Single-Cell RNA Sequencing Revealed Functional Conjunctival Keratinocytes Loss via TGF-β-Wnt/β-Catenin Signaling in Sjögren's Syndrome Related Dry Eye.

Purpose: The role of the conjunctiva in the pathophysiology of Sjögren's syndrome (SS) related dry eye disease (DED) remains obscure especially in the view of functional conjunctival epithelia. In order to illustrate effects of conjunctiva in SS, we investigated the interactions between parenchymal cells and immune cells in the conjunctiva with single-cell RNA sequencing technique. Methods: Freshly collected conjunctiva from a canonical SS model was prepared for 10 × Genomics single-cell RNA sequencing and T cell receptor (TCR) sequencing. Conjunctiva was collected for Western blot, immunofluorescence, multiplex immunohistochemical (mIHC), and flow cytometry. Phenol red thread test, lissamine staining, and qRT-PCR were applied to evaluate signs of DED. Results: DED phenotype was validated in the SS model. Loss of water-secreting keratinocyte was projected in scRNA-seq data and proved by mIHC test in SS mice. The proportion of Lgr4+ basal epithelial cells with poor ability to differentiate into mature keratinocyte increased, and Wnt/β-catenin signaling was upregulated in it under regulation of TGF-β derived from macrophages. Such macrophages promoted angiogenesis through secretion of VEGFA to activate endothelial cells. Immuno-fibroblasts had an increased population, which were implicated in specifically activated T cell chemotaxis. Conclusions: In SS conjunctiva, a TGF-β-Wnt/β-catenin axis downregulated the formation of functional keratinocytes accompanied by infiltration of pro-angiogenetic and pro-fibrotic macrophage and pro-inflammatory T cell.