Rheumatoid arthritis (RA) is a chronic inflammatory, destructive disease of multiple joints that may lead to disability in patients with the progressive form. Accumulating evidence suggests the involvement of a recently identified family of receptors of the immune system, Toll-like receptors (TLRs), in inflammation and destruction driven by multiple cellular players in RA. Expression of TLRs 2, 3, 4, and 7 is enhanced in (early) RA synovial tissue (1, 2), and exogenous as well as endogenous TLR agonists have been detected in the joints of patients with RA (3–7). TLRs generally exert proinflammatory and catabolic effects on nonimmune cells relevant to RA, such as fibroblasts and chondrocytes (8, 9). Furthermore, TLR-2 and TLR-4 on immune cells, such as blood-derived mononuclear cells and dendritic cells, from RA patients are hyperresponsive to their respective ligands (2, 10, 11). Importantly, very recent reports implicate TLR-2 and TLR-4 activation in the spontaneous release of proinflammatory cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) by RA synovial tissue (12, 13).
Some TLR agonists can profoundly enhance arthritis expression upon injection in vivo (12, 14). In animal models of RA, TLRs, in particular TLR-4, drive the expression of inflammatory cytokines and determine the severity of joint inflammation and destruction (12, 15). TLR-4 activation seems to mainly contribute to the chronic phase of arthritis, probably due to the presence of endogenous damage-associated agonists in this phase (12, 16). In this regard, in arthritis induced by repetitive intraarticular injections of bacterial TLR ligands derived from streptococcal cell walls (SCWs), the TLR-2–dependent acute phase shifts toward a TLR-4–driven chronic phase in which TLR-4 determines the antigen-specific production of the pathogenic cytokine IL-17 (17). General inhibition of endosomal TLRs (TLR-3, TLR-7, and TLR-9) was recently reported to ameliorate murine arthritis as well (18).
Despite the great body of evidence indicating the involvement of TLRs in RA, the specific roles and the relative contribution of TLR signaling pathways in the disease are not well understood. TLR signaling involves two main pathways: one through myeloid differentiation factor 88 (MyD88), which is used by all TLRs except TLR-3 and is shared by IL-1 and IL-18 receptors, and the other through TRIF, which is solely engaged by TLR-3 and TLR-4 (19). The MyD88 pathway induces early activation of the NF-κB and activation of the activator protein 1 (AP-1) transcription factors, whereas the TRIF pathway is distinguished by the activation of interferon regulatory factor 3 (IRF-3) and IRF-7, in addition to a late NF-κB response (20–22).
Previous studies in animal models of acute arthritis have indicated the critical role of MyD88 in SCW-induced joint inflammation (23). Studies on the role of MyD88 during chronic T cell–dependent arthritis and the function of TRIF in any in vivo arthritis model are, however, lacking. Such insight would enable the development of optimal strategies for interfering with pathogenic processes driven by multiple TLRs, while preserving the protective features of TLR response.
In the present study, we extensively investigated the specific functions and the relative contribution of MyD88 and TRIF pathways in different aspects of joint pathology in the mouse model of chronic, predominantly TLR-driven SCW-induced arthritis. We also studied the impact of MyD88 and TRIF signaling on antigen-specific adaptive immune responses, with emphasis on T cell IL-17 production, as key pathogenic events in the disease.
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Accumulating evidence indicates the involvement of TLRs in arthritis. Therefore, insight into the mechanisms by which TLRs exert their proinflammatory and destructive functions is relevant for the development of efficient therapeutic strategies. In the present study, we studied the specific roles of the two signaling pathways used by TLRs, MyD88 and TRIF, in chronic T cell–driven and IL-17–driven arthritis induced by repetitive exposure to SCW fragments.
Acute SCW-induced arthritis has previously been demonstrated to be mediated through TLR-2 and, hence, strongly dependent on the MyD88 pathway (23). In contrast, chronic SCW arthritis is independent of TLR-2 and is instead dependent on TLR-4, which controls the production of IL-17 by antigen-specific T cells during the T cell–dependent phase of the disease (17). The present data demonstrate that the MyD88 pathway is critical for the induction of joint swelling and systemic TNFα expression, as well as the antigen-induced adaptive immune responses, during chronic arthritis. An important role of MyD88 in passive (T cell–independent) arthritis induced by the transfer of arthritogenic K/BxN serum has been reported before (31). Here, we provide the first evidence for the involvement of MyD88 in a T cell–driven arthritis model through inducing a Th17 response.
Since MyD88 is also shared by the IL-1 receptor (IL-1R), its role might be related to IL-1R, TLRs, or both. Involvement of IL-1R–induced MyD88 activation in T cell proliferation and antibody production is consistent with a previous report on adaptive responses to a retinal antigen used to induce uveitis in mice (32). Our previous findings on the marked protection of IL-1α/β−/− mice from chronic SCW arthritis support this idea as well (27). On the other hand, since IL-1 deficiency only affects T cell proliferation and not the humoral response to SCW (27), whereas MyD88 deficiency affects both (Figures 3A and C), MyD88 activation is unlikely to be restricted to IL-1R and is at least partly initiated by TLRs. A TLR-induced MyD88-mediated pathology is also consistent with the significant role of TLR-4 in the SCW-induced antibody response and IL-17 production (17). Our current knowledge of the involvement of IL-1R, TLRs, and their shared MyD88 signaling indicates that IL-1R–induced MyD88 activation is responsible for T cell proliferation; while TLR-4–induced MyD88 activation is responsible for T cell IL-17 production, possibly through induction of IL-1β, IL-6, and IL-23, hence creating a Th17-skewing environment (17, 27).
In addition to inflammatory cell influx and bone erosion, chondrocyte cell death during chronic SCW arthritis was significantly dependent on MyD88 activation as well. Considering the irreversibility of the latter pathologic phenomenon, targeting MyD88 may be of high therapeutic value in arthritis. A dominant-negative form of MyD88 has been shown to inhibit spontaneous cytokine release from RA synovial membrane cells (33). Nevertheless, a complete blockade of MyD88 signaling in patients would probably increase the risk of noninvasive bacterial infections, as has been reported in MyD88-deficient individuals (34).
This is the first study to investigate the role of TRIF in experimental arthritis. Our data provide the first evidence of the involvement of TRIF in antigen-specific adaptive responses to a non–TLR-3–triggering antigen. Previous reports have indicated a role of TRIF in T cell and B cell proliferation induced by TLR-3 triggering (35, 36). Stimulation of TLR-3 also induces the differentiation of naive T cells into an IL-21–producing, but not an IL-17–producing, phenotype (37). Furthermore, TRIF signals were found to mediate antibody class switching in B cells toward IgG in response to viral double-stranded RNA (38). In the chronic SCW arthritis model, moderate reductions in T cell proliferation and antibody production in TRIF−/− mice were not sufficient to affect joint pathology. In fact, SCW-directed antibodies seem unlikely to be the main drivers of arthritis, since mice lacking Fcγ receptor types I, II, and III develop the disease to a similar extent as wild-type mice (27). In addition, MyD88 signaling, but not TRIF, is responsible for TLR-4–induced osteoclastogenesis from osteoblasts and is therefore involved in bone resorption (39). These findings may explain the central role of MyD88 and the redundancy of TRIF in TLR-driven arthritis.
TRIF-dependent induction of type I interferons and IL-27 has recently been implicated in the negative regulation of Th17 differentiation and the suppression of autoimmune encephalomyelitis (29, 30). Th17 cells are the main producers of IL-17, a potent stimulator of osteoclastogenesis and an inducer of osteoclastic bone resorption either alone or in cooperation with IL-1β and TNFα (40–42). The highly pathogenic role of IL-17 in inflammatory and destructive processes in RA has now been widely accepted (43, 44). The T cell response to the SCW antigen is of the Th17 type rather than the Th1 type, and TLR-4 is an important driver of IL-17 production, which is crucial for the erosive stage of SCW arthritis (Figure 6) (17). In the present study, prolongation of this T cell–dependent erosive stage led to increased bone erosion in the absence of TRIF signaling, an event coincident with increased IL-17 production. These observations suggest a protective role of TRIF signaling on arthritic bone damage and support a possible beneficial effect of TRIF-biased stimulation of TLR signaling under certain circumstances (i.e., Th17-driven disease). A more extensive study of the regulatory role of TRIF in (Th17-driven) arthritis models is still warranted. Furthermore, a TRIF-biased TLR-4 agonist has previously been described (45); however, the impact of its immunostimulatory properties on arthritis remains to be elucidated. In this context, a possible proinflammatory function of TRIF on, for example, RA fibroblast-like cells, which can be activated by TLR-3 stimulation (46), should be taken into consideration as well.
Taken together, our data indicate a central role of MyD88 signaling in chronic T cell–mediated IL-17–driven arthritis. TRIF, in contrast, is redundant for joint pathology and may even down-modulate bone erosion via inhibition of IL-17 production. We postulate that under conditions of disease, such as RA, in which Th17 cells are considered pathogenic, interference with TLR activation should preferably target only MyD88 and leave the TRIF pathway intact.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Abdollahi-Roodsaz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Abdollahi-Roodsaz, Koenders, Joosten, van den Berg.
Acquisition of data. Abdollahi-Roodsaz, Koenders, Helsen, Walgreen, van den Bersselaar, Arntz, Takahashi.
Analysis and interpretation of data. Abdollahi-Roodsaz, van de Loo, Koenders, Takahashi, Joosten, van den Berg.