Essential role for epithelial HIF‐xenophagy axis in control of Salmonella infection and dissemination

Abstract

The intestinal mucosa exists in a state of “physiologic hypoxia,” in which oxygen tensions of the epithelium and lumen are markedly lower than those encountered in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain mucosal homeostasis in this austere environment in part through the actions of dedicated oxygen‐sensitive transcription factors, including hypoxia‐inducible factors (HIFs). HIFs are composed of a labile α‐subunit that becomes stabilized most notably in hypoxic environments and a constitutive β‐subunit. Using an unbiased chromatin immunoprecipitation (ChIP) promoter array screen for HIF‐1 targets in IEC, we identified autophagy as a major pathway induced by hypoxia and HIF in cultured IECs. Autophagy is a conserved eukaryotic system for degradation of intracellular cargo through the lysosome. Validation of the ChIP screen revealed that HIF promotes expression of multiple autophagy genes in IEC cell lines and further validated in primary murine organoids. One important function of autophagy is to defend against intracellular pathogens, a process termed xenophagy, a crucial innate immune mechanism in the mucosa that serves to control invasion of intracellular bacteria in vivo and to limit extraintestinal dissemination of enteric pathogens. Loss of function analysis revealed that HIF is a central regulator of autophagic flux and that in vitro infection of IECs with SalmonellaTyphimurium, a model intracellular bacterial pathogen, results in stabilization of HIF with the subsequent increase in HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Studies in vivo revealed that Salmonella­ drives increases in HIF activity using HIF luciferase reporter mice (HIF∆ODD) in combination with streptomycin‐pretreatment model of murine colitis. Further studies in vivo demonstrated that intestinal epithelial‐specific deletion of HIF (Hif1b∆IEC) compromised IEC xenophagy and exacerbated bacterial dissemination to multiple extraintestinal organs, implicating xenophagy‐assisted clearance of Salmonella in mucosal innate immunity.