Regulation of Epithelial Innate Immunity through Hypoxia-mediated Autophagy

Abstract

The gastrointestinal epithelium comprises the primary barrier between omnipresent luminal antigens and the underlying immune cell repertoire, and is subject to profound metabolic fluctuations, particularly in inflammatory bowel disease (IBD). A role for the hypoxia-inducible transcription factors (HIFs) in orchestrating transcriptional changes to promote barrier function has been well defined. Given the importance of HIF signaling to epithelial homeostasis, we hypothesized that HIF-mediated transcriptional changes contribute to epithelial innate immunity. Promoter microarray screens of HIF-enriched genomic DNA, derived from chromatin immunoprecipitation (ChIP-on-chip), highlighted autophagy as a novel pathway coordinately regulated by HIF-1. A fundamental role for autophagy in IBD pathogenesis has recently been elucidated, defined largely by abrogated bacterial killing (xenophagy). However, little is known about transcriptional regulation of coordinated autophagy gene responses, and how these modulate intestinal epithelial cells (IEC) xenophagy. Human intestinal epithelial Caco-2 and T84 cells were used. Hypoxia was defined as pO2 20 torr and pCO2 35 torr. ChIP-on-chip: ChIP was performed with a polyclonal HIF-1a antibody using sheared chromatin from hypoxic Caco-2 cells. ChIP-enriched and input DNA were Cy-labeled and hybridized to a promoter microarray (Switchgear Genomics). The log2 ratio (input-Cy3/HIF-ChIP-Cy5) was analyzed to identify sequences specific for HIF-1a binding. ChIP-qPCR: ChIP-qPCR was used to quantify HIF binding to candidate loci using primer sets flanking putative HRE sites. Salmonella Typhimurium Invasion and O2 Consumption: Caco-2 cells were infected with late-log cultures of S. Typhimurium 14028 at an multiplicity of infection (MOI) of 100. The SDR OxoDish system (Presens) was used to measure the effect of Salmonella invasion on epithelial O2 consumption in real-time. Gentamicin kill assays: Caco-2 cells were infected with S. Typhimurium and incubated in antibiotic-free medium for 30 min, followed by media supplemented with 50 μg/mL gentamicin (Sigma) for 1h. Viable intracellular bacteria were quantified by colony counts following epithelial lysis in 1% Triton-X-PBS. A cohort of promoters for autophagy genes were specifically enriched for HIF binding by ChIP-chip. Transcript levels of candidate genes were induced by 6hrs under hypoxia or prolyl hydroxylase inhibition in Caco-2 and T84 cells, corroborating association between HIF binding and gene expression. Hypoxic Caco-2 cells displayed 3.5-fold higher LC3/p62-positive puncta by IF than normoxic cells. By immunoblot analysis, LC3-II and p62 protein levels were increased and decreased respectively in hypoxic lysates, indicating hypoxia-induced autophagic turnover. Hypoxia stimulation of autophagy increased Salmonella killing, confirming hypoxic induction of xenophagy. Moreover, invasive Salmonella induced a time- and MOI-dependent depletion of O2 with stabilization of HIF protein. To analyze baseline epithelial autophagy in the absence of HIF signaling in vivo, we measured IEC levels of LC3 and p62 protein in HIF-1b−/− mice. Consistent with a role for HIF, the ratio of LC3-I to LC3-II and total levels of p62 were increased in vil-Cre+/HIF-1bfl/fl mice relative to vil-Cre−/HIF-1bfl/fl controls. Modulation of HIF-mediated autophagy by inflammatory hypoxia and/or bacterial pathogens themselves defines a conserved innate response for host protection, and may represent a novel target for development of therapeutic strategies to modify epithelial autophagic targeting of bacteria.