Retinoid X receptor alpha (RXRa) mutant Pinkie and vitamin A deficient B6 mice develop similar spectrum of corneal inflammation

Abstract

Purpose : To test the hypothesis that attenuated retinoid X receptor alpha (RXRa) signaling in the gene mutated Pinkie strain and C57/BL6 (B6) mice with systemic vitamin A deficiency (VAD) develop a similar spectrum of corneal inflammation.

Methods : Cornea immune cells were characterized by flow cytometry, gene expression pattern in the cornea was evaluated by Nanostring immunological array, and immunostaining evaluated expression of cornified envelope (SPRR2) and neovascularization (CD31, LYVE-1) markers and metalloproteinases. Effects of 9-Cis retinoic acid (RA) and all-trans RA (ATRA) on suppression of cyto/chemokine production by stimulated purified monocytes was detected by multiplex immunoassay.

Results : Among the resident CD45⁺ cells in the normal B6 cornea, we found lymphoid (CD45⁺CD3⁺; 21.6%), and myeloid (78.2%) cells which include CD45⁺CD11b⁺Ly6G⁺ (16.9%), CD45⁺CD11b⁺Ly6C^lowCD64^high (13.9%, as macrophages) and CD11b⁺Ly6C^highCD64^low (19.08%, as classical monocytes). Classical monocytes were the predominant cell population increased in the cornea of Pinkie and VAD strains. Tears cytokines G-CSF, IL-1α, CXCL-1 and VEGF were significantly upregulated in Pinkie and VAD mice. Both 9-Cis and ATRA attenuated LPS stimulated production of G-CSF, IL-1α/β, IL-12 and CXCL9 by purified CD11b⁺Ly6C^high monocytes in a dose dependent manner. VAD mice lost weight and developed mild to moderate corneal opacity around 12-14 weeks of age. Systemic 9-Cis or ATRA prevented the VAD phenotype. Approximately 20% of Pinkie developed corneal opacification, vascularization and ulceration by age 20 weeks. The ulcerated corneas are densely infiltrated with CD11b⁺immune cells and have greater immunostaining for SPRR2, blood (CD31+) and lymphatic (LYVE-1+) vessels and MMP-9 compared to age-matched normal B6 corneas. Nanostring RNA expression profiling in the ulcerated cornea revealed high expression of S100a8, S100a9, Plau, Plaur, IL1β, COX2, CCL2, IL6, CCR4, ICAM1, Pro-Platelet basic protein (PPBP), CCL9, VEGFA, FGF7 and MMP-9.

Conclusions : Corneas from Pinkie with attenuated Rxra signaling and VAD B6 are infiltrated with inflammatory monocytes, have increased expression of proinflammatory and proangiogenic mediators and develop corneal neovasculariazation and opacification. These findings suggest that RXRa signaling modulates corneal inflammation and maintains corneal health and clarity.

This is a 2021 ARVO Annual Meeting abstract.