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BACKGROUND: Ongoing work from our group has demonstrated that neutrophil (PMN) recruitment during colitis induces mucosal hypoxia. Subsequently we demonstrated that the NADPH oxidase burst was primarily responsible for eliciting tissue hypoxia. Chronic granulomatous disease (CGD) patients exhibit mutations in neutrophil NADPH oxidase, resulting in an impaired ability to form reactive oxygen species, required for killing phagocytosed pathogens. Utilizing a murine CGD model we demonstrated an aggravated non-resolving inflammatory response to colitis coupled with diminished tissue hypoxia. CGD neutrophils fail to undergo apoptosis or externalize phosphatidylserine, resulting in diminished efferocytosis by macrophages and ultimately impaired resolution of inflammation. The principal aims of this project were to ascertain if and how pharmacological stabilization of HIF could overcome the resolution-deficit of NADPH oxidase-deficient mice. METHODS: Wild type (C57/B6) and mice deficient in the NAPDH oxidase subunit (gp91phox-/- or CGDx) were subjected to TNBS chemically-induced colitis. CGDx PMN were harvested from bone marrow, fluorescently labeled with cell tracker dye and adoptively transferred by tail vein injection into wild type mice. Efferocytosed PMN were identified by flow cytometric analysis gating on cell tracker dye positive macrophages (MHCii+, CX3CR1+, Ly6c+). RESULTS: Compared with wild type, CGDx mice failed to induce mucosal hypoxia and developed a more severe and non-resolving colonic inflammation. Histological examination revealed extensive mucosal damage coupled with augmented infiltration of neutrophils and monocytes. Fluorescent in situ hybridization revealed mucosal infiltration of bacteria and 16S rDNA qPCR revealed dissemination of bacteria to mesenteric lymph nodes. To overcome the HIF deficit in CGDx mice, we treated with a PHD-inhibitor and observed a restitution of inflammation resolution. Fewer bacteria were observed in both the mucosa and in the lymph nodes of PHD-inhibitor treated CGDx mice. Moreover, histological and injury scores were improved. In order to ascertain how HIF stabilization elicits resolution, CGDx neutrophils were fluorescently labeled and adoptively transferred into wild type mice. TNBS colitis was induced in the recipient mice in the presence and absence of PHD-inhibitor. Flow cytometric analysis, gating on macrophages, revealed that HIF stabilization resulted in enhanced efferocytosis of CGD neutrophils. Analysis of PMN apoptosis in vitro by both flow cytometric Annexin-V/propidium iodide staining and Caspase 3/7 cleavage revealed augmented PMN apoptosis in the presence of PHD-inhibition. Finally, blocking NADPH oxidase reduced apoptosis, an effect abrogated by pharmacological HIF stabilization. CONCLUSIONS: Pharmacological PHD-inhibition resulted in enhanced efferocytosis of CGDx PMN by macrophages and increased PMN apoptosis independent of NADPH oxidase activity.

P-228 Pharmacological HIF Stabilization Overcomes the Resolution-deficit of NADPH Oxidase-deficient Mice in TNBS Colitis