NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures

Abstract

ObjectiveLoss-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.DesignROS 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.ResultsTNFα 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 H2O2 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.ConclusionsNOX1 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.

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

“…Luminal sampling at steady state is important for homeostasis and building mucosal tolerance towards commensal microorganisms and is usually attributed to professional mucosal-resident phagocytes and to transcytosis (uptake and delivery without intracellular degradation) by specific epithelial M-cells, that expose immune cells in the lamina propria to luminal antigens (Chu et al, 2016; Grijmans et al, 2022; Rios et al, 2016; Schulz and Pabst, 2013; Yu et al, 2014). The phagocytic capacity presented in this work is not likely to be assigned to M-cells for several reasons: first, it is observed in three human epithelial cell lines and in organoids derived from different parts of the gastro-intestinal tract from ileum to sigmoid colon, while M-cells are mostly found in the small intestine and require specific differentiation conditions including TNFα and RANKL (Fasciano et al, 2019; Hsu et al, 2022; Staab et al, 2020) that were not used in this study. Second, we find that phagocytosis occurs by MUC2 positive and MUC2 negative cells indicating goblet and non-goblet cells.…”

“…An attractive approach will be to assess whether genes associated with IBD that may function in LAP indeed affect epithelial LAP. An immediate candidate from our work is NOX1, which has recently been shown to prevent inflammation, and its mutations were linked to ulcerative colitis (Hsu et al, 2022; Schwerd et al, 2018). Another interesting candidate is the ATG16L1 T300A polymorphism.…”

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

“…Luminal sampling at steady state is important for homeostasis and building mucosal tolerance towards commensal microorganisms and is usually attributed to professional mucosal-resident phagocytes and to transcytosis (uptake and delivery without intracellular degradation) by specific epithelial M-cells ( 34 , 56 – 59 ). The phagocytic capacity presented in this work is not likely to be assigned to M-cells for several reasons: first, it is observed in epithelial cell lines and in organoids derived from different parts of the gastrointestinal tract from ileum to sigmoid colon, while M-cells are mostly found in the small intestine and require specific differentiation conditions including TNFα and RANKL ( 60 – 62 ) that were not used in this study. Second, phagocytosis occurs by goblet and non-goblet cells.…”

New insights into bacterial mechanisms and potential intestinal epithelial cell therapeutic targets of inflammatory bowel disease

“…In the gastrointestinal tract, ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX/DUOX) is a key biological mechanism regulating pathogen killing, host-microbe interactions and tissue repair after injury (Aviello et al, 2019). ROS production via enterocyte NOX1 has been shown to transduce microbial signals that promote epithelial wound healing (Alam et al, 2014) and mutations in genes that induce ROS production, such as Nox1, are highly associated with IBD (Aviello et al, 2019;Hsu et al, 2022). Mechanistically, the absence of intestinal epithelial NOX1 combined with TNFα stimulation can alter the stem cell microenvironment and stem cell differentiation, promote the number of lymphoplasmacytes and thus accelerate the progression of colitis, which may provide new insights into the prevention and treatment of IBD (Hsu et al, 2022).…”

“…ROS production via enterocyte NOX1 has been shown to transduce microbial signals that promote epithelial wound healing (Alam et al, 2014) and mutations in genes that induce ROS production, such as Nox1, are highly associated with IBD (Aviello et al, 2019;Hsu et al, 2022). Mechanistically, the absence of intestinal epithelial NOX1 combined with TNFα stimulation can alter the stem cell microenvironment and stem cell differentiation, promote the number of lymphoplasmacytes and thus accelerate the progression of colitis, which may provide new insights into the prevention and treatment of IBD (Hsu et al, 2022). Superoxide dismutase 1 (SOD1) is one of the three superoxide dismutases responsible for the destruction of free superoxide radicals in the body.…”