A290 THE ATF6 ARM OF ER STRESS ANTAGONIZES METABOLIC STRESS-INDUCED DECREASES IN EPITHELIAL BARRIER FUNCTION TO COMMENSAL BACTERIA BY PROMOTING XENOPHAGY

The barrier function of the intestinal epithelium is a first-line of defense that is compromised in IBD. We have shown that epithelia treated with dinitrophenol (DNP: uncouples oxidative phosphorylation) have a dramatic impairment in their barrier function, and internalize non-invasive commensal E. coli. Mitochondria do not function in isolation; they are closely aligned with the endoplasmic reticulum (ER). To investigate the interaction of mitochondrial dysfunction, ER stress and autophagic processes as a key determinant of epithelial-bacterial interaction, transcellular permeability and the fate of commensal bacteria that gain access to the intracellular compartment as a consequence of metabolic stress. Human colonic biopsies were mounted in Ussing chambers, murine epithelial colonic organoids were re-suspended and grown as a monolayer, and human colon-derived epithelial cell lines were cultured on transwell filters or on plastic. Bacteria were added to the luminal buffer and the tissue treated with DNP ± the ER stressor, tunicamycin (TM), and bacterial internalization and translocation assessed. Mechanistic studies involved measuring ATP production, immunoblotting, assessment of LC3 activation for autophagy and gene knock-down (KD) by siRNA and CRISPR/cas9. DNP promoted the translocation and internalization of E. coli and, remarkably, TM reduced this barrier defect. TM did not prevent the DNP-evoked drop in ATP or the rate of epithelial update of inert beads, but E. coli, DNP and TM-treated cells had increased autophagy. The effect of TM was lost in cells lacking the autophagy protein, ATG16L1, suggesting that the ER-stress promoted killing of the internalized bacteria. Of the 3 major arms of the ER stress response, only KD of ATF6 ablated the TM antagonism of the DNP-evoked barrier effect; the increase in autophagy evoked by TM was absent in ATF6 KD epithelia. Pharmacological studies indicate that the kinase, DAPK1 is required from the ER stress-ATF6 signal to drive autophagy to kill the internalized E. coli. Conclusion: The ATF6 arm of ER stress antagonizes metabolic stress-induced decreases in epithelial barrier function to commensal bacteria by promoting xenophagy via upregulation of DAPK1. CAG, CIHRAIHS, HPI-NSERC, CNPq

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