Transient Mitomycin C treatment of human corneal epithelial cells impacts expression of autophagy and cytokine genes and increases phagocytosis

Abstract

Purpose : A single MMC treatment enhances reinnervation of the intraepithelial corneal nerves after debridement injury. RNAseq studies performed on mouse corneas revealed that MMC enhanced protease inhibitor expression in the epithelium. To confirm that the gene expression changes in mouse corneal epithelial cells was due to a direct impact of MMC on corneal epithelial cells, we performed RNAseq studies on transiently treated telomerase immortalized human corneal limbal epithelial (HCLEs) and primary human corneal epithelial (PHCLE) cells before and after MMC treatment.

Methods : HCLE and PHCLE cells were cultured in KSFM media to 70% confluence. Cells were treated with 0.0025% MMC for 3h followed by RNA extraction. For each variable, 4 replicate sets of RNA were isolated. mRNA expression profiling was performed in the NIAMS Genome Core Facility at the NIH. Single end, 50 base reads were mapped to the mouse genome mm10 using TopHat 2.1.0. For phagocytosis studies, fluorescent 1.0 mm latex beads were opsinized with BSA and added to cell cultures for 1 hr. Cells were washed, fixed and stained with phalloidin. The fluorescence intensity of the beads present per cell were corrected for difference in cell size.

Results : Comparing 5-fold up and down regulated genes, we determined genes that are altered in expression after MMC treatment in HCLE and PHCLE cells. There are 15 MMC signature genes with 11 upregulated and 4 down regulated. There are too few down regulated genes to determine shared gene ontology but the 11 up regulated genes show common gene ontology terms for cytokines with 4 of the genes falling into category [CXCL1, CXCL16, IL32, and ISG15]. In addition, MMC induced an increase in expression of autophagy and phagocytosis genes which was also shown by the phagocytosis studies.

Conclusions : MMC treatment impacts gene expression in the corneal epithelium both in vivo and in vitro in HCLE and PHCLE cells by inducing the up-regulation of numerous cytokines, autophagy and phagocytosis genes which lead to increased phagocytosis by corneal epithelial cells. The enhanced reinnervation seen in the mouse corneal epithelium after a single MMC treatment during debridement wounding may be due to its ability to alter immune cell recruitment via alterations in cytokine secretion and/or the improved ability to remove debris released by injured cells and severed axons.

This is a 2021 ARVO Annual Meeting abstract.