. 2021, 12, 676105. [CrossRef]
16. Young, S.P.; Kapoor, S.R.; Viant, M.R.; Byrne, J.J.; Filer, A.; Buckley, C.D.; Kitas, G.D.; Raza, K. The Impact of Inflammation on
Metabolomic Profiles in Patients with Arthritis. Arthritis Rheum. 2013, 65, 2015–2023. [CrossRef] [PubMed]
17. Pietzner, M.; Kaul, A.; Henning, A.-K.; Kastenmüller, G.; Artati, A.; Lerch, M.M.; Adamski, J.; Nauck, M.; Friedrich, N.
Comprehensive metabolic profiling of chronic low-grade inflammation among generally healthy individuals. BMC Med. 2017, 15,
210. [CrossRef] [PubMed]
18. Kapoor, S.R.; Filer, A.; Fitzpatrick, M.A.; Fisher, B.A.; Taylor, P.C.; Buckley, C.D.; McInnes, I.B.; Raza, K.; Young, S.P. Metabolic
Profiling Predicts Response to Anti–Tumor Necrosis Factor α Therapy in Patients With Rheumatoid Arthritis. Arthritis Rheum.
2013, 65, 1448–1456. [CrossRef]
19. Alonso, A.; for the IMID Consortium; Julià, A.; Vinaixa, M.; Domènech, E.; Fernández-Nebro, A.; Cañete, J.D.; Ferrándiz, C.;
Tornero, J.; Gisbert, J.P. Urine metabolome profiling of immune-mediated inflammatory diseases. BMC Med. 2016, 14, 133.
[CrossRef]
20. Galvã¡n-Peã±A, S.; O’Neill, L.A.J. Metabolic Reprograming in Macrophage Polarization. Front. Immunol. 2014, 5, 420.
[CrossRef]
21. Williams, N.C.; O’Neill, L.A.J. A Role for the Krebs Cycle Intermediate Citrate in Metabolic Reprogramming in Innate Immunity
and Inflammation. Front. Immunol. 2018, 9, 141. [CrossRef] [PubMed]
22. Tannahill, G.M.; Curtis, A.M.; Adamik, J.; Palsson-McDermott, E.M.; McGettrick, A.F.; Goel, G.; Frezza, C.; Bernard, N.J.; Kelly, B.;
Foley, N.H.; et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature 2013, 496, 238–242. [CrossRef]
[PubMed]
23. Hotamisligil, G.S. Inflammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] [PubMed]
24. Pucino, V.; Certo, M.; Bulusu, V.; Cucchi, D.; Goldmann, K.; Pontarini, E.; Haas, R.; Smith, J.; Headland, S.E.; Blighe, K.; et al.
Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring. Cell Metab.
2019, 30, 1055–1074.e8. [CrossRef] [PubMed]
25. Weyand, C.M.; Zeisbrich, M.; Goronzy, J.J. Metabolic signatures of T-cells and macrophages in rheumatoid arthritis. Curr. Opin.
Immunol. 2017, 46, 112–120. [CrossRef]
26. Kettunen, J.; Tukiainen, T.; Sarin, A.-P.; Ortega-Alonso, A.; Tikkanen, E.; Lyytikäinen, L.-P.; Kangas, A.J.; Soininen, P.; Würtz, P.;
Silander, K.; et al. Genome-wide association study identifies multiple loci influencing human serum metabolite levels. Nat. Genet.
2012, 44, 269–276. [CrossRef]
27. Kettunen, J.; Demirkan, A.; Würtz, P.; Draisma, H.H.; Haller, T.; Rawal, R.; Vaarhorst, A.; Kangas, A.J.; Lyytikäinen, L.-P.; Pirinen,
M.; et al. Genome-wide study for circulating metabolites identifies 62 loci and reveals novel systemic effects of LPA. Nat. Commun.
2016, 7, 11122. [CrossRef]
28. Okada, Y.; Wu, D.; Trynka, G.; Raj, T.; Terao, C.; Ikari, K.; Kochi, Y.; Ohmura, K.; Suzuki, A.; Yoshida, S.; et al. Genetics of
rheumatoid arthritis contributes to biology and drug discovery. Nature 2014, 506, 376–381. [CrossRef]
29. Fernando, M.M.A.; Stevens, C.R.; Walsh, E.C.; De Jager, P.L.; Goyette, P.; Plenge, R.M.; Vyse, T.J.; Rioux, J.D. Defining the Role of
the MHC in Autoimmunity: A Review and Pooled Analysis. PLOS Genet. 2008, 4, e1000024. [CrossRef]
30. Rantapää-Dahlqvist, S.; de Jong, B.A.W.; Berglin, E.; Hallmans, G.; Wadell, G.; Stenlund, H.; Sundin, U.; van Venrooij, W.J.
Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis.
Arthritis Rheum. 2003, 48, 2741–2749. [CrossRef]
31. van Delft, M.A.; Huizinga, T.W. An overview of autoantibodies in rheumatoid arthritis. J. Autoimmun. 2020, 110, 102392.
[CrossRef] [PubMed]
32. Chen, J.; Wright, K.; Davis, J.M.; Jeraldo, P.; Marietta, E.V.; Murray, J.; Nelson, H.; Matteson, E.L.; Taneja, V. An expansion of rare
lineage intestinal microbes characterizes rheumatoid arthritis. Genome Med. 2016, 8, 43. [CrossRef] [PubMed]
33. Chen, Y.; Ma, C.; Liu, L.; He, J.; Zhu, C.; Zheng, F.; Dai, W.; Hong, X.; Liu, D.; Tang, D.; et al. Analysis of gut microbiota
and metabolites in patients with rheumatoid arthritis and identification of potential biomarkers. Aging 2021, 13, 23689–23701.
[CrossRef] [PubMed]
34. Zhong, D.; Wu, C.; Zeng, X.; Wang, Q. The role of gut microbiota in the pathogenesis of rheumatic diseases. Clin. Rheumatol. 2018,
37, 25–34. [CrossRef]
35. Montgomery, A.B.; Kopec, J.; Shrestha, L.; Thezenas, M.-L.; Burgess-Brown, N.A.; Fischer, R.; Yue, W.W.; Venables, P.J. Crystal
structure of Porphyromonas gingivalis peptidylarginine deiminase: Implications for autoimmunity in rheumatoid arthritis. Ann.
Rheum. Dis. 2016, 75, 1255–1261. [CrossRef]
BioChem 2023, 3 58

36. Maeda, Y.; Kurakawa, T.; Umemoto, E.; Motooka, D.; Ito, Y.; Gotoh, K.; Hirota, K.; Matsushita, M.; Furuta, Y.; Narazaki, M.; et al.
Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine. Arthritis Rheumatol. 2016,
68, 2646–2661. [CrossRef]
37. Janssen, K.M.; Vissink, A.; de Smit, M.J.; Westra, J.; Brouwer, E. Lessons to be learned from periodontitis. Curr. Opin. Rheumatol.
2013, 25, 241–247. [CrossRef]
38. Wegner, N.; Wait, R.; Sroka, A.; Eick, S.; Nguyen, K.-A.; Lundberg, K.; Kinloch, A.; Culshaw, S.; Potempa, J.; Venables,
P.J. Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: Implications for
autoimmunity in rheumatoid arthritis. Arthritis Rheum. 2010, 62, 2662–2672. [CrossRef]
39. Marchesan, J.T.; Gerow, E.A.; Schaff, R.; Taut, A.D.; Shin, S.-Y.; Sugai, J.; Brand, D.; Burberry, A.; Jorns, J.; Lundy, S.K.; et al.
Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis. Arthritis Res. Ther.
2013, 15, R186. [CrossRef]
40. Konig, M.F.; Abusleme, L.; Reinholdt, J.; Palmer, R.J.; Teles, R.P.; Sampson, K.; Rosen, A.; Nigrovic, P.A.; Sokolove, J.; Giles,
J.T.; et al. Aggregatibacter actinomycetemcomitans—Induced hypercitrullination links periodontal infection to autoimmunity in
rheumatoid arthritis. Sci. Transl. Med. 2016, 8, 369ra176. [CrossRef]
41. Kulkarni, A.; Beckler, M.D.; Amini, S.S.; Kesselman, M.M. Oral Microbiome in Pre-Rheumatoid Arthritis: The Role of Aggregati-
bacter Actinomycetemcomitans in Bacterial Composition. Cureus 2022, 5, e32201. [CrossRef]
42. Looh, S.C.; Soo, Z.M.P.; Wong, J.J.; Yam, H.C.; Chow, S.K.; Hwang, J.S. Aggregatibacter actinomycetemcomitans as the Aetiological
Cause of Rheumatoid Arthritis: What Are the Unsolved Puzzles? Toxins 2022, 14, 50. [CrossRef]
43. Liu, X.; Zeng, B.; Zhang, J.; Li, W.; Mou, F.; Wang, H.; Zou, Q.; Zhong, B.; Wu, L.; Wei, H.; et al. Role of the Gut Microbiome in
Modulating Arthritis Progression in Mice. Sci. Rep. 2016, 6, 30594. [CrossRef] [PubMed]
44. Schroeder, B.O.; Bäckhed, F. Signals from the gut microbiota to distant organs in physiology and disease. Nat. Med. 2016, 22,
1079–1089. [CrossRef] [PubMed]
45. Vaghef-Mehrabany, E.; Alipour, B.; Homayouni-Rad, A.; Sharif, S.-K.; Asghari-Jafarabadi, M.; Zavvari, S. Probiotic supplementa-
tion improves inflammatory status in patients with rheumatoid arthritis. Nutrition 2014, 30, 430–435. [CrossRef] [PubMed]
46. Zhou, J.; Chen, J.; Hu, C.; Xie, Z.; Li, H.; Wei, S.; Wang, D.; Wen, C.; Xu, G. Exploration of the serum metabolite signature in
patients with rheumatoid arthritis using gas chromatography–mass spectrometry. J. Pharm. Biomed. Anal. 2016, 127, 60–67.
[CrossRef] [PubMed]
47. Kishikawa, T.; Maeda, Y.; Nii, T.; Motooka, D.; Matsumoto, Y.; Matsushita, M.; Matsuoka, H.; Yoshimura, M.; Kawada, S.;
Teshigawara, S.; et al. Metagenome-wide association study of gut microbiome revealed novel aetiology of rheumatoid arthritis in
the Japanese population. Ann. Rheum. Dis. 2020, 79, 103–111. [CrossRef]
48. Bahadori, B.; Uitz, E.; Thonhofer, R.; Trummer, M.; Pestemer-Lach, I.; McCarty, M.; Krejs, G.J. ω-3 Fatty Acids Infusions as
Adjuvant Therapy in Rheumatoid Arthritis. J. Parenter. Enter. Nutr. 2010, 34, 151–155. [CrossRef] [PubMed]
49. Häger, J.; Bang, H.; Hagen, M.; Frech, M.; Träger, P.; Sokolova, M.; Steffen, U.; Tascilar, K.; Sarter, K.; Schett, G.; et al. The Role of
Dietary Fiber in Rheumatoid Arthritis Patients: A Feasibility Study. Nutrients 2019, 11, 2392. [CrossRef]
50. La Barbera, L.; Macaluso, F.; Fasano, S.; Grasso, G.; Ciccia, F.; Guggino, G. Microbiome Changes in Connective Tissue Diseases
and Vasculitis: Focus on Metabolism and Inflammation. Int. J. Mol. Sci. 2022, 23, 6532. [CrossRef]
51. Luu, M.; Visekruna, A. Short-chain fatty acids: Bacterial messengers modulating the immunometabolism of T cells. Eur. J.
Immunol. 2019, 49, 842–848. [CrossRef] [PubMed]
52. Zhou, B.; Dong, C.; Zhao, B.; Lin, K.; Tian, Y.; Zhang, R.; Zhu, L.; Xu, H.; Yang, L. Bacteroides fragilis participates in the therapeutic
effect of methotrexate on arthritis through metabolite regulation. Front. Microbiol. 2022, 13, 1015130. [CrossRef] [PubMed]
53. Kohashi, O.; Kohashi, Y.; Takahashi, T.; Ozawa, A.; Shigematsu, N. Reverse Effect of Gram-Positive Bacteria vs. Gram-Negative
Bacteria on Adjuvant-Induced Arthritis in Germfree Rats. Microbiol. Immunol. 1985, 29, 487–497. [CrossRef] [PubMed]
54. Kohashi, O.; Kohashi, Y.; Takahashi, T.; Ozawa, A.; Shigematsu, N. Suppressive effect ofEscherichia coli on adjuvant-induced
arthritis in germ-free rats. Arthritis Rheum. 1986, 29, 547–553. [CrossRef]
55. Breban, M.A.; Moreau, M.C.; Fournier, C.; Ducluzeau, R.; Kahn, M.F. Influence of the bacterial flora on collagen-induced arthritis
in susceptible and resistant strains of rats. Clin. Exp. Rheumatol. 1993, 11, 61–64. [PubMed]
56. Sandberg, M.E.C.; Bengtsson, C.; Klareskog, L.; Alfredsson, L.; Saevarsdottir, S. Recent infections are associated with decreased
risk of rheumatoid arthritis: A population-based case-control study. Ann. Rheum. Dis. 2015, 74, 904–907. [CrossRef] [PubMed]
57. Jenkins, R.T.; Rooney, P.J.; Jones, D.B.; Bienenstock, J.; Goodacre, R.L. Increased intestinal permeability in patients with rheumatoid
arthritis: A side-effect of oral nonsteroidal anti-inflammatory drug therapy? Rheumatology 1987, 26, 103–107. [CrossRef]
58. Segal, A.; Isenberg, D.; Hajirousou, V.; Tolfree, S.; Clark, J.; Snaith, M.L. Preliminary evidence for gut involvement in the
pathogenesis of rheumatoid arthritis? Rheumatology 1986, 25, 162–166. [CrossRef]
59. Macaluso, F.; Guggino, G.; Rizzo, A.; Ferrante, A.; Ciccia, F. Histopathology of the gut in rheumatic diseases. Reumatismo 2018, 70,
178–186. [CrossRef]
60. Porzio, V.; Biasi, G.; Corrado, A.; De Santi, M.; Vindigni, C.; Vrti, S.; Bayeli, P.F.; Marcolongo, R. Intestinal Histological and
Ultrastructural Inflammatory Changes in Spondyloarthropathy and Rheumatoid Arthritis. Scand. J. Rheumatol. 1997, 26, 92–98.
[CrossRef]
61. Mielants, H.; De Vos, M.; Goemaere, S.; Schelstraete, K.; Cuvelier, C.; Goethals, K.; Maertens, M.; Ackerman, C.; Veys, E.M.
Intestinal mucosal permeability in inflammatory rheumatic diseases. II. Role of disease. J. Rheumatol. 1991, 18, 394–400. [PubMed]
BioChem 2023, 3 59

62. Zhang, X.; Zhang, D.; Jia, H.; Feng, Q.; Wang, D.; Liang, D.; Wu, X.; Li, J.; Tang, L.; Li, Y.; et al. The oral and gut microbiomes are
perturbed in rheumatoid arthritis and partly normalized after treatment. Nat. Med. 2015, 21, 895–905. [CrossRef] [PubMed]
63. Scher, J.U.; Sczesnak, A.; Longman, R.S.; Segata, N.; Ubeda, C.; Bielski, C.; Rostron, T.; Cerundolo, V.; Pamer, E.G.; Abramson, S.B.;
et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife 2013, 2, e01202. [CrossRef]
64. Pianta, A.; Arvikar, S.L.; Strle, K.; Drouin, E.E.; Wang, Q.; Costello, C.; Steere, A.C. Two rheumatoid arthritis–specific autoantigens
correlate microbial immunity with autoimmune responses in joints. J. Clin. Investig. 2017, 127, 2946–2956. [CrossRef]
65. Radu, A.-F.; Bungau, S.G.; Negru, P.A.; Marcu, M.F.; Andronie-Cioara, F.L. In-depth bibliometric analysis and current scientific
mapping research in the context of rheumatoid arthritis pharmacotherapy. Biomed. Pharmacother. 2022, 154, 113614. [CrossRef]
[PubMed]
66. Bartikoski, B.J.; De Oliveira, M.S.; Santo, R.C.D.E.; Dos Santos, L.P.; Dos Santos, N.G.; Xavier, R.M. A Review of Metabolomic
Profiling in Rheumatoid Arthritis: Bringing New Insights in Disease Pathogenesis, Treatment and Comorbidities. Metabolites
2022, 12, 394. [CrossRef] [PubMed]
67. Nair, N.; Plant, D.; Verstappen, S.M.; Isaacs, J.D.; Morgan, A.; Hyrich, K.L.; Barton, A.; Wilson, A.G. The MATURA investigators
Differential DNA methylation correlates with response to methotrexate in rheumatoid arthritis. Rheumatology 2020, 59, 1364–1371.
[CrossRef] [PubMed]
68. Urbaniak, B.; Plewa, S.; Klupczynska, A.; Sikorska, D.; Samborski, W.; Kokot, Z.J. Serum free amino acid levels in rheumatoid
arthritis according to therapy and physical disability. Cytokine 2019, 113, 332–339. [CrossRef] [PubMed]
69. Priori, R.; Scrivo, R.; Brandt, J.; Valerio, M.; Casadei, L.; Valesini, G.; Manetti, C. Metabolomics in rheumatic diseases: The
potential of an emerging methodology for improved patient diagnosis, prognosis, and treatment efficacy. Autoimmun. Rev. 2013,
12, 1022–1030. [CrossRef]
70. Ramalho, R.; Rao, M.; Zhang, C.; Agrati, C.; Ippolito, G.; Wang, F.-S.; Zumla, A.; Maeurer, M. Immunometabolism: New insights
and lessons from antigen-directed cellular immune responses. Semin. Immunopathol. 2020, 42, 279–313. [CrossRef]
71. Gosselt, H.R.; Muller, I.B.; Jansen, G.; Van Weeghel, M.; Vaz, F.M.; Hazes, J.M.W.; Heil, S.G.; De Jonge, R. Identification of
Metabolic Biomarkers in Relation to Methotrexate Response in Early Rheumatoid Arthritis. J. Pers. Med. 2020, 10, 271. [CrossRef]
[PubMed]
72. Salamoun, Y.M.; Polireddy, K.; Cho, Y.K.; Medcalf, M.R.; Funk, R.S. Methotrexate Disposition, Anti-Folate Activity, and
Metabolomic Profiling to Identify Molecular Markers of Disease Activity and Drug Response in the Collagen-Induced Arthritis
Mouse Model. Metabolites 2021, 12, 24. [CrossRef] [PubMed]
73. Wang, M.; Huang, J.; Fan, H.; He, D.; Zhao, S.; Shu, Y.; Li, H.; Liu, L.; Lu, S.; Xiao, C.; et al. Treatment of Rheumatoid Arthritis
Using Combination of Methotrexate and Tripterygium Glycosides Tablets—A Quantitative Plasma Pharmacochemical and
Pseudotargeted Metabolomic Approach. Front. Pharmacol. 2018, 9, 1051. [CrossRef] [PubMed]
74. Artacho, A.; Isaac, S.; Nayak, R.; Flor-Duro, A.; Alexander, M.; Koo, I.; Manasson, J.; Smith, P.B.; Rosenthal, P.; Homsi, Y.; et al.
The Pretreatment Gut Microbiome Is Associated With Lack of Response to Methotrexate in New-Onset Rheumatoid Arthritis.
Arthritis Rheumatol. 2021, 73, 931–942. [CrossRef] [PubMed]
75. Shi, J.; Fan, J.; Su, Q.; Yang, Z. Cytokines and Abnormal Glucose and Lipid Metabolism. Front. Endocrinol. 2019, 10, 703. [CrossRef]
[PubMed]
76. Priori, R.; Casadei, L.; Valerio, M.; Scrivo, R.; Valesini, G.; Manetti, C. 1H-NMR-Based Metabolomic Study for Identifying
Serum Profiles Associated with the Response to Etanercept in Patients with Rheumatoid Arthritis. PLoS ONE 2015, 10, e0138537.
[CrossRef]
77. Tatar, Z.; Migne, C.; Petera, M.; Gaudin, P.; Lequerre, T.; Marotte, H.; Tebib, J.; Guillot, E.P.; Soubrier, M. Variations in the
metabolome in response to disease activity of rheumatoid arthritis. BMC Musculoskelet. Disord. 2016, 17, 353. [CrossRef]
78. Takahashi, S.; Saegusa, J.; Onishi, A.; Morinobu, A. Biomarkers identified by serum metabolomic analysis to predict biologic
treatment response in rheumatoid arthritis patients. Rheumatology 2019, 58, 2153–2161. [CrossRef]
79. Teitsma, X.M.; Yang, W.; Jacobs, J.W.G.; Pethö-Schramm, A.; Borm, M.E.A.; Harms, A.C.; Hankemeier, T.; van Laar, J.M.; Bijlsma,
J.W.J.; Lafeber, F.P.J.G. Baseline metabolic profiles of early rheumatoid arthritis patients achieving sustained drug-free remission
after initiating treat-to-target tocilizumab, methotrexate, or the combination: Insights from systems biology. Arthritis Res. Ther.
2018, 20, 230. [CrossRef]
80. Murillo-Saich, J.D.; Diaz-Torne, C.; Ortiz, M.A.; Coras, R.; Gil-Alabarse, P.; Pedersen, A.; Corominas, H.; Vidal, S.; Guma, M.
Metabolomics profiling predicts outcome of tocilizumab in rheumatoid arthritis: An exploratory study. Metabolomics 2021, 17, 74.
[CrossRef]
81. Sweeney, S.R.; Kavanaugh, A.; Lodi, A.; Wang, B.; Boyle, D.; Tiziani, S.; Guma, M. Metabolomic profiling predicts outcome of
rituximab therapy in rheumatoid arthritis. RMD Open 2016, 2, e000289. [CrossRef]
82. Singh, A.; Behl, T.; Sehgal, A.; Singh, S.; Sharma, N.; Naved, T.; Bhatia, S.; Al-Harrasi, A.; Chakrabarti, P.; Aleya, L.; et al.
Mechanistic insights into the role of B cells in rheumatoid arthritis. Int. Immunopharmacol. 2021, 99, 108078. [CrossRef] [PubMed]
83. Chang, C.-K.; Chen, P.-K.; Chen, C.-C.; Chang, S.-H.; Chen, C.-H.; Chen, D.-Y. Increased Levels of Omega-3 Fatty Acids and DHA
Are Linked to Pain Reduction in Rheumatoid Arthritis Patients Treated with Janus Kinase Inhibitors. Nutrients 2021, 13, 3050.
[CrossRef]
BioChem 2023, 3 60

84. Cuppen, B.V.J.; Fu, J.; van Wietmarschen, H.A.; Harms, A.C.; Koval, S.; Marijnissen, A.C.A.; Peeters, J.J.W.; Bijlsma, J.W.J.; Tekstra,
J.; van Laar, J.M.; et al. Exploring the Inflammatory Metabolomic Profile to Predict Response to TNF-α Inhibitors in Rheumatoid
Arthritis. PLoS ONE 2016, 11, e0163087. [CrossRef] [PubMed]
85. Zamani, B.; Golkar, H.R.; Farshbaf, S.; Emadi-Baygi, M.; Tajabadi-Ebrahimi, M.; Jafari, P.; Akhavan, R.; Taghizadeh, M.;
Memarzadeh, M.R.; Asemi, Z. Clinical and metabolic response to probiotic supplementation in patients with rheumatoid arthritis:
A randomized, double-blind, placebo-controlled trial. Int. J. Rheum. Dis. 2016, 19, 869–879. [CrossRef] [PubMed]
86. Sun, H.; Guo, Y.; Wang, H.; Yin, A.; Hu, J.; Yuan, T.; Zhou, S.; Xu, W.; Wei, P.; Yin, S.; et al. Gut commensal Parabacteroides distasonis
alleviates inflammatory arthritis. Gut 2023. [CrossRef]
87. Zhao, Q.; Ren, H.; Yang, N.; Xia, X.; Chen, Q.; Zhou, D.; Liu, Z.; Chen, X.; Chen, Y.; Huang, W.; et al. Bifidobacterium
pseudocatenulatum-Mediated Bile Acid Metabolism to Prevent Rheumatoid Arthritis via the Gut–Joint Axis. Nutrients 2023, 15,
255. [CrossRef] [PubMed]
88. Alipour, B.; Homayouni-Rad, A.; Vaghef-Mehrabany, E.; Sharif, S.K.; Vaghef-Mehrabany, L.; Asghari-Jafarabadi, M.; Nakhjavani,
M.R.; Mohtadi-Nia, J. Effects of Lactobacillus casei supplementation on disease activity and inflammatory cytokines in rheumatoid
arthritis patients: A randomized double-blind clinical trial. Int. J. Rheum. Dis. 2014, 17, 519–527. [CrossRef]
89. Amdekar, S.; Singh, V.; Kumari, A.; Sharma, P.; Singh, R.; Vaghef-Mehrabany, E.; Homayouni-Rad, A.; Alipour, B.; Sharif,
S.-K.; Vaghef-Mehrabany, L.; et al. Lactobacillus casei and Lactobacillus acidophilus Regulate Inflammatory Pathway and Improve
Antioxidant Status in Collagen-Induced Arthritic Rats. J. Interf. Cytokine Res. 2013, 33, 1–8. [CrossRef]
90. Amdekar, S.; Singh, V.; Singh, R.; Sharma, P.; Keshav, P.; Kumar, A. Lactobacillus casei reduces the Inflammatory Joint Damage
Associated with Collagen-Induced Arthritis (CIA) by Reducing the Pro-Inflammatory Cytokines: Lactobacillus Casei: COX-2
Inhibitor. J. Clin. Immunol. 2011, 31, 147–154. [CrossRef]
91. Pružinská, K.; Slovák, L.; Dráfi, F.; Poništ, S.; Juránek, I.; Chrastina, M.; Švík, K.; Strojný, L.; Ambro, L.; Bauerová, K. Enhanced
Anti-Inflammatory Effect of the Combination of Lactiplantibacillus plantarum LS/07 with Methotrexate Compared to Their
Monotherapies Studied in Experimental Arthritis. Molecules 2022, 28, 297. [CrossRef] [PubMed]

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