Deficiency in Nod2 is associated with dysregulation of Th17-related responses in the eye

Abstract

Purpose : Mutation in NOD2 results in Blau syndrome, which is characterized by granulomatous uveitis in association with dermatitis and arthritis. NOD2 belongs to a family of innate immune receptors that function in host defense, but we recently identified a novel suppressive role for Nod2 in experimental autoimmune uveitis (EAU), a T cell-dependent model of uveitis. Nod2 deficiency resulted in greater expansion, but similar proportion, of CD4+ T cells in eyes. Here we sought to study the cellular mechanisms behind modulation of EAU by Nod2.

Methods : Mice deficient in Nod2 or congenic wild type (WT) C57BL/6J controls were immunized for EAU with a mixture of interphotoreceptor retinoid-binding protein (IRBP) and IRBP_1-20 peptide. Flow cytometric analysis of CD4+ T cells isolated from whole inflamed eyes harvested d21 post-immunization was used to evaluate Th1 (IFNγ+) and Th17 (IL-17+) effector subsets. Single cell suspensions isolated from inflamed eyes were re-stimulated in vitro with IRBP_1-20 peptide to assess antigen-recall cytokine responses by flow cytometry after intracellular cytokine staining for IL-17A, TNF, IFNγ, GM-CSF, and IL-22. Relative contributions of IL-17 and IFNγ to disease in vivo were evaluated by fundus imaging and histopathology following antibody neutralization (100 μg/dose), on days -1,0,2,5,9, and 13 relative to immunization.

Results : Nod2 deficiency resulted in increased percentages of ocular CD4+ T cells that produced IL-17 (6.1% vs. 1.52% in WT) or IFNγ (9.38% vs. 1.57% in WT). Antigen-recall studies of purified ocular cells revealed a dramatic increase in the proportion of IL-17+CD4+ cells in Nod2 KO compared to WT mice (12.3% vs. 3.5%). Further evaluation of IRBP-stimulated cytokine production of IL-17+CD4+ cells revealed enhanced production of all cytokines tested, including IFNγ (i.e. IL-17+IFNγ+ cells). Blockade of IL-17 in vivo significantly diminished EAU severity for both genotypes, but to a greater extent in Nod2 KO mice. In contrast, blockade of IFNγ significantly worsened EAU severity in WT mice, but did not alter disease severity in Nod2 KO mice.

Conclusions : These data suggest that the exacerbated uveitis accompanying Nod2 deficiency is mediated primarily through dysregulation of the Th17-related rather than Th1-related response.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.