Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis.

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-driven Th17 response.

Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell-mediated experimental arthritis and evaluated modulation of type II collagen (CII)-reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease-induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell-driven arthritis through induction of Ag-specific Th17 response.

Toll-like receptor 2 controls acute immune complex-driven arthritis in mice by regulating the inhibitory Fcγ receptor IIB.

OBJECTIVE: Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. METHODS: Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined. RESULTS: While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages. CONCLUSION: TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

Destructive role of myeloid differentiation factor 88 and protective role of TRIF in interleukin-17-dependent arthritis in mice.

OBJECTIVE: Increasing evidence indicates the involvement of Toll-like receptors (TLRs) in the progression of arthritis; however, the contribution of the two signaling pathways used by TLRs, which are mediated by myeloid differentiation factor 88 (MyD88) and TRIF, remains unclear. The objective of this study was to investigate the specific roles of MyD88 and TRIF in chronic experimental arthritis and the accompanying adaptive immune responses. METHODS: Chronic arthritis was induced in wild-type, MyD88(-/-) , and Trif(lps2) (TRIF(-/-) ) mice by repetitive intraarticular injections of streptococcal cell wall (SCW) fragments. SCW-specific T cell and B cell responses, joint swelling, and histopathologic changes were analyzed during chronic arthritis. RESULTS: Both MyD88 and TRIF pathways contributed to antigen-specific T cell proliferation and antibody production, with the MyD88 pathway playing the dominant role. The severity of joint swelling and synovial inflammation, as well as the histopathologic damage to cartilage and bone, was strongly dependent on MyD88 signaling, whereas TRIF was redundant. MyD88 signaling was critical for the development of pathogenic T cell response (i.e., interleukin-17 [IL-17] production) in response to SCW antigen. Interestingly, when the T cell-dependent phase was prolonged, TRIF signaling appeared to down-regulate bone erosion, an effect accompanied by an inhibitory effect on IL-17 production. CONCLUSION: This study reveals a central role of MyD88 and a counterregulatory function of TRIF in T cell-driven arthritis. The findings provide a rationale for a pathway-specific interference in order to block the pathogenic features and to preserve or stimulate the beneficial aspects of TLR signaling.

Shift from toll-like receptor 2 (TLR-2) toward TLR-4 dependency in the erosive stage of chronic streptococcal cell wall arthritis coincident with TLR-4-mediated interleukin-17 production.

OBJECTIVE: Toll-like receptors (TLRs) may activate innate and adaptive immune responses in rheumatoid arthritis (RA) through recognition of microbial as well as endogenous ligands that have repeatedly been found in arthritic joints. The objective of this study was to investigate the involvement of TLR-2 and TLR-4 in the development of chronic destructive streptococcal cell wall (SCW)-induced arthritis, in which interleukin-1 (IL-1)/IL-17-dependent T cell-driven pathologic changes replace the macrophage-driven acute phase. METHODS: Chronic SCW arthritis was induced by 4 repeated intraarticular injections of SCW fragments in wild-type, TLR-2(-/-), and TLR-4(-/-) mice. Clinical, histopathologic, and immunologic parameters of arthritis were evaluated. RESULTS: The TLR-2 dependency of joint swelling during the acute phase was shifted to TLR-4 dependency during the chronic phase. Persistent joint inflammation in the latter phase of the model was significantly suppressed in TLR-4(-/-) mice. In the chronic phase, TLR-4 actively contributed to matrix metalloproteinase (MMP)-mediated cartilage destruction and to osteoclast formation, since the expression of the MMP-specific aggrecan neoepitope VDIPEN and the osteoclast marker cathepsin K was significantly reduced in TLR-4(-/-) mice. Furthermore, TLR-4(-/-) mice expressed less IL-1beta, tumor necrosis factor alpha, IL-6, and IL-23, cytokines that are implicated in IL-17 production. Accordingly, SCW-specific IL-17 production was found to be dependent on TLR-4 activation, since T cells from arthritic TLR-4(-/-) mice produced markedly less IL-17 upon SCW stimulation, whereas interferon-gamma production remained unaffected. CONCLUSION: These data indicate the involvement of TLR-4 in the chronicity and erosive character of arthritis coincident with the antigen-specific IL-17 response. The high position of TLR-4 in the hierarchy of erosive arthritis provides an interesting therapeutic target for RA.

T cell dependence of chronic destructive murine arthritis induced by repeated local activation of Toll-like receptor-driven pathways: crucial role of both interleukin-1beta and interleukin-17.

OBJECTIVE: The pathogenesis of rheumatoid arthritis is often linked to bacterial infections. The present study was undertaken to develop a mouse model of chronic destructive arthritis induced by repeated intraarticular (IA) exposure to bacterial cell wall fragments and to investigate the cytokine dependence of this model. METHODS: Mice that were deficient in various cytokines were injected IA with cell wall fragments of Streptococcus pyogenes on days 0, 7, 14, and 21. The development of chronic destructive arthritis was compared between groups of mice lacking different cytokines, to assess which cytokines were crucial for development of chronic destructive arthritis. RESULTS: Repeated exposure of a joint to S pyogenes cell wall fragments resulted in the development of chronic destructive arthritis. In mice deficient in recombination-activating gene 2, streptococcal cell wall (SCW)-directed T cell reactivity was found and chronic arthritis did not develop, implicating T cells in the generation of chronic SCW-induced arthritis. Interleukin-17 (IL-17) receptor-deficient mice showed a reduction of joint destruction in the chronic stage, implicating a detrimental role of the recently discovered IL-17-producing T helper cells (Th17 cells). IL-23 expression was apparent during the late stages of arthritis. Joint swelling was no longer dependent on tumor necrosis factor alpha (TNFalpha) after the last flare, and pronounced cartilage damage was found after 28 days in TNFalpha-deficient mice. In contrast, IL-1beta-deficient mice were fully protected against joint swelling and cartilage and bone destruction during the late stages of disease. CONCLUSION: These findings indicate that the TNFalpha dependence of arthritis is lost during the erosive stage, when Th17 cells become crucial. IL-1beta dependence remains strong, consistent with its pivotal role in the generation of Th17 cells.

Interleukin-18 promotes joint inflammation and induces interleukin-1-driven cartilage destruction.

Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.10(7) pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1beta generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.

Association of interleukin-18 expression with enhanced levels of both interleukin-1beta and tumor necrosis factor alpha in knee synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: To examine the expression patterns of interkeukin-18 (IL-18) in synovial biopsy tissue of patients with rheumatoid arthritis (RA), and to determine whether expression of this primary cytokine is related to the expression of other cytokines and adhesion molecules and related to the degree of joint inflammation. METHODS: Biopsy specimens of knee synovial tissue either without synovitis (n = 6) or with moderate or severe synovitis (n = 11 and n = 12, respectively) were obtained from 29 patients with active RA. Paraffin-embedded, snap-frozen sections were used for immunohistochemical detection of IL-18, tumor necrosis factor alpha (TNFalpha), IL-1beta, IL-12, and IL-17. Furthermore, adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, and cell markers CD3, CD14, and CD68 were stained. RESULTS: IL-18 staining was detectable in 80% of the RA patients, in both the lining and sublining of the knee synovial tissue. IL-18 expression in the synovial tissue was strongly correlated with the expression of IL-1beta (in the sublining r = 0.72, in the lining r = 0.71; both P < 0.0001) and TNFalpha (in the sublining r = 0.59, P < 0.0007, and in the lining r = 0.68, P < 0.0001). In addition, IL-18 expression in the sublining correlated with macrophage infiltration (r = 0.64, P < 0.0007) and microscopic inflammation scores (r = 0.78, P < 0.0001), and with the acute-phase reaction as measured by the erythrocyte sedimentation rate (r = 0.61, P < 0.0004). Interestingly, RA synovial tissue that coexpressed IL-18 and IL-12 demonstrated enhanced levels of the Th1-associated cytokine IL-17. CONCLUSION: Our results show that expression of IL-18 is associated with that of IL-1beta and TNFalpha and with local inflammation in the synovial tissue of patients with RA. In addition, synovial IL-18 expression correlates with the acute-phase response. These data indicate that IL-18 is a primary proinflammatory cytokine in RA that drives the local production of IL-1beta and TNFalpha.