Hypoxia-Inducible Factor-1 α in Macrophages, but Not in Neutrophils, Is Important for Host Defense during Klebsiella pneumoniae-Induced Pneumosepsis

Abstract

Hypoxia-inducible factor- (HIF-) 1α has been implicated in the ability of cells to adapt to alterations in oxygen levels. Bacterial stimuli can induce HIF1α in immune cells, including those of myeloid origin. We here determined the role of myeloid cell HIF1α in the host response during pneumonia and sepsis caused by the common human pathogen Klebsiella pneumoniae. To this end, we generated mice deficient for HIF1α in myeloid cells (LysM-cre × Hif1α ^fl/fl) or neutrophils (Mrp8-cre × Hif1α ^fl/fl) and infected these with Klebsiella pneumoniae via the airways. Myeloid, but not neutrophil, HIF1α-deficient mice had increased bacterial loads in the lungs and distant organs after infection as compared to control mice, pointing at a role for HIF1α in macrophages. Myeloid HIF1α-deficient mice did not show increased bacterial growth after intravenous infection, suggesting that their phenotype during pneumonia was mediated by lung macrophages. Alveolar and lung interstitial macrophages from LysM-cre × Hif1α ^fl/fl mice produced lower amounts of the immune enhancing cytokine tumor necrosis factor upon stimulation with Klebsiella, while their capacity to phagocytose or to produce reactive oxygen species was unaltered. Alveolar macrophages did not upregulate glycolysis in response to lipopolysaccharide, irrespective of HIF1α presence. These data suggest a role for HIF1α expressed in lung macrophages in protective innate immunity during pneumonia caused by a common bacterial pathogen.

Figure 1

HIF1α is important for glucose metabolism and TNF production of alveolar macrophages. HIF1α protein expression in AMs derived via BAL from naïve LysM-cre × Hif1α^fl/fl mice (cKO) and littermate controls (WT) treated with IOX2 for 24 hours (a). Lactate (b) and cytokine (TNF and IL-6) production (c) by AMs stimulated in vitro with LPS or left untreated for 24 hours. Data are shown as bar graphs showing mean with standard error of the mean from 6 technical replicates of pooled AMs from 7 mice per group. Dotted line indicates the reliable lower limit of detection of the cytokine assays. Lactate production by LysM-cre × Hif1α^fl/fl AMs was compared to that of control AMs (Hif1α^fl/fl) of mice using t-tests. TNF and IL-6 production of LPS-stimulated cKO AMs and control AMs was compared using the Mann-Whitney test. ^∗P < 0.05; ^∗∗P < 0.01.

Figure 2

Macrophage HIF1α is important for host defense against K. pneumoniae in the lung. Bacterial loads (CFU/ml) in the lungs of LysM-cre × Hif1α^fl/fl mice and littermate controls 12 and 40 hours after intranasal inoculation with ~10⁴ CFU K. pneumoniae (a) or in distant organs 40 hours after inoculation (b). Data are shown as box-and-whisker diagrams of 13-16 mice per group from two independent experiments for each time point. Bacterial loads (CFU/ml) in the lungs of Mrp8-cre × Hif1α^fl/fl mice and littermate controls 12 and 40 hours after intranasal inoculation with ~10⁴ CFU K. pneumoniae (c) or in distant organs 40 hours after inoculation (d). Data are shown as box-and-whisker diagrams of 7-8 mice per group at each time point. Bacterial loads of the LysM-cre × Hif1α^fl/fl mice were compared to those of littermate control (Hif1α^fl/fl) mice using the Mann-Whitney test. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 3

Pathology of the lungs after K. pneumoniae infection. Cytokine levels (TNF, IL-1β, IL-6, and IL-10) in the lungs of 12-16 mice per group 12 and 40 hours after infection with K. pneumoniae (a). The extent of inflammation in the lungs of LysM-cre × Hif1α^fl/fl mice and littermate controls scored on haematoxylin and eosin- (H&E-) stained tissue sections as total pathology score of the infected lungs from 7-8 mice per group at 12 and 40 hours after inoculation (b). Data are shown as box-and-whisker diagrams, and the LysM-cre × Hif1α^fl/fl mice were compared to littermate control (Hif1α^fl/fl) mice using the Mann-Whitney test. Representative photographs of H&E-stained tissue sections of the infected lungs from LysM-cre × Hif1α^fl/fl mice and littermate controls at 12 (c) and 40 hours (d).

Figure 4

HIF1α deficiency affects TNF production capacity of AMs and IMs. Percentage TNF-positive cells and the mean fluorescent intensity (MFI) of the TNF staining of AMs (a) and IMs (b) from LysM-cre × Hif1α^fl/fl (Cre+) and control mice (WT) after stimulation of lung suspensions for 2.5 hours with heat-killed K. pneumoniae (K. pneu) or medium control (MED). Data are shown as bar graphs showing mean with standard error of the mean from 10 mice per group. MFIs and percentage positive cells were compared using Student's t-tests followed by the Holm-Sidak multiple comparison test. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 5

HIF1α deficiency affects glucose metabolism but not mitochondrial mass of AMs and IMs. Glucose uptake after 2.5 hours of stimulation with heat-killed K. pneumoniae (K. pneu) or medium control (MED), as measured by the MFI of 2NBDG, of AMs and IMs within lung suspensions of LysM-cre × Hif1α^fl/fl mice and littermate controls (a). Mitochondrial mass as measured by the MFI of the MitoTracker Green probe in AMs and IMs of LysM-cre × Hif1α^fl/fl mice and littermate control after 3 hours of incubation in medium (b). As a negative control, lung suspensions were kept at 4°C. Data are shown as bar graphs showing mean with standard error of the mean of 10 mice per group for the glucose uptake assay and 6 mice per group for the mitochondrial mass. Groups were compared using Student's t-tests with the Holm-Sidak multiple comparison test where appropriate. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.