We used KO mice or inhibitors to identify the critical immune receptors responsible for zymosan-induced arthritis and cathepsin activity. Our results indicate that Dectin-1 is one of the major receptors responsible for ZIA. Dectin-2 played a lesser role and TLR2, MyD88 and CR3 played non-essential roles. Injection of purified fungal cell wall compounds including curdlan, laminarin and mannan support the dominant role of the Dectin-1 pathway. We also assessed the function of two NLR family members, NOD1 and NOD2, in ZIA. Our studies identify differential roles for NOD1 and NOD2 in ZIA. Surprisingly, NOD2 KO mice were protected against ZIA, thereby identifying NOD2 as a novel contributor to ZIA. These results thus establish functional, cooperative roles for NOD2 and Dectin-1 in a mouse model of inflammatory arthritis.
Our results identify an entirely novel role for NOD2 in amplifying inflammatory responses within the joint to zymosan. At this point, the mechanism by which NOD2 is involved in the joint’s responsiveness to zymosan is unclear. NOD2 senses muramyl dipeptide as a structural component of bacterial PGN. Within the class of poly-saccharides classified as β
-(1,3)-glucans, there are a number of structural variants. Glucans are the major structural components of the cell wall of fungi, but plants and certain bacteria such Brucella
are sources of β
-glucans as well [35
]. We could speculate that NOD2 evolved to sense β
-glucans on such bacteria or that molecular mimicry occurs between the structurally related β
-glucans and peptidoglycan; however, this remains to be shown. Emerging reports have since expanded roles for NOD2 responsiveness to other agonists in the setting of viral [37
] and toxoplasmotic [38
] infections and to the chemotherapeutic agent Vadimezan [39
], thereby suggesting that NOD2 responsiveness extends to a broader range of agonists than previously appreciated. The apparent differences in structure amongst these agonists, however, would not support molecular mimicry or a direct interaction between NOD2 and its agonists, but rather a more downstream role as a signaling intermediary.
NOD2 has been shown to play a “fine-tuning” role in inflammatory models triggered by TLR activation, suggesting cross-talk between NOD2 and TLR pathways. For example, the response to cell wall fragments from Streptococcus pneumoniae
induces both inflammatory and anti-inflammatory responses. The inflammatory responses are dependent on TLR2 and are NOD2-independent while the anti-inflammatory responses are dependent on TLR2 and NOD2, which involves IL-10 secretion and IL-10-regulated target gene expression [8
]. The simultaneous activation of NOD2 by muramyl dipeptide (MDP) and TLRs by different ligands causes the TLR response to be downregulated in an interferon regulatory factor 4-dependent manner [7
]. The cross-talk is thought to occur through converging pathways leading to NF-κ
B activation. Since our data here do not support essential roles for MyD88 or TLR2, we conclude that the contribution of NOD2 in this model is probably not due to any influence on TLR signaling. Indeed, our prior report supports the independence of NOD2 and TLR2 signaling in arthritis [5
Whether or not NOD2 pathways can intersect and alter the function of zymosan-activated Dectin-1 pathways within the joint has not been previously addressed until this report. There are numerous sequellae of Dectin-1 activation, including the generation of ROS, release of chemokines, cytokines, anti-inflammatory cytokines and inflammatory lipid mediators, and induction of phagocytosis (reviewed in [17
]). Testing the role of NOD2 in fine-tuning any one of these response may be informative. It is interesting to speculate that cross-talk between NOD2 and Dectin-1 may occur via CARD9, an essential downstream mediator of Dectin-1, which also directly interacts with NOD2 [27
]. CARD9 was found to be important for NOD2-dependent activation of p38 and JNK pathways, but not NF-κ
B activation [27
]. Studies are underway to test the hypothesis that NOD2 and Dectin-1 pathways converge at CARD9 in response to zymosan and upregulate various inflammatory mediators in the joint.
The mechanisms involved in responsiveness to zymosan in vivo are complex. Even for cell culture models showing zymosan responses that are dependent on TLR2, the component of zymosan that is detected by TLR2 has not been definitively identified, although several glycolipids are candidates [17
]. Dectin-1 recognizes zymosan and β
-1,3-linked glucans with the minimal unit being a 10-mer [40
], as well as whole fungi such as C. albicans
and S. cerevisiae
. Among the various components of zymosan we tested in isolation for their ability to trigger arthritis, the β
-(1,3) glucan, curdlan, demonstrated the most inflammatory potential within the joint. Curdlan-induced arthritis was abolished by co-treatment with anti-Dectin-1 blocking antibody. Laminarin has been shown to exhibit a high degree of specificity for binding to Dectin-1, but is not thought to activate Dectin-1 [41
]. Local injection of mannan did not result in any measurable joint inflammation, indicating that activation of the mannose receptor on its own is not sufficient for induction of arthritis. These data, combined with the anti-Dectin-1 blocking experiments, support our conclusion that Dectin-1 is one of the primary receptors involved in ZIA. Notably, activation of Dectin-1 by curdlan alone did not result in the same extent of arthritis as zymosan did, again suggesting a role for other receptors capable of detecting zymosan.
Our data on TLR2-deficient mice are consistent with previous reports where no differences were noted in ZIA in TLR2-deficient mice 3 days after i.a. injection [42
]. However, our report does describe differences in arthritis at day 1 and day 24, suggesting a role for TLR2 very early and very late in disease. The lack of a role for TLR2 at day 3 may seem surprising given evidence that some zymosan-induced inflammatory responses in macrophages in vitro are abrogated in TLR2 or Myd88-deficient cells [17
]. However, at the level of the intact animal and in specific organs, receptor density, tissue distribution and differential expression of other participating molecules such as NOD2 may play a more significant role. Cellular composition and architecture in each individual organ may lead to different inflammatory responses from those exhibited in homogeneous cells in isolation. For example, while we find zymosan responses in the joint to be independent of TLR2 but dependent on Dectin-1, administration of zymosan directly into lungs by intratracheal aerosolization triggers inflammation independent of both TLR2 and Dectin-1 [43
], indicating that different organs handle localized exposure to zymosan in unique ways and other receptors participate in the recognition of zymosan. Systemic exposure of autoimmune-prone mice to zymosan has also revealed striking differences in disease pathology. Zymosan, via Dectin-1, potentiates arthritis in SKG mice, a strain with a genetic predisposition to produce arthritogenic autoreactive T cells [44
]. Surprisingly, treatment of non-obese diabetic (NOD) mice, which are genetically prone to autoimmune type I diabetes, with zymosan results in suppression of autoimmunity [45
]. The mode of zymosan delivery, the particular organ exposed, and the underlying specificities of host T cells are factors that influence the outcome of both T-cell-based models of autoimmunity and models of inflammation based on activating PRRs.
Other C-type lectin receptors such as Dectin-2 have been shown to recognize zymosan and carbohydrate moieties of fungi. Like Dectin-1, the signal transduction pathway of Dectin-2 also involves Src kinases, CARD9 and activation of NF-κ
B. However, unlike Dectin-1, Dectin-2 lacks an intracellular ITAM motif and does not initiate signals but couples with Fc receptor gamma (FcRγ
) chain signaling. We found that blockade of Dectin-2 with a neutralizing antibody did significantly diminish ZIA, but not to the same extent as we observed with the blocking studies for Dectin-1. We are aware that a caveat to these studies is the potencies of the blocking antibodies, which could be a variable as opposed to examination of Dectin-1 and Dectin-2 function using KO mice where complete inhibition would be achieved. NOD1 does not appear to play any significant contribution to ZIA. Despite the two NLRs activating homologous signal transduction pathways downstream of RIP2 and NF-κ
B, such differential roles for NOD2 and NOD1 in response to other agonists have been reported [46
]. This would suggest that there are unique or opposing functions for NOD1 versus NOD2 responses.
Although the mechanism by which NOD2 and Dectin-1 modulate zymosan-triggered arthritis is not entirely clear at this point, their relevance is supported by our in vivo studies which demonstrate that NOD2 and Dectin-1 are both required for ZIA. It will be of interest to determine whether NOD2 plays a role in zymosan responses in other organs or if this is a joint-specific response. Unraveling the role of NOD2 in inflammatory models may provide insights into NOD2’s function in normal joint homeostasis and provide insights into the mechanism of arthritis seen in patients with NOD2 mutations.