Hypoxia Selectively Inhibits Respiratory Burst Activity and Killing of Staphylococcus aureus in Human Neutrophils

Abstract

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α–dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.

Hypoxic regulation of neutrophil function and consequences for Staphylococcus aureus infection

“…Studies of neutrophils isolated from human volunteers or mice exposed to hypoxia have shown an increase in ROS production [44,56,65], although, of note, all cell isolation and in vitro assays were conducted under normoxic conditions. Whilst HIF-1a manipulation did not affect ROS release [55,60], in vitro assays conducted under hypoxia have consistently shown decreased intracellular and extracellular superoxide anion production, restored by re-oxygenation [54] and further increased under hyperbaric oxygen conditions. Moreover, intermittent hypoxia and its clinical correlate obstructive sleep apnoea appear to prime neutrophils for augmented superoxide anion generation [68].…”

“…Extravasated neutrophils undergo shape change to a polarised morphology, which is essential for directional movement. McGovern et al showed no change in IL-8-induced shape change, or chemotaxis towards IL-8, bacterial formylated peptide (fMLF) or LPS in human neutrophils cultured under hypoxia [54], and, likewise, Peyssonnaux et al found no difference in chemotaxis towards fMLF through an endothelial monolayer between wildtype, HIF-1a null and vHL-null murine neutrophils [55]. In contrast, Wang and Liu showed enhanced chemotaxis of neutrophils isolated from hypoxic subjects [56], whereas Rotstein et al showed reduced chemotaxis of human neutrophils towards fMLF and zymosan-activated serum under hypoxia in an agarose gel migration assay [57].…”

“…Further evidence that hypoxia-induced signalling pathways can increase phagocytosis is provided by neutrophils from patients with heterozygous mutations in vHL protein, which display enhanced phagocytosis of Streptococcus pneumoniae under normoxia, further augmented by hypoxia [42]. The same group found that neutrophils from healthy volunteers incubated under hypoxia had enhanced CD11b expression but that phagocytosis of S. pneumoniae was not increased [54]. However, the majority of studies suggest that neutrophil phagocytosis is increased by hypoxia.…”

“…Together these data suggest that ROS production depends on oxygen availability, and that the decrease seen under hypoxic conditions is likely due to a lack of molecular oxygen, which is not recapitulated by modulation of HIF-1a signalling. Microbicidal activity of ROS is highly context-dependent and varies with bacterial species; ROS production contributes significantly to S. aureus killing whereas E. coli killing is predominantly oxidase-independent [54,69]. In addition to ROS, neutrophils produce antimicrobial reactive nitrogen species, a process which does appear to be under HIF-1a control.…”

“…A number of in vitro studies have suggested that impaired ROS generation in hypoxia accounts for decreased bactericidal activity of neutrophils against S. aureus; the degree of phagocytosis has not been found to be diminished [54,86]. McGovern et al showed that hypoxia markedly reduced both ROS generation and S. aureus killing, and, interestingly, observed an intracellular bacterial survival advantage when S. aureus was incubated with neutrophils under hypoxia [54].…”