Several studies have shown that the composition of intestinal microbiota is perturbed in patients with inflammatory arthritis such as new-onset as well as chronic RA5,6,7,9,40. The development of autoimmunity in RA starts several years before the appearance of the clinical signs, a process that commensal microbiota may be able to promote41,42,43. Therefore, it is important to understand whether the observed perturbations in the intestinal microbiota precede the onset of inflammatory arthritis or are merely a consequence of disease. While this remains to be determined in humans at-risk of developing inflammatory arthritis, we here show that in experimental arthritis, marked changes in the intestinal microbiota occur in the preclinical immune-priming phase and precede the onset of inflammatory arthritis. A recent study showed that the composition of microbiota prior to arthritis onset differs between the CIA–susceptible and –resistant mice44. This study found that the family Lactobacillaceae was more abundant in CIA-susceptible mice, whereas Desulfovibrionaceae and Lachnospiraceae were more abundant in CIA–resistant mice44. Moreover, by comparing the fecal bacterial composition in the immunized mice before and after the onset of arthritis, it was shown that the families Bacteroidaceae, Lachnospiraceae and S24-7 significantly increased as arthritis initiated44. Our studies focused on the preclinical phase of arthritis and show that this phase is characterized by decreased S24-7 and Bacteroidaceae and increased Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae. Increased Lachnospiraceae has previously also been demonstrated in RA patients with varying disease duration7. Our data demonstrate that the increase in Lachnospiraceae occurs before the onset of arthritis and is induced by the immunization during the preclinical phase. Another study compared the microbiome of healthy non-immunized mice and mice with CIA. This study found that Clostridiales, Lachnospiraceae and Ruminoccocus gnavus were more abundant in the mice with established CIA compared to the healthy mice45. Interestingly, we found these taxa to be increased in the preclinical phase and before the onset of arthritis. Altogether, these observations suggest that early perturbations of the intestinal microbiota during the immune-priming phase may contribute to the initiation of inflammatory arthritis.

We previously showed that treatment with Tobramycin resulted in a near-complete elimination of the genera Helicobacter and Flexispira (both belonging to the family Helicobacteraceae) and suppressed arthritis in IL-1Ra-deficient mice46. In addition, a strong and highly significant reduction in the genus Clostridium was observed, which suggests that these bacteria may promote intestinal Th17 cell differentiation and arthritis46. In the present study, we observed a strong and significant increase in the order Clostridiales after immunization with collagen. However, a large part of the order Clostridiales could not be assigned to a higher taxonomic level and the exact effects on the genus Clostridium remain to be clarified.

Previous studies showed that Prevotella copri is expanded in patients with new-onset RA6,8. In addition, it was shown that transfer of Prevotella-dominated microbiota of RA patients to SKG mice results in increased numbers of intestinal Th17 cells and exacerbates arthritis compared with mice receiving fecal microbiota from healthy controls8. Identification of the exact Prevotella species altered upon immunization in CIA mice was not possible using 16S rRNA gene sequencing due to inherently limited resolution of this technique. Furthermore, none of the observed OTUs related to Prevotella in our database could be assigned to P. copri. Therefore, we performed P. copri-specific qPCR using species specific primers6 to detect P. copri with a higher sensitivity (primer sequences in Suppl. Table S2). While our positive control (DNA isolated from anaerobically grown P. copri) showed a positive signal on qPCR with a Ct value of 17.7 cycles, P. copri DNA was detected at very low levels (Ct value of >33 cycles) in fecal DNA of the mice and was not different between naïve and immunized mice. Therefore, the abundance of P. Copri was not altered in our mice by CII immunization.

Loss of intestinal microbial diversity and richness coincides with autoimmune diseases such as diabetes, RA and PsA in patients5,6,47. Our data suggest that the preclinical phase of experimental arthritis is not accompanied by significant changes in the microbial diversity and richness. It is tempting to speculate that the reduced microbial diversity reported in human autoimmune disease may be a consequence of the ongoing inflammation at the time of sampling. While our study was able to identify robust changes in the intestinal microbiota that precede the onset of inflammatory arthritis, it is important to expand and strengthen such studies with shotgun sequencing coupled with transcriptomics and metabolomics approaches to understand the full functional capacity of the microbiome during the preclinical phase of arthritis. More importantly, similar studies are warranted in humans at risk of developing autoimmune arthritis before the onset of inflammation to unravel the relevance of the microbiome in predisposition or the inductive phase of the disease.

Intestinal LP is a major source of Th17 cells in the body29,48. Previous studies have shown that alterations of the gut commensal bacteria have strong effects on Th17 cell differentiation29,48. Commensal microbiota have been shown to promote the initiation of spontaneous arthritis in IL-1Ra−/− mice as well as in K/BxN mice, which bear a transgenic auto-reactive T cell receptor27,28. In non-transgenic animal models, treatment with a cocktail of vancomycin, neomycin and metronidazole before the induction of antigen-induced arthritis resulted in milder disease32. On the other hand, gut microbiota-induced IL-1 and IL-6 can trigger the respective cytokine receptors on B cells to promote differentiation of IL-10-producing regulatory B cells and restrict antigen-induced arthritis32. However, it is not clear whether modulation of intestinal microbiota after the onset of arthritis can still alter the T cell phenotype and modify the progression of arthritis.

In this study, we employed a widely-used model of inflammatory arthritis induced in non-transgenic mice to assess the role of intestinal microbiota during already established disease. We show here for the first time that elimination of intestinal microbiota during established CIA attenuates inflammatory arthritis. This is in line with earlier reports that oral antibiotic treatments with sulfasalazine and minocycline reduce RA disease activity in patients with disease duration of <1 year49,50. Elimination of microbiota after the onset of arthritis resulted in suppression of intestinal SAA1, SAA2 and IL-22 expression and a specific reduction of LP Th17 cells accompanied with reduced Th17 cell abundance in joint-draining lymph nodes of the CIA mice. The potential of intestinal T cells to migrate to peripheral lymph nodes, as reported before, may explain these observations51. Our data also show for the first time a direct correlation between the abundance of LP Th17 cells and the severity of arthritis, which further supports a continued pro-inflammatory gut-joint axis during established arthritis.

Previous studies identified SFB as potent inducers of Th17 cell differentiation promoting arthritis development in the T cell-mediated K/BxN model28,29. Elimination of SFB by ABX treatment in our study is consistent with reduced LP Th17 cells and suppressed expression of SAA1 and SAA2, which were recently reported to act on Th17 cells to further enhance their differentiation and effector function30. Our data suggest that the observed Th17 cell reduction upon antibiotic treatment in our study is at least partially due to the elimination of SFB, although the effect of other microbiota cannot be excluded. Interestingly, although SAA1 and SAA2 were the most abundant SAA isoforms in the intestine, SAA3 appeared to be the prominent isoform in the target tissue, i.e. synovium, and its expression was similarly affected by the microbiota. While SAA1/SAA2 were shown to promote intestinal Th17 cells, it is not known if SAA has a similar effect on Th17 cells residing in the synovium. Several studies have shown the relevance of SAA as a marker of RA disease activity and its association with disease-relevant autoantibodies and acute phase proteins52,53,54,55. In addition, several RA therapies have been shown to influence circulating SAA levels during treatment52,53,54,55. Furthermore, SAA is also a good indicator of cardiovascular and renal involvement in patients with RA56. Besides being a valuable marker for disease activity, SAA has been shown to mediate inflammatory and angiogenic mechanisms, likely through TLR257,58. Our data demonstrate that increased systemic level of SAA precedes the onset of arthritis and can be used as a disease marker even before its clinical onset.

In line with the essential role of IL-22 in intestinal SAA production30, we observed a significant reduction of intestinal IL-22 expression after antibiotic treatment. IL-22 was shown to be highly expressed in synovium of RA patients and serum levels of IL-22 are significantly higher in RA patients compared with healthy controls and correlate with disease activity59,60. In addition, elevated levels of IL-22 in early disease seem to predict erosive progression, suggesting that IL-22 has a role in the pathophysiology of RA61. In patients with PsA, IL-22-producing CD4+ T cells were increased in peripheral blood as well; however, IL-22-positive cells could not be detected in synovial fluid and tissue of PsA patients62.

Although our studies support suppression of intestinal and joint-adjacent Th17 differentiation as an underlying mechanism for attenuation of arthritis upon elimination of intestinal microbiota, involvement of microbiota-induced regulation of other immune cells is not excluded. In this regard, a recent study showed that continuous treatment with ampicillin and vancomycin limited follicular T helper (Tfh) cell differentiation and germinal center formation in K/BxN mice, resulting in reduced production of arthritogenic autoantibodies and less severe arthritis63. These effects appeared to be independent of IL-17, which was found to be dispensable for the disease development in this study, although an earlier study showed that IL-17 blockade could reduce anti-GPI antibody levels and suppress K/BxN arthritis28,63. We observed no effect of ABX treatment on serum levels of anti-mouse collagen type II antibodies in our experiments, suggesting that during ongoing CIA, intestinal microbiota affect Th17 cell differentiation and arthritis without influencing the autoantibody response. Therefore, intestinal microbiota appear to influence different disease mechanisms depending on the disease phase and processes being studied. In addition, by mimicking the effector phase of the K/BxN arthritis using KRN serum-transfer model, we show that intestinal microbiota do not play an important role in K/BxN arthritis anymore once the disease-inducing auto-antibodies have been developed (Supplementary Fig. S6).

In agreement with our findings, IL-17 producing T cells can promote arthritis independent of their influence on antibody production, since transfer of IL-17-producing KRN transgenic T cells into a B-cell-deficient host can enhance arthritis in an IL-17-dependent manner64. In fact, Th17 cells contribute to several pro-inflammatory and tissue-destructive processes during inflammatory arthritis and may represent a relevant target to control the disease12,13. The role of Th17 cells in arthritis is likely to expand beyond its prototypic cytokine IL-17A. The actual requirement of Th17 cells for the microbiota-induced aggravation of arthritis requires, however, further investigation.

It has been described that Th17 cells can transition into a stage in which they produce both IL-17 and IFNγ, after which they may lose their expression of IL-1765. A recent study showed that these IFNγ-producing cells, called ex-Th17 cells or non-classical Th1 cells, are not constrained by regulatory T cells66. In addition, these cells were shown to accumulate in arthritic joint of arthritis patients66, suggesting a role in the pathogenesis of RA. We analyzed the proportions of IL-17+ IFNγ+ double-positive TCRβ+CD4+ cells. We observed a non-significant reduction in the percentage, but not absolute number, of IL-17+ IFNγ+ TCRβ+CD4+ in intestinal lamina propria by antibiotic treatment (Mean ± SEM of control: 1.049% ± 0.2594; ABX: 0.4699% ± 0.1297). Furthermore, while there was no significant effect of antibiotic treatment on the percentage of IL-17+ IFNγ+ TCRβ+CD4+ cells in pLNs, we observed a non-significant reduction in the numbers of these cells (Ctrl: 537.9 ± 228.6; ABX: 240.6 ± 76.38). We cannot exclude the possibility that the Th1 cells, which were significantly reduced in pLNs after ABX-treatment, also included exTh17 cells only producing IFNγ.

Multiple studies showed that Th17 levels are high in treatment-naïve patients with early RA67,68,69. While elevated levels of Th17 cells have been found in RA and PsA patients, reports on the proportion of Th17 cells in patients with established RA are inconsistent62,67,70,71,72,73,74,75. Based on the higher efficacy of IL-17 blockade in PsA compared with RA, it is plausible that the PsA microbiome has a higher potential to induce mucosal Th17 and IL-17 responses. On the other hand, it is possible that stratification of patients with RA based on their gut microbiota and its Th17-inducing potential improves the efficacy of IL-17 inhibition in RA. A side-by-side comparison of matched cohorts of PsA and RA patients will be required to address the exact differences between RA and PsA microbiomes and their relevance for the efficacy of biologic treatments.

In summary, our study suggests that perturbations in the intestinal microbiota precede the onset of arthritis. In addition, we show that elimination of intestinal microbiota during established CIA reduces Th17 cell abundance in intestinal mucosa and joint-draining lymph nodes and attenuates arthritis without affecting autoantibody production. While our study does not advocate the use of antibiotics as a treatment for RA or related inflammatory arthropathies, it supports the notion that inflammatory signals provided by the gut microbiota continue to promote arthritis after its onset. The striking correlation between the abundance of intestinal Th17 cells and the severity of arthritis supports this link. Understanding the exact mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic strategies for inflammatory arthritis.