Abstract
Purpose :
Interleukin 35 (IL-35) is an immunosuppressive cytokine that inhibits experimental autoimmune uveitis (EAU), a model of human uveitis (Nature Medicine. 20:633-41). It is comprised of IL-12p35 and Ebi3 and each subunit possesses intrinsic immunoregulatory activities (Nature Communication 8:719, 2017). In this study, we have investigated whether IL-12p35 can be used to suppress uveitis.
Methods :
We generated an IL-12p35 reporter knock-in mouse strain that expresses yellow fluorescent protein (yfp) by inserting the yfp-gene cassette in-front of mouse il12a exon 1 gene using CRISPR-Cas9 technology. After backcrossing the F1 founder to C57BL/6J, we derived the IL-12p35/YFP-knock-in reporter strain which is hemizygous at the il12a locus (p35-KI). EAU was induced by immunization of C57B/6J or p35-KI mice with IRBP635-655 peptide/CFA and EAU development was assessed by fundoscopy, OCT and ERG. RNA from the retina and draining lymph nodes (dLNs) were analyzed using a Nanostring autoimmune panel containing 770 targets. Inflammatory cells in the dLN and retina were analyzed by intracellular cytokine analysis. Eyes were fixed for H&E and histological analysis.
Results :
Immunohistochemical analysis of eyes of unimmunized p35-KI mouse localized IL-12p35 expression in the retinal pigmented epithelium and tissues of the uvea including the iris, ciliary body, and photoreceptor cells. Compared to WT C57BL/6J, the p35-KI mice developed a more severe acute uveitis. Importantly, the p35-KI mouse strain develops chronic uveitis characterized by significantly impaired vision and intraocular inflammation that persists more than 120 days post-immunization. Furthermore, exacerbated uveitis in the p35-KI mouse correlates with reduced levels of Treg cells and expansion of proinflammatory T lymphocytes.
Conclusions :
Data presented in this study show that the hemizygous p35-KI mice which express 1 copy of IL-12p35 develop severe uveitis compared to C57BL/6J mice that express 2 copies of IL-12p35, suggesting that the exacerbated uveitis derived from reduced IL-35 expression. Moreover, our immunohistology studies of p35-KI reporter eyes which reveal that retina and pigment epithelial cells constitutively express IL-12p35, suggest that the immunosuppressive IL-35 cytokine might contribute to mechanisms of intraocular immunity and maintenance of ocular immune privilege.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.