Abstract
OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H(2)O(2) and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.