Analysis of the inflammatory response dynamics in colonic epithelial organoids

Abstract

Introduction Microscopic colitis (MC) is a chronic inflammatory bowel disease (IBD) characterized by intestinal epithelial barrier disruption due to an excessive immune response to environmental antigens. The dynamics of inflammation in colonic epithelial organoid models remain largely unexplored, particularly the timing of cytokine-induced responses and their resolution [1]. Aim To investigate the dynamics of the inflammatory response in colonic epithelial organoids. Methods Colonic epithelial organoids were derived from control individual biopsies and stimulated with TNF-α and IFN-γ at a single time point. Samples were collected at 2, 4, 8, 12, 24, 36, and 48 hours post-treatment to assess temporal molecular changes. Expression of TNF-α, CXCL9, TJP1, and OCLN was analyzed using qRT-PCR. Results The analysis of pro-inflammatory gene expression (TNF-α and CXCL9) indicates that the inflammatory response in colonic organoids remains elevated and stable for at least 12 hours post-cytokine stimulation. Both genes are significantly upregulated at 2h (TNF-α: FC = 23.1, p = 1.5 × 10⁻³; CXCL9: FC = 4991.2, p = 5.5 × 10⁻⁴), peaking at 4h–8h (TNF-α: FC = 30.5, p = 3.1 × 10⁻⁵; CXCL9: FC = 31288.6, p = 8.3 × 10⁻⁷), and remaining high at 12h (TNF-α: FC = 21.6, p = 5.3 × 10⁻⁴; CXCL9: FC = 21567.4, p = 2.7 × 10⁻⁵). By 24h, inflammation starts to subside (TNF-α: FC = 10.5, p = 7.0 × 10⁻⁵; CXCL9: FC = 14499.0, p = 5.8 × 10⁻⁴), though a sustained response is observed. At 48h, inflammatory marker expression returns to baseline (TNF-α: FC = 9.2, p = 1.4 × 10⁻²; CXCL9: FC = 3111.2, p = 5.8 × 10⁻⁴), similar to 2h levels. Concurrently, the downregulation of OCLN after 4h (FC = 0.8, p = 3.1 x 10-2), 12h (FC = 0.7, p = 2.0 x 10-2) and 48h (FC = 0.7, p = 4.8 x 10-3) of stimulation with inflammatory cytokines had tendency reflect epithelial barrier disruption. Conclusions This study demonstrates that the inflammatory response in colonic epithelial organoids remains elevated and stable for at least 12 hours following cytokine stimulation, with TNF-α and CXCL9 peaking at 4h–8h and gradually declining by 24h–48h. The observed downregulation of OCLN at multiple time points suggests epithelial barrier disruption as a consequence of prolonged inflammation.