Hypoxia: an alarm signal during intestinal inflammation

Abstract

Intestinal epithelial cells that line the mucosal surface of the gastrointestinal tract are positioned between an anaerobic lumen and a highly metabolic lamina propria. As a result of this unique anatomy, intestinal epithelial cells function within a steep physiologic oxygen gradient relative to other cell types. Furthermore, during active inflammatory disease such as IBD, metabolic shifts towards hypoxia are severe. Studies in vitro and in vivo have shown that the activation of hypoxia-inducible factor (HIF) serves as an alarm signal to promote the resolution of inflammation in various mouse models of disease. Amelioration of disease occurs, at least in part, through transcriptional upregulation of nonclassic epithelial barrier genes. There is much interest in harnessing hypoxia-inducible pathways, including stabilizing HIF directly or via inhibition of prolyl hydroxylase enzymes, for therapy of IBD. In this Review, we discuss the signaling pathways involved in the regulation of hypoxia and discuss how hypoxia may serve as an endogenous alarm signal for the presence of mucosal inflammatory disease. We also discuss the pros and cons of targeting these pathways to treat patients with IBD.

Figure 1. Potential sources of hypoxia in mucosal inflammation

During episodes of inflammation, a number of factors influence the supply and demand of oxygen to the tissues, as well as influencing oxygen delivery. Noted here are edema, vasculitis and vasoconstriction, which separate epithelial cells from the blood supply and limit oxygen availability. In addition, local depletion of oxygen through processes such as the polymorphonuclear cells oxygen burst can usee large quantities of oxygen and result in localized hypoxia, where red depicts normoxia and blue represents hypoxia.

Figure 2. Detection of hypoxia in the mucosa

a) The in vivo evidence for hypoxia associated with inflammation (so called “inflammatory hypoxia”) are provided using nitroimidazole-based dye retention in vitro and in mice. These molecules (R-NO₂) are taken into cells passively and reduced to highly reactive nitrogen intermediates (R-NO₂-[dot]). In the absence of adequate oxygen to regenerate the native compound, these intermediates react with cellular proteins to form adducts (R-NH₂), which can be visualized using labeled antibodies. b) In the colons of mice with no inflammation (control) small amounts of nitroimidazole adduct is detected along the luminal aspect of the colon (red), suggesting a degree of physiological hypoxia in the normal colon. c) During episodes of inflammation, such as seen here in a mouse model of colitis, intense and deep tissue hypoxia is prevalent, particularly in areas overlying lesions.

Figure 3. Structural features of hypoxia-inducible factor (HIF) and mechanism of HIF stabilization

A) The alpha subunit of HIF (HIF-1, HIF-2 and HIF-3) consist of a C-terminal basic helix-loop-helix domain (bHLH, gray), a Per-Arnt-SIM (PAS, purple), a C- and N-terminal oxygen-dependent degradation domain (ODD) region (orange) and a N-terminal transactivation domain (TAD, blue). Also shown are the proline (Pro) and asparagines (Asn) sites for hydroxylation by proly-hydroxylases (PHD) 1–3 and functional inhibitor of HIF (FIH), respectively. B) Depiction of HIF hydroxylation by the combination of alpha-ketoglutarate (αKG), molecular oxygen (O₂) and the PHD enzyme in normoxia. When O₂ becomes limiting (hypoxia), the alpha subunit is stabilized, binds to the HIF-1 beta subunit within the nucleus where it becomes transcriptionally active upon binding to the hypoxia-response element (HRE) consensus sequence on DNA.

Figure 4. Activation of hypoxia-inducible factor (HIF) and NF-κB in IEC’s during inflammation

Episodes of inflammation and hypoxia activate a number of pathways in intestinal epithelial cells. The transcriptional activation of both HIF (blue) and NF-κB (red) is stabilized by proly-hydoxylase enzymes (PHDs) and functional inhibitor of HIF (FIH). Activation of these pathways has been shown to promote epithelial barrier function and to decrease epithelial apoptosis, with an overall epithelial protective phenotype. As noted, the various PHD enzymes cross-regulate both HIF and NF-κB in a non-redundant manner, including the regulation of activating transcription factor-4 (ATF-4) and Notch (see text). Green arrows indicate hydroxylation reactions and black arrows indicate transcriptional activation. Activation of HIF through these hydroxylation reactions (blue) promotes barrier function while activation of NF-κB (red) diminishes epithelial apoptosis.