539 The role of autophagy in IFN-γ effects on global gene expression in keratinocytes

Abstract

Background: Vitiligo is a common acquired skin disease with characteristic pigment disorder and there is no effective treatment. IFN-γ has been regarded as a crucial factor in the progression of vitiligo. Autophagy also plays an important role in maintaining the function of skin cells and defects in autophagy cause defective pigmentation; however, the relationship between autophagy and IFN-gamma effects in keratinocytes remains unclear. Objective: The aim of the study was to explore whether autophagy could regulate the IFN-gamma effects in keratinocytes. Methods: The induction and activity of autophagy in keratinocytes was determined by western blotting, immunofluorescence and electron microscopy after IFN-γ stimulation.n RNA-Seq analysis was performed to investigate IFN-γ effects on global RNA expression in keratinocytes from autophagy competent (Atg7 f/f, “WT”) and autophagy incompetent (Atg7 f/f K14: Cre mice, “KO”) mice of young and old age. Expression of specific genes was confirmed by real-time q-PCR. Results: Western-blotting showed that the ratio of LC3II/LC3I increased after the stimulation of IFN-γ in keratinocytes. Immunofluorescence showed an increase in the number of autophagy puncta with IFN-γ stimulation. Electron microscopy showed an increase in the number of autolysosomes with IFN-γ stimulation. They suggested that IFN-γ could induce autophagy in keratinocytes. Ingenuity pathway analysis of differentially expressed genes (DEGs) revealed pathways and functions activated by IFN-γ under different genotypes. Pathways like “Dendritic Cell Maturation” and “p38 MAPK Signaling” showed significant differences in z-scores between WT and KO groups. Conclusions: Our results suggested that IFN-γ could induce autophagy in keratinocytes; The ability to perform autophagy can influence the interferon gamma response; Activation of autophagy by its agonist rapamycin can modify the interferon gamma response depending on the age of the cell donor.