Role of Hypoxia Inducible Factors in Alveolar Macrophage Physiology

Abstract

The rapid transit from hypoxia to normoxia in the lung that follows the first breath in newborn mice coincides with alveolar macrophage (AM) terminal differentiation. Indeed, in silico analysis showed that the expression of genes involved in glycolysis and adaptation to hypoxia is gradually downregulated during AM maturation after birth, suggesting an adaptation to increased oxygen concentrations. However, whether sensing of oxygen fluctuations contributes to post-birth AM maturation and function has not been previously explored. For this purpose, we generated mice whose AMs show a deficient ability to sense oxygen after birth by deleting von Hippel-Lindau (Vhl) gene, which codifies for the master negative regulator of hypoxia inducible factors (HIF), under the control of the CD11c promoter (CD11c∆Vhl mice). VHL-deficient AMs were more glycolytic and showed a decreased oxygen consumption, compared to control WT AMs in steady state. We found that VHL-deficient AMs showed an immature-like phenotype and an altered transcriptional identity. In addition, the absence of VHL impaired AM self-renewal capacity in vivo, and also upon growth factor stimulation ex vivo. Unlike AMs transplanted from control Vhlfl/fl mice, AMs from CD11c∆Vhl mice did not reverse pulmonary alveolar proteinosis when transplanted into Csf2rb-/- mice, which spontaneously develop lung proteinosis. This result correlated with the increased lipid accumulation found in AMs lacking VHL, which also showed a decreased lipid oxidation capacity. In order to demonstrate how all these AM features are regulated by HIF, we generated mice with a deletion in Hif1a, Hif2a or both in addition to the Vhl deletion, all under the control of the CD11c promoter. HIF-1 and HIF-2 depletion differentially affected AM phenotypic maturation. However, both isoforms similarly restrain AM self-renewal. HIF-1 was essential for the glycolytic shift in VHL-deficient AMs. Finally, we found that HIF-2 depletion rescued the ability of AMs to remove the surfactant excess in vivo, pointing to its specific role regulating lipid metabolism in AMs. Thus, these results highlight the relevance of HIF regulation for AM maturation and function and contribute to clarify the molecular requirements for the adaptation of AMs to its evolving niche during maturation.