In addition to skin and joints, the eye is one of the three organs most consistently affected in Blau syndrome, which results in granuloma formation in affected tissues. Thus, understanding the function of NOD2 within this specific tissue is critical to clarifying this disease. Investigation of a gene that so clearly links innate immunity to disease could also further our understanding of more common forms of uveitis. To gain insight into functional consequences of NOD2 activation on eye inflammation, we investigated the role of IFN-γ, a cytokine involved in granuloma formation, in a previously established mouse model of MDP-induced uveitis. Taken together, our findings reveal a novel role for IFN-γ as a downstream mediator of NOD2 in ocular inflammation and demonstrate that IFN-γ has the capacity to act as a regulator of NOD2 function in vivo, thereby synergizing with MDP-induced ocular inflammation.
The role of IFN-γ as a participant in NOD2 inflammation has not been previously studied in vivo. Our findings identify IFN-γ as a downstream modulator of NOD2 inflammatory functions in the eye and systemically. Thus, in contrast to our earlier findings with IL-1β, IFN-γ appears to be a regulator of NOD2-driven inflammation in the eye and peritoneum. MDP activated IFN-γ production within the eye in a NOD2-dependent fashion, consistent with an earlier report indicating that MDP stimulation results in IFN-γ production in freshly isolated hepatocytes.
28 Moreover, our findings demonstrate that in the absence of IFN-γ, ocular inflammation is diminished. IFN-γ is a cytokine regulated transcriptionally by NF-κB. Because most of the inflammatory effects of NOD2 are believed to be mediated by NF-κB activation, it seems likely that this could be the mechanism by which IFN-γ could be produced in response to MDP. NOD2 is expressed in immune cells, including neutrophils and macrophages, and in nonlymphoid tissue including endothelial cells within the choroid and retina,
23 thereby suggesting that activation of NOD2 within the vascular endothelium—or perhaps activation in neutrophils, macrophages, NK cells or possibly even T cells—could result in IFN-γ production, though this remains to be tested. Indeed, in the case of
Salmonella or
Listeria infection wherein innate immunity and NOD2 play pivotal roles in host defense, IFN-γ–producing phagocytes predominate in the early course of infection.
29 30 Future investigation should elucidate the mechanisms by which IFN-γ is regulated by NOD2 within the eye.
Our findings identify IFN-γ as a promoter of NOD2 ocular inflammation, which would be contradictory to the role of IFN-γ in other models of uveitis in which T cells are critical regulators. Although autoimmune uveitis can occur with IFN-γ and IL-17 responses,
31 IFN-γ has been considered protective.
32 33 It is thought that the protective effects of IFN-γ result in part from its suppressive actions on IL-17–driven ocular inflammation. Notably, we have not been able to detect IL-17 protein production in the eye in response to MDP at 2 or 6 hours after treatment (data not shown). However, consistent with the role for IFN-γ, we have observed a significant increase in protein production of other Th1-associated cytokines, IL-12p40 and IL-27 (data not shown). The role for NOD2 in modulating the IL-17 axis has not been completely established. Activation of NOD2 expressed within dendritic cells promotes IL-17 production in human memory T cells in vitro.
34 Our own studies in a mouse model of proteoglycan-induced arthritis support a role for MDP in promoting a Th1 response known to contribute to disease in this model.
35 36 37 38 We have observed that MDP treatment exacerbates disease along with the antigen-mediated production of Th1 cytokines, IFN-γ and IL-12p40, but not Th17 cytokines such as IL-17, IL-6, TGF-β.
39
It is also possible that immune status influences the nature of inflammatory responses. Contrary to earlier reports examining the role of IL-17, MDP-induced uveitis is likely to occur independently of T cells because it is an acute inflammatory response characterized primarily by innate immune cells such as neutrophils and macrophages. Interestingly, we demonstrated that IFN-γ deficiency exacerbated EIU in mice, which is consistent with an earlier report wherein IFN-γ played a role in the development of ocular inflammation.
40 Moreover, administration of exogenous IFN-γ suppressed EIU,
40 whereas our findings demonstrated a role for IFN-γ in exacerbating MDP-induced uveitis. This suggested that even innate-driven ocular inflammatory responses can differ with respect to the role of IFN-γ and that TLR4 and NOD2 may result in opposing mechanisms of uveitis.
The capacity of IFN-γ to regulate the amplitude of TLR responses in macrophages has been described; however, whether IFN-γ regulates NLR responses in a similar fashion has not been examined. A single report indicated that IFN-γ- and MDP-stimulated macrophages produce more NO in vitro,
41 42 yet there has been no evidence to demonstrate that IFN-γ can enhance NOD2-induced cellular inflammatory responses in vivo. Our studies here indicate that IFN-γ and MDP synergize in vivo in the exacerbation of ocular inflammation. The mechanisms by which IFN-γ synergizes with NOD2 are not known. Others and we have shown that NOD2 expression in a variety of cell types can be enhanced by IFN-γ,
28 42 43 including human ocular endothelial cells,
23 suggesting that the increased expression of NOD2 could promote inflammation. Our findings here also indicate that IFN-γ appears to preferentially enhance leukocyte-endothelial interactions involving adherence and cellular infiltration, a process abolished in the absence of CD11b expression. We have found that the expression of CD11b and ICAM-1, the endothelial ligand for CD11b, in eye tissue is increased slightly less than twofold in response to cotreatment with MDP and IFN-γ (data not shown), indicating that changes in expression of these two adhesion molecules are not limiting factors in MDP-induced uveitis or MDP-IFN-γ synergy. IFN-γ by itself is a relatively weak inducer of NF-κB or MAPK. Rather, most of the more well-described biological activities of IFN-γ are mediated by the JAK-STAT signaling pathway. However, it is possible that in NOD2 activation, the NF-κB or MAPK pathways may be preferentially enhanced by IFN-γ. Or perhaps the expression of factors regulated by IFN-γ, such as IP-10, MIP-1α and MIP-1β, RANTES, and MCP-1, could provide unique signals for cellular recruitment that are not otherwise induced by NOD2. Given that NOD2 and TLR pathways share some common signaling mediators, it is intriguing to speculate whether an analogous mechanism by which IFN-γ amplifies TLR responses could occur for NOD2 responses. In enhanced TLRs, one theory is that IFN-γ synergizes with TLRs in part by suppressing a TLR-induced feedback inhibition, which is a response that involves IL-10 and STAT3.
25 Further investigation of how inflammatory mediators are differentially regulated in the setting of IFN-γ and NOD2 signaling will be informative.
Our findings here along with the studies described support a role for NOD2 in promoting ocular inflammatory responses, yet they would be contradictory to the role of NOD2 in animal models of inflammatory bowel disease. In this case, NOD2 is considered to function as a negative regulator of PGN-induced responses such that in its absence, Th1 responses and colitis are exacerbated.
44 The authors’ interpretation is that loss of function as a consequence of Crohn-associated mutations in
NOD2 results in inappropriate intestinal inflammatory responses to TLRs over time and predisposes to disease. This differs from mutations in
NOD2 causing Blau syndrome, which is an autosomal dominant disease in which infection does not appear to play a role. In contrast to polymorphisms associated with Crohn disease, the mutations causing Blau syndrome have been suggested to result in constitutive NOD2-driven NF-κB activity.
45 IFN-γ is a cytokine regulated by NF-κB, and our data here indicate it is a downstream mediator of NOD2 inflammation, making it intriguing to speculate that in Blau syndrome, dysregulation of NOD2 activation could result in excessive IFN-γ production. Excessive IFN-γ production could in turn further enhance NOD2-induced inflammation. This hypothesis would be consistent with the known role of IFN-γ as a regulator of granuloma formation, a hallmark feature of Blau syndrome and Crohn disease. Although our model of MDP-induced eye inflammation is a valuable tool for exploring the inflammatory effects of NOD2 activation in vivo, we recognize that the ocular inflammation in our mouse model is transient and differs from sustained, granulomatous inflammation seen in Blau syndrome. Intriguingly, we have also found a role for NOD2 in promoting joint disease in a chronic model of immune-mediated arthritis.
39 Clearly, how different mutations in
NOD2 alter its function and cause different diseases is not entirely understood. Future investigation of NOD2 functions in human ocular tissue would be informative.
In conclusion, inflammatory responses must be modulated appropriately for the specific tissue environment, especially within the eye, where it is vital to avoid inflammation that can compromise vision. Thus, the investigation of NOD2 inflammatory functions within the eye, which is a tissue clearly affected in Blau syndrome, should provide important insight into underlying mechanisms of this disease. Our studies here contribute to the understanding of inflammatory functions of NOD2 involving IFN-γ.
The authors thank C. Wayne Smith of Baylor University and Richard Flavell of Yale Medical School for their donations of CD11b KO and NOD2 mice, respectively. They also thank Monica Jann, AiLien Truong, and Hari Sawkar for their technical contributions.