O-032 Neutrophil-epithelial Interactions Elicit a Protective Hypoxic Microenvironment during Colitis

Abstract

BACKGROUND: Mucosal infiltration of neutrophils (PMN) and formation of crypt abscesses is a pathological hallmark of ulcerative colitis. We hypothesized that activated neutrophils influence the transcriptional profile of adjacent intestinal epithelia. METHOD(S): Wild type (C57/B6), HIF reporter mice (ODD-Luciferase) and mice deficient in the NAPDH oxidase subunit (phox-/- or CGDx) were subjected to TNBS chemically-induced colitis. PMN were depleted by intraperitoneal Gr-1 antibody injection. Pharmacological HIF stabilization was achieved via daily subcutaneous injection of AKB-4924. Disease progression was monitored via weight loss. RESULT(S): Microarray profiling of transcriptional changes induced in epithelia following exposure to activated PMN, revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize epithelial hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the contribution of PMNs and the oxidative burst to "inflammatory hypoxia". Phox-/- mice (CGDx), mirror human chronic granulomatous disease patients and lack functional NADPH oxidase. Similar to patients, CGDx mice developed exacerbated colitis with enhanced PMN infiltration and diminished inflammatory hypoxia. Pharmacological intervention with a HIF-stabilizing compound (AKB-4924) resulted in restoration of mucosal HIF-signaling concomitant with abrogation of colitis-severity in the CGDx mice. Moreover, we demonstrated that PMN infiltration elicited transcriptional changes within the epithelium synonymous with a resolution signature. This transcriptional profile was deficient in CGDx mice during colitis but augmented by AKB-4924 in both wild type and CGDx mice. The severe CGDx inflammatory phenotype appeared to be due to an accumulation of bacteria in the colonic crypts, visualized by fluorescent in situ hybridization. Potential bacterial dissemination was supported by 16S rDNA qPCR and serum LPS measurements. Treatment with AKB-4924 enhanced mucosal barrier function of both wild type and CGDx mice, determined following an oral gavage of FITC-dextran. Finally, we observed an increase in goblet cell number in both wild type and CGDx mice following treatment with AKB-4924. CONCLUSION(S): In conclusion, we propose that transcriptional imprinting of host tissue by infiltrating PMNs modulates host responses to inflammation. Additionally, the oxidative burst of PMN contributes fundamentally to localized O2 depletion, resultant mucosal hypoxia and effective inflammatory resolution. CGDx mice develop severe non-resolving colitis, coupled with a failure to initiate "inflammatory hypoxia." Finally, pharmacological HIF stabilization is capable of providing mucosal protection to CGDx mice, likely through enhanced goblet cell mucus production, leading to reduced bacterial dissemination.