Nod2 controls susceptibility to autoimmune uveitis through control of a Th17-driven response

Abstract

Purpose : The innate immune receptor Nod2 may provide a clue to understanding the pathogenesis of uveitis. Although NOD2 is the genetic cause of Blau syndrome, which includes uveitis, it is also required for host defense. We recently uncovered an unanticipated suppressive role for Nod2 in a T cell-dependent model of uveitis, experimental autoimmune uveitis (EAU), which was further explored here.

Methods : EAU was induced in mice deficient in Nod2 or Rip2 and wildtype (WT) C57BL/6J controls by immunization with interphotoreceptor retinoid-binding protein (IRBP). Uveitis was evaluated by fundus imaging and histology. CD4+ T cells were depleted with antibody (clone GK1.5, 600µg/mouse, on days -1,0,1,6,11,16 post-immunization). Cultured splenocytes were stimulated with IRBP to evaluate antigen-recall cytokine responses (Luminex) and T cell composition (intracellular cytokine staining and flow cytometry). IL-17 was neutralized with antibody (clone 501040, 100µg/mouse, on days 0, 2, 5, 9, 13).

Results : Nod2 knockout (KO) mice developed earlier and more severe EAU (p<0.05 vs. WT; n=15 mice/group; 3.5 vs 1.25 respectively). As most functions of Nod2 have been attributed to its recognition of muramyl dipeptide (MDP) and interaction with the kinase Rip2, EAU was evaluated in Rip2 KO mice. Rip2 KO mice initially displayed reduced uveitis (between d10-d12; n=10-12 mice/group, p<0.05), but this was not sustained thereafter. In keeping with this, replacement of CFA with MDP elicited minimal EAU in WT (p<0.05 vs. CFA; n=8 mice/group), indicating minimal involvement for Rip2 or MDP. Depletion studies showed that as in WT, CD4+ T cells were required for EAU in Nod2 KO mice. Antigen-stimulation assays demonstrated that IRBP-induced IL-17A was significantly elevated in Nod2 KO vs. WT cells, along with IL-2, TNFa and KC (p<0.05), but Th1-associated IFNγ production was not altered. Flow cytometry analysis showed presence of both Th1 and Th17 cells in WT and Nod2 KO. The T cell infiltrate within eyes was markedly increased in Nod2 KOs, with a striking increase in frequency of Th17 cells; and IL-17A neutralization reverted the Nod2-KO associated phenotype (p<0.05 vs. isotype control; n=15-18 mice/group).

Conclusions : These data reveal a novel Rip2-independent function for Nod2 in conferring protection against EAU through a mechanism involving control of the Th17 response.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.