Abstract
Purpose :
Vitamin A is essential in maintaining ocular surface health. The Lrat-/-Rbp-/- double knockout (DKO) mouse strain fed a vitamin A deficient (VAD) diet develops severe systemic vitamin A deficiency. The purpose of this study was to investigate if the VAD DKO strain develops ocular surface inflammation and disease.
Methods :
DKO of the retinol binding protein Rbp4 and retinol esterifying gene Lrat was created on a B6 (WT) background and fed control (Co) or VAD diets. Retinol was measured in the lacrimal gland by HPLC mass spectrometry. Corneal barrier function was measured by Oregon Green dextran (OGD) permeability and conjunctival density/volume by WGA lectin immunostaining. ScRNA-seq defined conjunctival immune cell populations and transcriptional profiles. Nanostring array assessed gene expression in conjunctival immune cells and conjunctival immune cell phenotype and percentage was evaluated by flow cytometry.
Results :
DKO on a VAD diet had a 40% decrease in lacrimal gland retinol concentration compared to WT and DKO Co. DKO VAD had significantly greater corneal OGD staining (count and intensity). Conjunctival goblet cell density and volume was significantly reduced (74% and 97%, respectively) in the DKO VAD. scRNA-seq detected 20 immune cell populations in the conjunctiva with increases in macrophages (11%), monocytes (6%), gamma delta (GDT, 9%) and conventional (2.8%) T cells and a 12% decrease in eosinophils. These findings were consistent with flow cytometry that showed significant increases in CD11b+ cells, total GDT+ cells, IFN-g+GDT and IL-17+GDT cells. Expression of Csf1, Flt1, Il10rb, Il1b, Ccl22, Tgfb1, Il22ra increased vs. WT; Csf3, Cxcl1, Cxcl9 and Nod2 were increased vs. WT and DKO Co and Il10rb, Stat3, Pdgfa, Tlr1 were decreased vs. both groups (FDR adj P < 0.001).
Conclusions :
The tenuous vitamin A status in the DKO strain worsens when fed a VAD deficient diet. The DKO on a VAD diet develops severe cornea and conjunctival epithelial disease and inflammation associated with increases in macrophages and GDT cells. These findings indicate that retinoids are essential in maintaining ocular surface epithelial health and suppressing inflammation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.