Uveitis, or intraocular inflammation, is an immune-mediated disease that occurs as recurrent episodes of inflammation or as chronic inflammation. The complications of uveitis can include cataracts, glaucoma and retinal detachment, and it is one of the leading causes of blindness. Although the characteristics of uveitis can be confined to the eye, it can be associated with systemic immune-mediated disease such as ankylosing spondylitis, juvenile idiopathic arthritis, reactive arthritis, Behçet syndrome, inflammatory bowl disease, and Blau syndrome.22,36,37
The heterogeneity of uveitis suggests that multiple pathways contribute to its pathogenesis. Microbial triggers are considered to be crucial players in some inflammatory eye diseases.3
Therefore, the innate immune recognition system and its pattern recognition receptors (PRRs) within the eye and their interactions with their microbial PAMPs may play important roles in the underlying mechanisms involved in uveitis. The discovery of a single nucleotide change that virtually guarantees the development of uveitis as a component of Blau syndrome is a seminal finding that clearly identifies a gene, NOD2
, whose contribution to ocular inflammation must be clarified.
Here, we report the novel finding that NOD2 functions in ocular inflammation in mice. We show that activation of NOD2 by MDP elicits a transient increase in rolling and adhering leukocytes within the vasculature of the eye and a mild infiltration within the iris tissue. The capacity of MDP to induce ocular inflammation in mice was dependent on the stereoisomeric configuration of the isoglutamine moiety because the L-isomer did not induce significant ocular inflammation. This is consistent with an earlier study in vitro that showed stimulation of NOD2-transfected cell lines with L-MDP did not activate a NF-κ
B luciferase reporter.33
We demonstrated that the adhesion molecule L-selectin participates in the initiation of the MDP-induced inflammatory response within the vasculature because L-selectin knockout mice showed fewer rolling cells. Importantly, Nod2
knockout mice did not show an intravascular response to MDP, which indicates that NOD2 plays an essential role in MDP-induced uveitis. NOD2 knockout mice also failed to show a cellular inflammatory response to MTP treatment. These findings reveal an in vivo function for NOD2 in ocular inflammation and indicate that NOD2 specifically mediates ocular inflammatory responses to the minimal motifs of PGN, the muramic acid peptides MDP and MTP. It does not, however, mediate responses to the entire PGN molecule because Nod2
knockout mice retained their ability to elicit a cellular inflammatory response to PGN. Although the role of NOD2 has been studied indirectly in other mouse models of inflammation such as colitis, this work is the first, to our knowledge, to describe an in vivo model of ocular inflammation that depends on NOD2.
Treatment with bacterial cell wall components such as peptidoglycan complexes or MDP has been shown to elicit acute anterior uveitis in rabbits.31,32,38,39
In the present study, a direct function of NOD2 was shown for the first time to be essential for ocular inflammation induced by an intravitreal treatment of MDP or MTP in mice. Although intravitreal and systemic administration of MDP in the rabbit resulted in ocular inflammation, we found that intravitreal injection of MDP in mice was more effective than systemic treatment. PGN-induced uveitis did not completely require NOD2 because Nod2
knockout mice were still capable of increased rolling and sticking leukocytes within the iris vasculature. This finding is consistent with the observation that TLR2 mediates the recognition of PGN35
and that NOD2 is capable of being activated independently of TLRs.40,41
Our results also emphasize that it is unlikely that MDP or MTP is capable of inducing ocular inflammation independently of NOD2 (e.g., by TLRs) because MDP- and MTP-induced ocular inflammations were completely abolished in the Nod2
knockout mice. Given the few studies of the functional consequences of NOD2 activation on inflammatory cascades or adhesion molecules in the eye, this mouse model should help to clarify specific ocular activities of NOD2.
In addition to its role in host defense, the importance of NOD2 in regulating inflammation is further emphasized by the fact that mutations in NOD2
cause Blau syndrome, an autosomal dominant, multiorgan, granulomatous inflammation of the eye, skin, and joint.22,23
Distinct polymorphisms of NOD2
have also been strongly linked to the onset of Crohn disease,26,42
an inflammatory disorder of the intestine and that can involve the uvea. Mutations in other NLR family members also cause auto-inflammatory diseases, many of which are associated with uveitis. Notably, the three common residues affected by the Blau mutations correspond to the position of pathogenic mutations in the closely related and NLR family member Cryopyrin/Nalp3, which causes a triad of diseases: chronic infantile neurologic cutaneous and articular syndrome, familial cold autoinflammatory syndrome, and Muckle-Wells syndrome.14,43,44
Because of the functional and mutational similarities of NOD2 and NALP3, it has been hypothesized that the underlying mechanisms might be similar.
MDP-induced uveitis in the mouse results in a relatively mild nature of inflammation, wherein the inflammatory response occurs predominantly within the vasculature. MDP is a small molecule and is rapidly cleared, with less than 10% recoverable MDP in the urine 2 hours after intravenous or subcutaneous injection in mice.45
This might explain why MDP elicits such an acute and mild inflammation compared with that of the more robust endotoxin-induced uveitis. However, in both uveitis models, neutrophil response predominates. Repeated treatments or a slow-release depot of MDP might elicit a more robust or chronic ocular inflammatory response. It is possible that altered function of NOD2 attributed to the Blau mutations might also account for the more severe nature of uveitis than the transient inflammation we observed in response to activation of the endogenous form of NOD2. In vitro assays with transfected HEK cells have shown that Blau mutations result in increased basal NF-κ
which would suggest a gain of function in the mutant NOD2. It is intriguing to speculate whether chronic activation of NOD2 might drive perpetual production of inflammatory mediators that promote neutrophil recruitment and granuloma formation. The functional consequences of Blau mutations of NOD2 are a topic for further investigation.
In addition to the role of NOD2 in mediating MDP responsiveness, NOD2 appears to cooperate with other TLRs. This is best exemplified in the setting of “synergy,” wherein MDP, together with LPS, enhances cytokine production that is greater than the additive effects of either treatment alone.47
Such synergist effects result in increased sensitization of mice to LPS-induced lethality. The essential role of NOD2 in MDP synergy was recently demonstrated in Nod2
knockout mice, which showed increased resistance to LPS-induced lethality because of cotreatment with MDP.18
Although NOD2 is capable of enhancing TLR responses, in certain situations it also appears to regulate TLR responses negatively.48
Unpublished data indicate that treatment with MTP-phosphatidylethanolamine reduced LPS-induced ocular inflammation in rats (Kozak Y, et al. IOVS
2006;47:ARVO E-Abstract 2917). Taken together, these data support a possible function for NOD2 in ocular host defense and for modulation of subsequent vascular inflammatory responses in the eye to other PAMPs.
In conclusion, we demonstrate an essential role for NOD2 in uveitis in mice. There are a number of fundamental questions regarding the role of NOD2 in uveitis that must be explored, and these studies provide the foundation for future studies that will be critical to our understanding of how NOD2 contributes to the pathogenesis of uveitis.