Sean P. Colgan

Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function

Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the influences of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut.

Targeting the A2B adenosine receptor during gastrointestinal ischemia and inflammation

Extracellular adenosine functions as an endogenous distress signal via activation of four distinct adenosine receptors (A1, A2A, A2B and A3). Conditions of limited oxygen availability or acute inflammation lead to elevated levels of extracellular adenosine and enhanced signaling events. This relates to a combination of four mechanisms: i) increased production of adenosine via extracellular phosphohydrolysis of precursor molecules (particularly ATP and ADP); ii) increased expression and signaling via hypoxia-induced adenosine receptors, particularly the A2B adenosine receptor; iii) attenuated uptake from the extracellular towards the intracellular compartment; and iv) attenuated intracellular metabolism. Due to their large surface area, mucosal organs are particularly prone to hypoxia and ischemia associated inflammation. Therefore, it is not surprising that adenosine production and signaling plays a central role in attenuating tissue inflammation and injury during intestinal ischemia or inflammation. In fact, recent studies combining pharmacological and genetic approaches demonstrated that adenosine signaling via the A2B adenosine receptor dampens mucosal inflammation and tissue injury during intestinal ischemia or experimental colitis. This review outlines basic principles of extracellular adenosine production, signaling, uptake and metabolism. In addition, we discuss the role of this pathway in dampening hypoxia-elicited inflammation, specifically in the setting of intestinal ischemia and inflammation.

Identification of Hypoxia-Inducible Factor HIF-1A as Transcriptional Regulator of the A2B Adenosine Receptor during Acute Lung Injury

Although acute lung injury (ALI) contributes significantly to critical illness, resolution often occurs spontaneously through endogenous pathways. We recently found that mechanical ventilation increases levels of pulmonary adenosine, a signaling molecule known to attenuate lung inflammation. In this study, we hypothesized a contribution of transcriptionally controlled pathways to pulmonary adenosine receptor (ADOR) signaling during ALI. We gained initial insight from microarray analysis of pulmonary epithelia exposed to conditions of cyclic mechanical stretch, a mimic for ventilation-induced lung disease. Surprisingly, these studies revealed a selective induction of the ADORA2B. Using real-time RT-PCR and Western blotting, we confirmed an up to 9-fold induction of the ADORA2B following cyclic mechanical stretch (A549, Calu-3, or human primary alveolar epithelial cells). Studies using ADORA2B promoter constructs identified a prominent region within the ADORA2B promoter conveying stretch responsiveness. This region of the promoter contained a binding site for the transcription factor hypoxia-inducible factor (HIF)-1. Additional studies using site-directed mutagenesis or transcription factor binding assays demonstrated a functional role for HIF-1 in stretch-induced increases of ADORA2B expression. Moreover, studies of ventilator-induced lung injury revealed induction of the ADORA2B during ALI in vivo that was abolished following HIF inhibition or genetic deletion of Hif1a. Together, these studies implicate HIF in the transcriptional control of pulmonary adenosine signaling during ALI.

Hypoxia-inducible factors as molecular targets for liver diseases.

Liver disease is a growing global health problem, as deaths from end-stage liver cirrhosis and cancer are rising across the world. At present, pharmacologic approaches to effectively treat or prevent liver disease are extremely limited. Hypoxia-inducible factor (HIF) is a transcription factor that regulates diverse signaling pathways enabling adaptive cellular responses to perturbations of the tissue microenvironment. HIF activation through hypoxia-dependent and hypoxia-independent signals have been reported in liver disease of diverse etiologies, from ischemia-reperfusion-induced acute liver injury to chronic liver diseases caused by viral infection, excessive alcohol consumption, or metabolic disorders. This review summarizes the evidence for HIF stabilization in liver disease, discusses the mechanistic involvement of HIFs in disease development, and explores the potential of pharmacological HIF modifiers in the treatment of liver disease.

P-219 Regulation of Epithelial Innate Immunity through Hypoxia-mediated Autophagy

BACKGROUND: Mitochondrial dysfunction is central to many chronic diseases. Inflammatory bowel disease (IBD), proposed to be an energy deficient disease of the intestinal epithelium, is associated with mitochondrial abnormalities of the epithelium before the onset of inflammation. We and others previously demonstrated that levels of the mitochondrial protein prohibitin (PHB) are decreased in mucosal biopsies of active and inactive IBD and in experimental models of colitis. Prohibitin, the major component protein of the inner mitochondrial membrane, regulates respiratory chain assembly and function. In this study, we sought to use an intestinal epithelial cell-specific PHB conditional knockout mouse model to examine the role of prohibitin in maintaining mucosal homeostasis. METHODS: Transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the villin promoter (vil-CreERT2) and PHB-floxed (PHBfl/fl) mice were crossed to generate a mouse line with inducible intestinal epithelial cell-specific deletion of the PHB gene (PHBDIEC). 8-week old PHBDIEC mice were i.p. injected with the estrogen analog tamoxifen for four consecutive days to activate deletion of PHB from the intestinal epithelium. PHBfl/fl littermates were injected with vehicle. Tamoxifen injections were repeated every 4 weeks. 12 weeks after initial tamoxifen injection mice were sacrificed and assessed for mucosal barrier function, mitochondrial function, and inflammation as measured by TNFa and IL-1b mRNA expression, Cox2 and p65 protein expression, histology, myeloperoxidase activity, and spleen weight. RESULTS: PHBDIEC mice gained less weight starting 6 weeks after initial tamoxifen injection which by 12 weeks was 20% below weight gained by PHBfl/fl littermates. PHBDIEC mice exhibited increased water content of stool, gut permeability as measured by FITC-dextran translocation, spleen weight, colonic myeloperoxidase activity, colonic TNFa and IL-1b mRNA expression, and Cox2 and p65 protein expression. Transmission electron microscopy revealed that mitochondria in intestinal epithelial cells of PHBDIEC mice exhibited structural abnormalities including electron dense spherical inclusion bodies, disorganization and dissolution of cristae, and dilation/swelling. Colonic expression of mitochondrial unfolded-protein response proteins ClpP, Hsp60 and PKR were increased in PHBDIEC mice. Histological examination revealed that the epithelium of PHBDIEC mice exhibited areas of high-grade dysplasia and lymphoid polyps. CONCLUSION(S): Intestinal epithelial cell-specific PHB deficiency causes mitochondrial dysfunction and intestinal inflammation. PHB is crucial in maintaining normal mucosal barrier function and epithelial cell homeostasis. Loss of PHB expression in intestinal inflammation may be an early event rather than a consequence of disease development.