Despite the link between NOD2 and arthritis very few studies have examined NOD2 function within the joint. In murine experimental models of colitis, NOD2 functions to negatively regulate TLR2 signaling events in the setting of a significant bacterial load. Given that the joint is a sterile environment, we investigated TLR2-NOD2 interactions within the joint to determine if NOD2 negatively regulates inflammation in this organ as well.
We found that mice deficient in NOD2 developed significantly less PGN-induced arthritis than their WT controls, indicating that NOD2 promotes inflammation in the joint rather than suppressing inflammation. We show that PGN-induced arthritis does indeed involve TLR2 signaling events as mice deficient in either TLR2 or its signaling mediator, MyD88, demonstrated impaired PGN-induced arthritis. Taken together, these findings indicate that both NOD2 and TLR2 when activated within the joint promote inflammation. The possibility of NOD2 exerting a regulatory role on TLR2-induced inflammation was further explored in our experiments using the synthetic TLR2 agonist, Pam3CSK4, or the NOD2 agonist, MDP, to dissect the possible cooperation between NOD2 and TLR2 pathways. These data support a role for both NOD2 and TLR2 in promoting joint inflammation, albeit completely independently. It seems reasonable to postulate that joint inflammation triggered by PGN involves cellular recognition of PGN at the cell surface by TLR2 and then subsequent activation of NOD2 within the cell by PGN-derived MDP. In support of this theory, we have observed that co-treatment with PGN and MDP did not have an additive effect with regard to joint inflammation 3 days later (data not shown), suggesting both receptor systems had reached saturation and addition of more MDP could not be sensed.
The exact nature of how NOD2 influences the function of TLRs has yet to be completely resolved. The synergistic cooperation between NOD2 and TLRs that has been observed could be protective to the host in the elimination of pathogens. However, overproduction of cytokines can be harmful leading to tissue injury as in the case of colitis or inflammatory arthritis. While global suppression of TLR responses by NOD2 has been demonstrated (18
), our studies in an arthritis model would demonstrate the opposite that in fact, NOD2 does not participate in TLR2 suppression in the joint. This is not necessarily in opposition to other reports of NOD2 demonstrating its ability to regulate TLR-triggered inflammatory responses. Rather it indicates that the tissue type, the context of inflammation, or doses and timing, may influence the inflammatory effects of NOD2.
Our finding that NOD2 promotes PGN-induced arthritis would be consistent with a recent report by Saha et al wherein mice that were administered PGN systemically developed joint inflammation (25
). However, several distinctions are also noted. Consistent with SCW-induced arthritis (23
) our data support an essential role for TLR2 and MyD88 in arthritis induced by locally administered PGN whereas Saha et al report a TLR2-independent mechanism (25
). The importance of either TLR2 or NOD2 in PGN-induced arthritis is inferred from studies with KO mice. However, KO mice may have compensatory mechanisms that complicate our interpretation of experimental data. For example, TLR4 deficient mice have a defect in neutrophil development (28
). In our own data, analysis of TLR2 KO mice would support the interpretation that TLR2 is the dominant receptor for PGN-induced arthritis. However, the use of NOD2 KO mice would argue that TLR2 plays a minimal role and NOD2 is the key determinant of PGN-induced arthritis, presumably after it has been degraded to MDP. While our study does not allow identification of whether or not TLR2 or NOD2 is the dominant receptor, it is clear that NOD2 is contributing to arthritis in a positive, enhancing manner.
In contrast to systemically
administered PGN-induced arthritis, wherein neutrophils played an essential role in joint inflammation (25
), our data indicate that neutrophils are not the major cellular mediator of arthritis induced by intra-articular PGN as neutrophil depletion had no marked effect on arthritis. The non-essential role of neutrophils would be consistent with a report of SCW-induced arthritis, which likely involves macrophages and other tissue resident cells (23
). Indeed by immunohistochemistry we detect minimal neutrophils present within the joint after i.a. injection of PGN and mice deficient in CD11b, an adhesion molecule considered necessary for neutrophil extravasation and infiltration into inflamed tissues, are still capable of developing PGN-induced arthritis (data not shown). NOD2 is thought to be predominantly expressed in myeloid cells. However, NOD2 is expressed in several other cell types within tissues including vascular endothelial cells, epithelial cells, osteoclasts and macrophages and fibroblasts in the synovium (22
). Together with our finding, this would underscore the importance of NOD2 on cellular functions within the joint itself in the initiation and perpetuation of the inflammation and joint destruction within the joint. In contrast, i.v. administered PGN may upregulate NOD2 in or around joints spaces thereby acting on different cell types. This process has been shown to involve PGLYRP-2 expression and the induction of cytokine and chemokine expression, neutrophil recruitment and subsequent arthritis (25
). Neutrophils may be involved in this model at other points in the pathway, such as upstream at the point of degrading PGN into MDP.
In conclusion, our studies here used different mouse models of arthritis to explore the interplay between NOD2 and TLR2 in inflammation within the joint. We provide evidence that in addition to TLR2, NOD2 functions within the joint promote arthritis. Each receptor system functions independently and NOD2 did not exert a negative regulatory effect on TLR2 signaling.