In vitro studies demonstrated that NOD2-expressing cell lines responded to PGN and that NOD2 requires intact muramyl peptides with two MDP or three MTP amino acids.
33 These observations prompted us to further define the functional role of NOD2 in vivo within the eye. We first assessed the ability of synthesized MTP (wherein the third amino acid is a lysine residue) or purified PGN of
Staphylococcus aureus to induce ocular inflammation in mice. Because this had not been previously demonstrated, the dose and time of the vascular inflammatory response induced by an intravitreal treatment of either PGN or MTP were optimized, as assessed by intravital microscopy (data not shown). We then went on to investigate the possible requirements for NOD2 in MTP- or PGN-induced ocular inflammation.
Nod2 knockout mice were treated with intravitreal injections of MTP, and the ocular inflammatory response was assessed by intravital microscopy 6 hours after treatment
(Fig. 4) . We found that MTP elicits a significant increase in the number of rolling and adhering leukocytes within the eye of WT mice. In contrast,
Nod2 knockout mice show an abolished response to MTP, indicating that this response is dependent on NOD2. We then tested the role for NOD2 in response to PGN. Although PGN is degraded into metabolites that include MDP and MTP, PGN itself is also recognized by TLR2.
35 Mice were treated with intravitreal injections of PGN, and inflammation was assessed by intravital microscopy 6 hours later
(Fig. 5) . We found that though PGN induced a significant ocular inflammatory response, this response was not entirely dependent on NOD2 because the number of rolling cells was only partially reduced and no difference was detected in the number adhering cells. This finding suggests that NOD2 may contribute in part to PGN responses such as leukocyte rolling. Thus, NOD2 is required for ocular inflammation induced specifically by the minimal peptide fragments of PGN, and it demonstrates that
Nod2 knockout mice are fully capable of mounting an intraocular inflammatory response to other stimuli of innate immune responses.