Hypoxia Inducible Factor (HIF)-Dependent Regulation of TH1 CD41 T Cells in Inflammatory Bowel Disease

Abstract

Hypoxia and inflammation share an interdependent relationship. During inflammatory bowel disease (IBD) the inflamed intestinal mucosa becomes hypoxic due to increased metabolic demand and a paucity of available oxygen. Stabilization of hypoxia-inducible factors (HIF) occur within the cellular milieu of the inflamed intestine and directly affects the leukocyte transcriptome. While effector TH1 CD4+ T cells infiltrating the intestinal lamina propria have been implicated in driving induction and perpetuation of the inflammatory processes in Crohns' Disease (CD), the effect of hypoxia signaling on effector TH1 T cells remains unknown. To address this, we examined the role of hypoxia signaling on the regulation of TH1 CD4+ T cells by differentiating murine TH1 T cells ex vivo and exposed them to ambient hypoxia in vitro (1% P02). The effect of hypoxia on transcriptional machinery and cytokine production was assessed by QPCR, flow cytometry, western immunoblotting and ELISA. Using the CD45Rbhigh murine model of ileocolitis we assessed the localization of T cells infiltrating hypoxic lesions within the inflamed mucosa and the stabilization of T cell-HIF. Lastly, using targeted pharmacological treatment or T cells with and intrinsic genetic deletion of HIF, we further assessed the impact of HIF stabilization or deletion on CD45Rbhigh ileocolitis. Using the CD45Rbhigh murine model of ileocolitis, we show that lamina propria-infiltrating CD4+ T cells are exposed to regions of fulminant hypoxia and express stabilized hypoxia-inducible factor compared to colonic CD4+ T cells from uninflamed mice. Building on these preliminary studies we differentiated murine TH1 CD4+ T cells ex vivo and exposed them to ambient hypoxia in vitro (1% P02). Experimental hypoxia induced a marked repression of the transcription factor Tbet (Tbx21; the master regulator of TH1 T cells) at both the mRNA and protein levels. This was paralleled with a functional effect as hypoxia further suppressed IFNc secretion (the predominant Tbet-responsive TH1 effector cytokine). These findings were also mirrored with pharmacological stabilization of HIF using the prolyl-hydroxylase inhibitor, DMOG. In an effort to define the molecular mechanism employed by hypoxia to repress Tbet, both lentiviral knock-down and genetic deletion of T cell-intrinsic HIF, revealed a redundant role HIF-1a whereas hypoxic-repression of Tbet and IFNc was attenuated in HIF-2a deficient T cells. Lastly, in a proof of principal in vivo experiment, pharmacological stabilization of HIF attenuated all indices of CD45Rbhigh colitis while conversely, selective T cell-HIF-2a deletion exacerbated disease compared to WT counterparts. Based on these preliminary studies, we hypothesize that HIF-2a critically regulates mucosal TH1 CD4+ T cell function and may highlight a novel targeted therapeutic option for the treatment of inflammatory bowel disease.