Changes in conjunctival mononuclear phagocytes and suppressive activity of regulatory macrophages in desiccation induced dry eye.

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 TLFCCR2 MΦs. Monocytes showed the highest expression of Ace, Cx3cr1, Vegfa, Ifngr1,2, and Stat1 and TLFCCR2 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.