Abstract
Dysregulation of the intestinal epithelial barrier in genetically susceptible individuals may lead to both intestinal and extraintestinal autoimmune disorders. There is emerging literature on the role of microbiota changes in the pathogenesis of systemic rheumatic diseases such as rheumatoid arthritis, spondyloarthropathies, and connective tissue diseases. Although the role of the gastrointestinal tract in the pathogenesis of spondyloartropathies is well defined and many studies underline the importance of gastrointestinal inflammation in modulating local and systemic inflammation, the data are inconclusive regarding the effect of dysbiosis on rheumatoid arthritis and connective tissue diseases. This review aims to summarize current data on the role of the gastrointestinal involvement and intestinal microbiota in the pathogenesis of systemic rheumatic disease.
Introduction
The gastrointestinal tract regulates the trafficking of macromolecules between the environment and the host through an epithelial barrier mechanism . The intestinal epithelial barrier, together with the gut-associated lymphoid tissue and neuroendocrine network, plays a fundamental role in controlling the equilibrium between tolerance and immunity to non-self-antigens . Dysregulation of this organ in genetically susceptible individuals may lead to both intestinal and extraintestinal autoimmune disorders . The gastrointestinal tract is colonized by many trillions of microbes that represent the so-called microbiota . These microorganisms are not mere bystanders because they play metabolic, trophic, and protective roles and participate in normal human development and homeostasis . The perturbation of this interaction results in dysbiosis that in turn may influence the susceptibility of the host to many immune-mediated diseases. There is emerging literature on the role of microbiota changes in the pathogenesis of systemic autoimmune disease such as type 1 diabetes, celiac disease, ankylosing spondylitis (AS), and rheumatoid arthritis (RA). This review aims to summarize the current data on the role of the gastrointestinal involvement and intestinal microbiota in the pathogenesis of systemic rheumatic disease.
Rheumatoid arthritis
RA is a chronic autoimmune inflammatory disorder affecting 0.5–1% of the population . Although the cause of RA remains elusive, it has been demonstrated that in genetically predisposed subjects carrying the (Human Leukocyte Antigen) HLA–DRB1*04 cluster, environmental factors such as the oral and gut microbiota may lead to the abnormal activation of the innate and adaptive immunity, which involve cellular and humoral immune responses . This activation leads to the formation of autoantibodies [rheumatoid factors (RF) and antibodies against citrullinated peptides/proteins (ACPA)] and invasion of T cells and B cells into the synovium ( Fig. 1 ). Loss of intestinal integrity and increased intestinal permeability have been demonstrated in RA patients, particularly those receiving non steroid anti-inflammatory drugs (NSAIDs) and those with active joint disease . Alteration of intestinal permeability may facilitate antigenic absorption and contribute to the persistence of the disease; however, more studies are required to further clarify this specific issue.
Gastrointestinal microbiota in rheumatoid arthritis patients
Many studies have asserted the role of gastrointestinal microbiota in the pathogenesis of RA. This is suggested by the evidence that the oral bacteria Porphyromonas gingivalis (PG) is capable of inducing the local production of citrullinated protein . PG is a leading pathogen of chronic periodontitis and produces a unique bacterial enzyme, PG peptidyl-arginine deiminase (PPAD), which has the ability to convert arginine residues in proteins to citrulline . Because protein citrullination alters the protein structure, PPAD may be involved in the alteration of the host signaling network and immune evasion ( Fig. 1 ).
Unlike in the case of the oral microbiome, conflicting data emerge from the studies of the intestinal microbiome in animal models of RA and in humans. Data from murine studies support the role of microbiota in influencing arthritis susceptibility. Liu X et al. performed 16S rRNA sequencing to characterize the gut microbiota of DBA1 mice that did or did not develop collagen-induced arthritis and demonstrated marked and significant divergence in the distribution of microbiota after arthritis induction. Mice susceptible to collagen-induced arthritis (CIA) showed enriched operational taxonomic units (OTUs) affiliated with the genus Lactobacillus prior to arthritis onset. With disease development, the abundance of OTUs affiliated with the families Bacteroidaceae , Lachnospiraceae , and S24-7 significantly increased in CIA-susceptible mice. Notably, germ-free mice conventionalized with the microbiota from CIA-susceptible mice showed a higher frequency of arthritis induction than those conventionalized with the microbiota from CIA-resistant mice. In this study, the serum concentration of interleukin (IL)-17 and the proportions of CD8 + T cells and Th17 lymphocytes in the spleen were significantly higher in the susceptible group. Using genetic approaches, Block et al. demonstrated that gut microbiota regulates arthritis through follicular helper T (Tfh) cells, which are defective in antibiotic-treated mice, but not Th17 cells. In particular, the authors investigated the contribution of Th17 and Tfh cells in the induction of arthritis in a K/BxN autoimmune arthritis model that is dependent on segmented filamentous bacteria for the induction of the autoimmune phenotype. The authors particularly studied how microbiota modulates the differentiation of Th17 and Tfh cells. Using genetic approaches, they demonstrated that IL-17 is dispensable for arthritis and that antibiotic treatment inhibits disease in IL-17-deficient animals; this suggests that the gut microbiota regulates arthritis independent of Th17 cells. In contrast, conditional deletion of Bcl6 in T cells blocked Tfh cell differentiation and arthritis development. Furthermore, Tfh cell differentiation was defective in antibiotic-treated mice. Taken together, these findings indicate that gut microbiota regulates arthritis through Tfh but not Th17 cells ( Fig. 1 ).
Data from human studies are more mixed. On the one hand, there is much evidence that supports the association between RA and the intestinal microbiota; bacteria such as Mycoplasma fermentans , Escherichia coli , and Proteus mirabilis have been reported to initiate the disease. On the other hand, it has been demonstrated that gastrointestinal and urogenital infections are associated with a significantly lowered risk of RA and that patients with RA exhibit decreased gut microbial diversity compared with controls . In particular, it has been proved that expansion of rare taxa such as Actinobacteria along with decrease in abundant taxa occur in patients with RA compared with controls . In this study, three genera, Collinsella , Eggerthella , and Faecalibacterium , were found to be associated with RA; abundance of Collinsella was strongly correlated with the production of the proinflammatory cytokine IL-17A . The role of Collinsella in altering gut permeability and disease severity has been confirmed in experimental arthritis . A more recent study evaluated human fecal Lactobacillus community and its relationship with RA . Samples obtained from RA patients and healthy individuals were analyzed and demonstrated that fecal microbiota of RA patients contained significantly more Lactobacillus than that of the control group. In a recently published paper, compositional and functional alterations in RA-associated gut and oral microbiomes were observed, which were partly relieved by disease modifying anti-rheumatic drugs (DMARD) treatment . To delineate the features of the RA-associated gut microbiome, the authors performed a microbiome genome-wide association study identifying 117,219 gene markers that were differentially enriched in RA patients and controls and clustered these genes into metagenomic linkage groups (MLGs) on the basis of their correlated abundance variation among samples. Different from that of the controls, the RA-enriched MLGs formed a large cluster containing strains such as Clostridium asparagiforme , Gordonibacter pamelaeae , Eggerthella lenta , and Lachnospiraceae bacterium and small clusters or single MLGs containing strains such as Bifidobacterium dentium , Lactobacillus sp., and Ruminococcus lactaris . The RA gut was rich in gram-positive bacteria and depleted of gram-negative bacteria, including some Proteobacteria and gram-negative Firmicutes of the Veillonellaceae family. In accordance with the relative depletion of gram-negative bacteria in RA, enrichment of the reductive acetyl-CoA pathway was present in individuals with RA together with modules for converting acetate to methane. Conversely, modules involved in lipopolysaccharide biosynthesis, lipopolysaccharide transport, and secretion systems (type II, type IV, and type VI) were more abundant in samples from healthy controls. Furthermore, several correlations with prognostic or diagnostic factors were found. For instance, the abundance of RA-enriched MLGs such as C . asparagiforme and Bacteroides sp. was positively correlated with titers of immunoglobulin A (IgA) and unclassified Lactobacillus sp. (most likely L . salivarius ) was positively correlated with titers of the major serum immunoglobulin, IgG. A positive correlation between Enterococcus faecalis and platelet count was also detected. Bacteroides plebeius , Streptococcus australis , Veillonella sp., and Haemophilus parainfluenzae were found to be negatively correlated with titers of the RA-specific autoantibodies ACPA and rheumatoid factor (RF). Furthermore, gut MLGs fit the clinical indices, suggesting that the gut MLGs to some extent could reflect the clinical variations among these subjects. Interestingly, molecular mimicry between RA-associated antigens such as Collagen XI and HLA–DRB1*0401 and gut microbial genes from Clostridium , Eggerthella , Bacteroides , and Citrobacter was also observed. The above-mentioned studies were performed on patients with established RA. However, early identification of RA is a crucial step for controlling the progression of the disease, and studying the bacterial composition in early stages of disease may provide interesting information on its pathogenesis. Fecal microbiota in patients with early RA and patients with Fibromyalgia has been recently studied and compared . In this study, RA patients showed significantly less bifidobacteria and bacteria from the Bacteroides-Porphyromonas-Prevotella group, Bacteroides fragilis subgroup, and Eubacterium rectale-Clostridium coccoides group.
Probiotics are microorganisms that can normalize gut microbiota and are potentially useful to alleviate RA symptoms. Effects of probiotic supplementation on disease activity and inflammatory cytokines in patients with RA was recently studied by Vaghef-Mehrabany . In this study, the disease activity score was significantly decreased by the probiotic supplementation. Tumor necrosis factor (TNF)-α, IL-6, and IL-12 were also significantly decreased in the probiotic group, whereas serum level of the regulatory cytokine IL-10 was increased by the supplementation.
Rheumatoid arthritis
RA is a chronic autoimmune inflammatory disorder affecting 0.5–1% of the population . Although the cause of RA remains elusive, it has been demonstrated that in genetically predisposed subjects carrying the (Human Leukocyte Antigen) HLA–DRB1*04 cluster, environmental factors such as the oral and gut microbiota may lead to the abnormal activation of the innate and adaptive immunity, which involve cellular and humoral immune responses . This activation leads to the formation of autoantibodies [rheumatoid factors (RF) and antibodies against citrullinated peptides/proteins (ACPA)] and invasion of T cells and B cells into the synovium ( Fig. 1 ). Loss of intestinal integrity and increased intestinal permeability have been demonstrated in RA patients, particularly those receiving non steroid anti-inflammatory drugs (NSAIDs) and those with active joint disease . Alteration of intestinal permeability may facilitate antigenic absorption and contribute to the persistence of the disease; however, more studies are required to further clarify this specific issue.
Gastrointestinal microbiota in rheumatoid arthritis patients
Many studies have asserted the role of gastrointestinal microbiota in the pathogenesis of RA. This is suggested by the evidence that the oral bacteria Porphyromonas gingivalis (PG) is capable of inducing the local production of citrullinated protein . PG is a leading pathogen of chronic periodontitis and produces a unique bacterial enzyme, PG peptidyl-arginine deiminase (PPAD), which has the ability to convert arginine residues in proteins to citrulline . Because protein citrullination alters the protein structure, PPAD may be involved in the alteration of the host signaling network and immune evasion ( Fig. 1 ).
Unlike in the case of the oral microbiome, conflicting data emerge from the studies of the intestinal microbiome in animal models of RA and in humans. Data from murine studies support the role of microbiota in influencing arthritis susceptibility. Liu X et al. performed 16S rRNA sequencing to characterize the gut microbiota of DBA1 mice that did or did not develop collagen-induced arthritis and demonstrated marked and significant divergence in the distribution of microbiota after arthritis induction. Mice susceptible to collagen-induced arthritis (CIA) showed enriched operational taxonomic units (OTUs) affiliated with the genus Lactobacillus prior to arthritis onset. With disease development, the abundance of OTUs affiliated with the families Bacteroidaceae , Lachnospiraceae , and S24-7 significantly increased in CIA-susceptible mice. Notably, germ-free mice conventionalized with the microbiota from CIA-susceptible mice showed a higher frequency of arthritis induction than those conventionalized with the microbiota from CIA-resistant mice. In this study, the serum concentration of interleukin (IL)-17 and the proportions of CD8 + T cells and Th17 lymphocytes in the spleen were significantly higher in the susceptible group. Using genetic approaches, Block et al. demonstrated that gut microbiota regulates arthritis through follicular helper T (Tfh) cells, which are defective in antibiotic-treated mice, but not Th17 cells. In particular, the authors investigated the contribution of Th17 and Tfh cells in the induction of arthritis in a K/BxN autoimmune arthritis model that is dependent on segmented filamentous bacteria for the induction of the autoimmune phenotype. The authors particularly studied how microbiota modulates the differentiation of Th17 and Tfh cells. Using genetic approaches, they demonstrated that IL-17 is dispensable for arthritis and that antibiotic treatment inhibits disease in IL-17-deficient animals; this suggests that the gut microbiota regulates arthritis independent of Th17 cells. In contrast, conditional deletion of Bcl6 in T cells blocked Tfh cell differentiation and arthritis development. Furthermore, Tfh cell differentiation was defective in antibiotic-treated mice. Taken together, these findings indicate that gut microbiota regulates arthritis through Tfh but not Th17 cells ( Fig. 1 ).
Data from human studies are more mixed. On the one hand, there is much evidence that supports the association between RA and the intestinal microbiota; bacteria such as Mycoplasma fermentans , Escherichia coli , and Proteus mirabilis have been reported to initiate the disease. On the other hand, it has been demonstrated that gastrointestinal and urogenital infections are associated with a significantly lowered risk of RA and that patients with RA exhibit decreased gut microbial diversity compared with controls . In particular, it has been proved that expansion of rare taxa such as Actinobacteria along with decrease in abundant taxa occur in patients with RA compared with controls . In this study, three genera, Collinsella , Eggerthella , and Faecalibacterium , were found to be associated with RA; abundance of Collinsella was strongly correlated with the production of the proinflammatory cytokine IL-17A . The role of Collinsella in altering gut permeability and disease severity has been confirmed in experimental arthritis . A more recent study evaluated human fecal Lactobacillus community and its relationship with RA . Samples obtained from RA patients and healthy individuals were analyzed and demonstrated that fecal microbiota of RA patients contained significantly more Lactobacillus than that of the control group. In a recently published paper, compositional and functional alterations in RA-associated gut and oral microbiomes were observed, which were partly relieved by disease modifying anti-rheumatic drugs (DMARD) treatment . To delineate the features of the RA-associated gut microbiome, the authors performed a microbiome genome-wide association study identifying 117,219 gene markers that were differentially enriched in RA patients and controls and clustered these genes into metagenomic linkage groups (MLGs) on the basis of their correlated abundance variation among samples. Different from that of the controls, the RA-enriched MLGs formed a large cluster containing strains such as Clostridium asparagiforme , Gordonibacter pamelaeae , Eggerthella lenta , and Lachnospiraceae bacterium and small clusters or single MLGs containing strains such as Bifidobacterium dentium , Lactobacillus sp., and Ruminococcus lactaris . The RA gut was rich in gram-positive bacteria and depleted of gram-negative bacteria, including some Proteobacteria and gram-negative Firmicutes of the Veillonellaceae family. In accordance with the relative depletion of gram-negative bacteria in RA, enrichment of the reductive acetyl-CoA pathway was present in individuals with RA together with modules for converting acetate to methane. Conversely, modules involved in lipopolysaccharide biosynthesis, lipopolysaccharide transport, and secretion systems (type II, type IV, and type VI) were more abundant in samples from healthy controls. Furthermore, several correlations with prognostic or diagnostic factors were found. For instance, the abundance of RA-enriched MLGs such as C . asparagiforme and Bacteroides sp. was positively correlated with titers of immunoglobulin A (IgA) and unclassified Lactobacillus sp. (most likely L . salivarius ) was positively correlated with titers of the major serum immunoglobulin, IgG. A positive correlation between Enterococcus faecalis and platelet count was also detected. Bacteroides plebeius , Streptococcus australis , Veillonella sp., and Haemophilus parainfluenzae were found to be negatively correlated with titers of the RA-specific autoantibodies ACPA and rheumatoid factor (RF). Furthermore, gut MLGs fit the clinical indices, suggesting that the gut MLGs to some extent could reflect the clinical variations among these subjects. Interestingly, molecular mimicry between RA-associated antigens such as Collagen XI and HLA–DRB1*0401 and gut microbial genes from Clostridium , Eggerthella , Bacteroides , and Citrobacter was also observed. The above-mentioned studies were performed on patients with established RA. However, early identification of RA is a crucial step for controlling the progression of the disease, and studying the bacterial composition in early stages of disease may provide interesting information on its pathogenesis. Fecal microbiota in patients with early RA and patients with Fibromyalgia has been recently studied and compared . In this study, RA patients showed significantly less bifidobacteria and bacteria from the Bacteroides-Porphyromonas-Prevotella group, Bacteroides fragilis subgroup, and Eubacterium rectale-Clostridium coccoides group.
Probiotics are microorganisms that can normalize gut microbiota and are potentially useful to alleviate RA symptoms. Effects of probiotic supplementation on disease activity and inflammatory cytokines in patients with RA was recently studied by Vaghef-Mehrabany . In this study, the disease activity score was significantly decreased by the probiotic supplementation. Tumor necrosis factor (TNF)-α, IL-6, and IL-12 were also significantly decreased in the probiotic group, whereas serum level of the regulatory cytokine IL-10 was increased by the supplementation.
Spondyloarthritis
Spondyloarthritis (SpA) is a generic term used for indicating inflammatory diseases that involve both the joints and entheses (the sites where the ligaments and tendons attach to the bones) . The prototype of these diseases is AS. Other diseases include reactive arthritis, psoriatic arthritis (PsA), and arthritis associated with inflammatory bowel disease. Involvement of the gastrointestinal tract in the pathogenesis of SpA is strongly suggested because subclinical gut inflammation is present in up to 60% of patients with SpA . SpA-associated gut inflammation has been demonstrated to be an important prognostic factor in SpA because it is associated with more extensive bone marrow edema of the sacroiliac joints and a higher risk of progression to AS and of developing Crohn’s disease (CD) .
Histologic changes in the gut of SpA patients
Historically, gut inflammation in SpA patients has been classified into two categories: acute inflammation, which resembles a self-limiting bacterial enterocolitis, and chronic inflammation, which displays altered intestinal architecture with varying degrees of infiltration of inflammatory mononuclear cells that are sometimes aggregated in lymphoid follicles, resembling the ileo-colitis observed in CD . More recently, other relevant intestinal alterations have been demonstrated in AS gut: (i) goblet cell hyperplasia and increased mucin production , (ii) activation of paneth cells (PC) and hyperplasia of PC , particularly in PsA patients, (iii) detachment of epithelial cells from the basal membrane , and (iv) occurrence of vasculitic lesions that are mainly represented by intense hemorrhagic extravasation of the lamina propria. Interestingly, similar tissue damages have been described as a consequence of the response of epithelial cells to bacterial toxins . The relative contribution of the genetic factors involved in SpA pathogenesis in influencing these histologic changes cannot be excluded and requires further study. The diagnosis of intestinal inflammation in SpA is essentially made using endoscopy as reliable biomarkers are lacking. Recently, however, elevated serum calprotectin and C-reactive protein (CRP) were demonstrated to be independently associated with microscopic bowel inflammation . During intestinal inflammation, fecal calprotectin is elevated because of the migration of neutrophils to the inflamed intestinal mucosa . In this study, the authors suggested a screening approach where initially serum calprotectin, CRP, and, if necessary, fecal calprotectin are assessed. The model using this scenario provided an area of 74.4% under the Receiver Operating Characteristic (ROC) curve for the detection of bowel inflammation .
Immune alterations in the gut of SpA patients
In addition to the presence of histologic alterations, the intestine of SpA patients is characterized by the abnormal activation of the innate and adaptive immune system and specific immunologic signatures ( Fig. 2 ). Subclinical gut inflammation in patients with PsA seems to be characterized by the overexpression of IL-9 and presence of fully developed Th9, Th17, and Th22 responses . In particular, in PsA, gut PC express a specific IL-9 receptor, and stimulation of isolated epithelial cells with IL-9 up-regulates the expression of the antimicrobial peptide α-defensin 5 and cytokines, such as IL-23 and IL-9, which suggests the possibility of a functional autocrine loop that involves IL-9/IL-9R . Th9 cells also increase in the peripheral blood and synovial tissues in PsA and express α4β7+, indicating the intestinal origin of these cells . Conversely, in patients with AS, IL-23 over-expression dominates and is mainly detected in the presence of PC without a clearly defined Th17, Th1, and/or Th9 polarization and Treg expansion .
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