During inflammation, human neutrophils engage beta 2-integrins to migrate from the blood circulation to inflammatory sites with high cytokine but low oxygen concentrations. We tested the hypothesis that the inhibition of prolyl hydroxylase domain-containing enzymes (PHDs), cytokines, and beta 2-integrins cooperates in HIF pathway activation in neutrophils. Using either the PHD inhibitor roxadustat (ROX) (pseudohypoxia) or normobaric hypoxia to stabilize HIF, we observed HIF1 alpha protein accumulation in adherent neutrophils. Several inflammatory mediators did not induce HIF1 alpha protein but provided additive or even synergistic signals (e.g., GM-CSF) under pseudohypoxic and hypoxic conditions. Importantly, and in contrast to adherent neutrophils, HIF1 alpha protein expression was not detected in strictly suspended neutrophils despite PHD enzyme inhibition and the presence of inflammatory mediators. Blocking beta 2-integrins in adherent and activating beta 2-integrins in suspension neutrophils established the indispensability of beta 2-integrins for increasing HIF1 alpha protein. Using GM-CSF as an example, increased HIF1 alpha mRNA transcription via JAK2-STAT3 was necessary but not sufficient for HIF1 alpha protein upregulation. Importantly, we found that beta 2-integrins led to HIF1 alpha mRNA translation through the phosphorylation of the essential translation initiation factors eIF4E and 4EBP1. Finally, pseudohypoxic and hypoxic conditions inducing HIF1 alpha consistently delayed apoptosis in adherent neutrophils on fibronectin under low serum concentrations. Pharmacological HIF1 alpha inhibition reversed delayed apoptosis, supporting the importance of this pathway for neutrophil survival under conditions mimicking extravascular sites. We describe a novel beta 2-integrin-controlled mechanism of HIF1 alpha stabilization in human neutrophils. Conceivably, this mechanism restricts HIF1 alpha activation in response to hypoxia and pharmacological PHD enzyme inhibitors to neutrophils migrating toward inflammatory sites.
