Title: O-024 Hypoxia-Inducible Factors are Essential for the Recruitment of Granulocytic Cells to Intestinal Inflammatory Foci and in Colon Tumors
Abstract: Inflammatory foci and tumors recruit a vast array of immune cells that are essential in the resolution or potentiation of the disease. Epithelial cells are critical in this recruitment, however the regulatory mechanisms in epithelial cells that control recruitment are not clear. Hypoxia can result from several pathogenic conditions, such as ischemia, infection and cancer. Regulation of hypoxia-mediated genes is transcriptionally dependent on hypoxia-inducible factor (HIF)-1a and HIF-2a. Our recent data reveals that in mice HIF-2a but not HIF-1a in the colonic epithelium is critical to initiate an inflammatory response and chronic activation of HIF-2a leads to colon cancer. Here we hypothesize that epithelial HIF-2a controls the recruitment of immune cells. Apcmin/+ mice with intestinal epithelium-specific activation of HIF-2a (Hif-2aLSL/LSL/Apcmin/+) or intestinal epithelium-specific deletion of HIF-2a (Hif-2aΔIE/Apcmin/+) were generated. Colon tissues from these mice were assessed by histologic analyses, immunohistochemistry, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Enhanced colon carcinogenesis with increased tumor cell proliferation were observed in Hif-2aLSL/LSL/Apcmin/+ mice, whereas decreased colitis-associated colon cancer with increased cell apoptosis were noticed in Hif-2aΔIE/Apcmin/+ mice. Interestingly, the expression of myeloid cell marker CD11b and granulocytic cell marker Ly6G were decreased by disruption of HIF-2a in colon tumors. Activation of HIFs by intestinal-specific deletion of the von Hippel-Lindau tumor suppressor protein (VHL) (VhlΔIE) increased CD11b+Ly6G+ cells in colon tumors. In addition to colon tumors, the expression of CD11b and Ly6G are increased by deletion of VHL and decreased by disruption by HIF-2a in mouse models of colitis, further demonstrating the importance of hypoxia signaling in the recruitment of these cells. Further analysis found that CD11b+Ly6G+ cells in blood and colonic lamina propria from VhlΔIE mice are increased by DSS-induced colitis. Depletion of CD11b+Ly6G+ cells by transplanting bone marrow from Lys-Cre; MclF/F mice that are lacking CD11b+Ly6G+ cells are protected from DSS-induced colitis. To assess if hypoxia led to a recruitment of granulocytic cells, primary neutrophils were isolated and migration studies were assessed using colons from VhlΔIE and Hif-2aLSL/LSL mice. This data demonstrated that HIF activation in the colon dramatically recruited neutrophils in vitro. CD11b+Ly6G+ cells are recruited by chemokines that are increased in human inflammatory bowel diseases (IBD) and colon tumors. Microarray analysis showed that the expression of a chemokine Cxcl1 in colitis and colon tumor was dependent on HIF-2a, which was confirmed by qPCR and ELISA. Promoter reporter analysis showed that Cxcl1 was directly regulated by HIF-2a. Our findings reveal a novel hypoxia mediated epithelial-immune cells interaction that is critical for inflammatory response and colon tumorigenesis.