GZMA Suppressed GPX4-mediated Ferroptosis to Improve Intestinal Mucosal Barrier Function in Inflammatory Bowel Disease

Abstract

Abstract Background Intestinal epithelial cells (IECs) is a critical component in maintaining intestinal homeostasis. Our previous study demonstrated a decreased population of colonic CD8+CD39+ T cells in pediatric-onset colitis and inflammatory bowel disease (IBD), and further revealed an enrichment of granzyme A (GZMA) in CD8+CD39+ T cells. However, the function of GZMA in IECs remained to be identified. Methods ELISA was performed to detect GZMA expression. WB, q-PCR and IF were performed to detect the expression of CDX2, OCLN, ZO-1 and ferroptosis-related genes expression. In vitro permeability assay was performed to assess the impact of GZMA in intestinal permeability. Intestinal organoid assay was used to explore the effect of GZMA on intestinal organoid differentiation. Luciferase and a subcellular fractionation approach were applied to reveal the mechanism underlying GZMA mediated ferroptosis in vitro. In vivo experiment in mice was conducted to validate the role of GZMA in alleviating ferroptosis in IECs, thereby promoting cell differentiation. Results Herein, we, for the first time, demonstrated that GZMA contributed to IECs differentiation characterized by enhanced CDX2 expression, leading to induce Occludin(OCLN) and Zonula Occludens-1(ZO-1) expression, which was attributed to ferroptosis inhibition caused by GZMA. Mechanically, GZMA activated cAMP/PKA/CREB pathway in IECs, leading to CREB nuclear translocation and initiate GPX4 transactivity. Most importantly, treatment of GZMA could alleviate DSS-induced colitis in mice confirmed by immunofluorescence. Conclusion These findings suggested that GZMA contributed to intestinal epithelial cell differentiation through ferroptosis via the PDE4/PKA/CREB signaling pathway, targeting GZMA could be a promising strategy to patients with IBD.