Hypoxia and Reactive Oxygen Species Modulate the Interaction Between Human Neutrophils and Staphylococcus aureus?

Rationale & Hypothesis: Neutrophils are crucial to host defense, and their impaired function leads to more susceptibility to infection with a range of pathogens, with Staphylococcus aureus (S. aureus) a common and sometimes life-threatening problem in this setting. Sites of infection are characterized by a low oxygen level caused by consumption of oxygen by both bacteria and infiltrating cells, contributing to profound tissue hypoxia, which may alter bactericidal propensity of neutrophils. Limited evidence suggests the resulting lack of oxygen to fuel the ‘oxidative burst’ may contribute to failure to control staphylococcal sepsis. We hypothesized that hypoxia modulates the host-pathogen interaction by influencing the generation of reactive oxygen species (ROS) in human neutrophils. Objective: To determine the impact of hypoxia/ROS generation on neutrophil function, staphylococcal growth and pathogen killing. Methodology: Neutrophils from healthy volunteers were subjected in parallel to normoxia (21%O2) and hypoxia (0.8%O2; Ruskinn hypoxic workstation). The kinetics of bacterial growth (S. aureus strains SH1000, Wood 46 and JE2) in bacterial (BHI) and cell culture (RPMI) media was measured by quantitative microbiology (Miles-Misra). Neutrophil-mediated killing of S. aureus was assessed by gentamicin protection assay. Diphenyleneiodonium chloride (DPI) was used to inhibit the neutrophil oxidative burst. Neutrophil-staphylococcal interactions were imaged by SpinningDisk microscopy with DCF (ROS) and pHrodo (pH) staining. Transmission Electron Microscopy was used to assess morphology of neutrophil phagosomes during staphylococcal infection. Findings: Diminished neutrophil apoptosis and up-regulation of hypoxia-regulated genes (BNIP3 and GLUT1) confirmed establishment of a hypoxic environment. Hypoxia limited the growth of S. aureus in nutrient rich BHI, but bacteria grew slowly in the RPMI medium, irrespective of oxygen tension. Treatment with DPI impaired neutrophil-mediated killing of S. aureus, profoundly altering phagosomal morphology and pH. Using bafilomycin on DPI- treated neutrophils resulted in restoration of pH of intracellular bacteria to a level observed in controls, however with no effect on phagosomal morphology or killing of S. aureus. Importantly, levels of hypoxia typical of sites of infection markedly reduced the ability of neutrophils to kill S. aureus and led to changes in phagosomal morphology which was confirmed by using TEM. Use of DMOG and cycloheximide showed that observed impairment of killing of S. aureus is HIF- independent. Conclusion: Hypoxia/inhibition of the oxidative burst profoundly alter the host-pathogen interaction with ingested pathogens persisting within abnormal phagosomes in viable phagocytes, which is pH independent. This may contribute to staphylococcal persistence and dissemination.