P0183 GSDMD regulates interactions between macrophages and intestinal epithelial cells in inflammatory bowel disease

Abstract Background Our previous studies have demonstrated the significant role of the GSDMD-mediated pyroptotic pathway in inflammatory bowel disease (IBD). However, there remains some controversy surrounding the research findings concerning GSDMD in IBD. Methods Employing lentiviral transduction techniques, we manipulated GSDMD expression levels in THP-1 and HT-29 cells, assessing the impact of GSDMD on macrophage M1 polarization and epithelial barrier function. Further, co-culturing transduced THP-1 and HT-29 cells enabled the examination of changes in the intestinal barrier. Moreover, we generated conditional knockout mice with Gsdmdflox/floxLyz2-Cre and Gsdmdflox/floxVillin1-Cre, inducing experimental colitis with dextran sodium sulfate (DSS) to evaluate mucosal macrophage phenotypes and intestinal barrier integrity. Additionally, primary macrophages and intestinal epithelial cells were co-cultured, and downstream inflammatory factors were screened through transcriptome sequencing. Results Knockdown of GSDMD in THP-1 cells significantly reduces CD86 expression, indicating a decrease in M1 polarization efficiency, whereas overexpression of GSDMD yields opposite results. In HT-29 cells, overexpression of GSDMD followed by LPS treatment leads to a significant decrease expression of Claudin-1, Occludin, and Zo-1, whereas knockdown of GSDMD shows the opposite outcome. ELISA results demonstrate a correlation between GSDMD expression levels and levels of LDH, IL-1β, IL-18, TNF-α, and IL-6. In the inflammatory co-culture model of THP-1 and HT-29 cells with double knockdown of GSDMD, the severity of intestinal barrier damage and release of inflammatory factors is minimal. Conversely, in the inflammatory co-culture model of THP-1 and HT-29 cells with double overexpression of GSDMD, the intestinal barrier damage and release of inflammatory factors are maximal. Following DSS-induced colitis, compared to the Gsdmdflox/flox group, two conditional knockout mice exhibit reduced inflammatory evaluation. Furthermore, the protein expression of Zo-1, Occludin, and Claudin-1 in two conditional knockout mice is significantly higher than that in the Gsdmdflox/flox group. Flow cytometry results show that the proportion of M1 macrophages in the mucosal lamina propria is significantly lower in two conditional knockout mice compared to the Gsdmdflox/flox group. Additionally, primary macrophage and intestinal epithelial cell co-culture experiments, coupled with transcriptomic sequencing, revealed IL-1β as a downstream mediator of GSDMD-mediated macrophage-epithelial cell interplay. Conclusion It was confirmed that GSDMD serves as a key target in the pathological interplay between macrophages and epithelial cells.