Epigenetic regulation of intestinal innate immunity during differentiation of Caco-2 cells under inflammatory stress

The intestinal epithelium supports antimicrobial programs and epithelial barrier function; while epigenetic mechanisms are increasingly implicated in these processes, how the differentiation stage shapes epigenetic regulation of key innate immune genes under inflammatory stress remains unclear. We investigated differentiation-stage-dependent epigenetic modulation of intestinal innate immunity-associated genes, focusing on α-defensin 5 (DEFA5) and SRY-box transcription factor 9 (SOX9), in a controlled Caco-2 differentiation framework. Human colon-derived Caco-2 cells were cultured for 2-14 days and transiently exposed to dextran sulfate sodium (DSS) as an in vitro epithelial inflammatory stressor. Promoter DNA methylation and gene expression of DEFA5, SOX9, odd-skipped-related transcription factor 1 (OSR1), and receptor-interacting protein 140 (RIP140) were assessed. DSS exposure at the undifferentiated stage was associated with sustained suppression of DEFA5 and SOX9 accompanied by increased DNMT1/3B expression, together with altered expression of SOX9-associated transcription factors (OSR1 and RIP140). Transient DNMT1 inhibition with GSK-3484862 during early differentiation partially rescued DSS-imposed suppression of DEFA5 and SOX9 and shifted promoter methylation readouts toward the control direction. Overall, early-stage inflammatory exposure imposes a persistent epigenetic constraint on Paneth-associated innate immune gene programs in this in vitro model, providing insights into how epithelial differentiation stage may influence sustained innate immune dysregulation.