EPITHELIAL DUOX2 ACTIVATION INCREASES GUT PERMEABILITY AND BACTERIAL TRANSLOCATION THAT IS RESCUED WITH BUTYRATE SUPPLEMENTATION

Abstract BACKGROUND Inflammatory bowel diseases (IBD) are chronic pathologies characterized by dysbiosis, defects in epithelial barrier function, and increased redox stress. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, which produce metabolites important in maintaining gut homeostasis. The epithelial NADPH oxidase dual oxidase 2 (DUOX2) catalyzes the production of hydrogen peroxide (H2O2) and is the only gene consistently altered in IBD patients in association with dysbiosis and before the onset of disease. However, the functional consequence of DUOX2 activation in IBD is not understood. Using models of dysbiosis leading to DUOX2 activation, we aimed to dissect the contribution of DUOX2 activity to the defects in epithelial barrier function observed in IBD. METHODS villin-TLR4 (vTLR4) mice, which express a constitutively active form of TLR4 in colonic epithelial cells (CECs) and have increased activation of DUOX2, vTLR4-DUOX2-KO mice, and their wild-type (WT) littermates were euthanized. Livers were collected for permeability assessment by bacterial translocation. Colonic tissue was collected for RNA-sequencing and isolation of CECs for gene expression determinations by qPCR. Separately, vTLR4 mice and littermates were treated with butyrate in drinking water. Tissue was harvested for the same determinations and for measurement of epithelial H2O2 production via Amplex Red. Colonoids from WT mice were treated with butyrate and stimulated with IFNγ or heat-killed adherent invasive Escherichia coli. H2O2 production and gene expression of Duox2 were determined. RESULTS RNA-sequencing revealed that abrogation of DUOX2 activity downregulated cytokine-mediated signaling and cytokine production pathways, including tumor necrosis factors and key chemotactic proteins such as CXCL1 and CXCL2. Similarly, expression of Tnfa, Cxcl1, and Cxcl2 was significantly downregulated to WT levels in vTLR4-DUOX2-KO mice. vTLR4 mice had increased bacterial translocation, which was rescued by deletion of DUOX2. Butyrate treatment inhibited epithelial H2O2 response to stimuli in vitro, which was accompanied by a reduction in Duox2 transcripts. In vivo, butyrate treatment of vTLR4 mice significantly reduced epithelial production of H2O2, bacterial translocation to the liver, and led to a downregulation of Tnfa, Cxcl1, and Cxcl2 transcripts. CONCLUSIONS Chronic DUOX2 activity leads to defects in epithelial barrier function resulting in increased bacterial translocation. This leads to an increase in proinflammatory cytokines and chemokines. The microbial metabolite butyrate blocked DUOX2 activity, rescuing intestinal permeability and reducing bacterial translocation and inflammatory cytokine gene expression. We posit that therapies aimed at controlling DUOX2 activity through microbiome or metabolome-targeted approaches may be beneficial in IBD.