Reduced neutrophilic inflammation and lung injury during inflammation resolution in mice with myeloid cell deficiency of HIF-2α (A) Expression profile of Hif2a in murine peripheral blood and bone marrow neutrophils. Neutrophils isolated by magnetic negative selection from peripheral blood (PB) or bone marrow (BM) or bone marrow mononuclear cells (MCs) were cultured in normoxia (Nx) or hypoxia (Hx) for 5 hours or lysed when freshly isolated (T0). cDNA was amplified using custom Hif2a primers, and a representative gel image of n = 3 is shown. (B-C) Murine peripheral blood neutrophils express (B) HIF-1α and (C) HIF-2α. Representative western blots of lysates from neutrophils cultured in normoxia (Nx) or hypoxia (Hx) or stimulated in normoxia with LPS (10 ng/mL) or DMOG (100 µM) for 5 hours. (D-M) Hif2a^flox/flox;LysMCre^−/− controls (WT) and littermate Hif2a^flox/flox;LysMCre^+/− (KO) mice were challenged with nebulized LPS (3 mg). BAL was performed at 24, 48, 72, and 120 hours. (D-G) Total cell counts were determined by hemocytometer and (H-K) neutrophil counts by cytospin analysis. (L-M) IgM levels were determined in BAL fluid by enzyme-linked immunosorbent assay. Data are mean and SEM for controls (open bars) and HIF-2α–deficient mice (filled bars), n = 5. (N-Q) Wild-type (WT) C57BL/6 mice (C57) were irradiated (12 fractions of 1Gy), injected with bone marrow from Hif2a^flox/flox;LysMCre^−/− (WT) or Hif2a^flox/flox;LysMCre^+/− (KO) mice, and allowed to reconstitute for 5 weeks before challenge with nebulized LPS. Bronchoalveolar lavage was performed at 6 and 48 hours. (N-O) Total cell counts determined by hemocytometer and (P-Q) neutrophil counts determined by cytospin. Data are mean and SEM for WT→C57 (open bars) and KO→C57 mice (filled bars), minute n = 5.