A number of recent studies have implicated tissue hypoxia in both acute and chronic inflammatory diseases, particularly as they relate to mucosal surfaces involving epithelial cells. In this context, a protective role for the transcriptional regulator hypoxia-inducible factor (HIF) was demonstrated through conditional deletion of epithelial HIF-1α in a murine model of colitis (J. Clin. Invest. 2004; 114:1098−1106. Here, we hypothesized that pharmacologic activation of HIF would similarly provide a protective adaptation to murine colitic disease. For these purposes, we used a novel prolyl hydroxylase (PHD) inhibitor (FG-4497) which readily stabilizes HIF-1α and subsequently drives the expression downstream HIF target genes (e.g. erythropoietin). Our results show that the FG-4497-mediated induction of HIF-1α provides an overall beneficial influence on clinical symptoms (weight loss, colon length, tissue TNFα) in murine TNBS colitis, most likely due to their barrier protective function and wound healing during severe tissue hypoxia at the site of inflammation. Taken together these findings emphasize the role of epithelial HIF-1α during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1 α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

Inflammatory diseases influence tissue metabolism, altering regulation of extracellular adenine nucleotides, with a resultant protective influence of adenosine. Ecto-5′-nucleotidase (CD73) is a central surface enzyme generating extracellular adenosine. Thus, we hypothesized that CD73 is protective in mucosal inflammation as modeled by trinitrobenzene sulfonate (TNBS) colitis. Initial studies revealed a >3-fold induction of CD73 mRNA levels after TNBS colitis. Additionally, the severity of colitis was increased, as determined by weight loss and colonic shortening, in cd73−/− mice relative to cd73+/+ controls. Likewise, enteral administration of the selective CD73 inhibitor α,β-methylene ADP to cd73+/+ mice resulted in a similar increase in severity of TNBS colitis. Gene array profiling of cytokine mRNA expression, verified by real-time PCR, revealed a >90% down-regulation of IFN-αA in cd73−/− mice and α,β-methylene ADP-treated cd73+/+ mice, compared with cd73+/+ mice. Exogenous administration of recombinant IFN-αA partially protected TNBS-treated cd73−/− mice. Cytokine profiling revealed similar increases in both IFN-γ and TNF-α mRNA in colitic animals, independent of genotype. However, IL-10 mRNA increased in wild-type mice on day 3 after TNBS administration, whereas cd73−/− mice mounted no IL-10 response. This IL-10 response was restored in the cd73−/− mice by exogenous IFN-αA. Further cytokine profiling revealed that this IL-10 induction is preceded by a transient IFN-αA induction on day 2 after TNBS exposure. Together, these studies indicate a critical regulatory role for CD73-modulated IFNαA in the acute inflammatory phase of TNBS colitis, thereby implicating IFN-αA as a protective element of adenosine signaling during mucosal inflammation.