Abstract
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.
Highlights
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium
Human colonic biopsies were mounted in Ussing chambers; bacteria were added to the luminal buffer, and biopsies were treated with DNP Ϯ the endoplasmic reticulum (ER) stressor tunicamycin (blocks protein glycosylation [21], which is often used to elicit ER stress in cell lines [19])
DNP treatment of colonic enteroid monolayers resulted in increased transcytosis of E. coli that was reduced by tunicamycin (Fig. 1B)
Summary
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. Nonsteroidal anti-inflammatory drugs (NSAID) can cause relapses in IBD [7], and their use damages mitochondria contributing to decreased enteric barrier function [8, 9]. Mitochondrial damage induced by uncoupling oxidative phosphorylation with the Hϩ ionophore, dinitrophenol (DNP), to model the mitochondrial dysfunction observed in IBD resulted in increased internalization into and transcytosis of Escherichia coli across epithelial monolayers [13,14,15]. Mutation in the X-box– binding protein-1 (XBP1) gene (downstream of IRE1) is a susceptibility trait for Crohn’s disease [17], and increased ER stress can occur in the epithelium of patients with IBD [17, 19, 20]. We hypothesized that uncoupling oxidative phosphorylation (i.e. mitochondrial dysfunction) combined with ER stress would severely compromise epithelial integrity
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