Abstract
PURPOSE: To evaluate the effects of dry eye on conjunctival immune cell number and transcriptional profiles with attention to mononuclear phagocytes. METHODS: Expression profiling was performed by single-cell RNA sequencing on sorted conjunctival immune cells from non-stressed and C57BL/6 mice subjected to desiccating stress (DS). Monocle 3 modeled cell trajectory, scATAC-seq assessed chromatin accessibility and IPA identified canonical pathways. Inflammation and goblet cells were measured after depletion of MRC1(+) MΦs with mannosylated clodronate liposomes. RESULTS: Mononuclear phagocytes (monocytes, MΦs, DCs) comprised 72 % of immune cells and showed the greatest changes with DS. Distinct DS induced gene expression patterns were seen in phagocytes classified by expression of Ccr2 and [Timd4, Lyve1, Folr2 (TLR)]. Expression of phagocytosis/efferocytosis genes increased in TLF(+)CCR2(-) MΦs. Monocytes showed the highest expression of Ace, Cx3cr1, Vegfa, Ifngr1,2, and Stat1 and TLF(-)CCR2(+) cells expressed higher levels of inflammatory mediators (Il1a, Il1b, Il1rn, Nfkb1, Ccl5, MHCII, Cd80, Cxcl10, Icam1). A trajectory from monocyte precursors branched to terminate in regulatory MΦs or in mDCs via transitional MΦ and cDC clusters. Activated pathways in TLF(+) cells include phagocytosis, PPAR/RXRα activation, IL-10 signaling, alternate MΦ activation, while inflammatory pathways were suppressed. Depletion of MRC1(+) MΦs increased IL-17 and IFN-γ expression and cytokine-expressing T cells, reduced IL-10 and worsened goblet loss. CONCLUSIONS: Dryness stimulates distinct gene expression patterns in conjunctival phagocytes, increasing expression of regulatory genes in TLF(+) cells regulated in part by RXRα, and inflammatory genes in CCR2(+) cells. Regulatory MΦs depletion worsens DS induced inflammation and goblet cell loss.