Neutrophil Intracellular Bactericidal Activity and Apoptosis Under Hypoxic Conditions

Neutrophils, major phagocytes, play a crucial role in infection control through their intracellular sterilization capabilities (36). However, under hypoxic conditions, the impaired bacterial clearance capacity of neutrophils can enable pathogens to exploit host immune cells, leading to intracellular infection (37). To investigate neutrophil intracellular bactericidal activities under various oxygen conditions (normoxia, hypoxia, and hypoxia + CaO2), we assessed the eradication of intracellular bacteria. Within 2 hours, up to 80% of intracellular bacteria were efficiently eliminated under normoxia and hypoxia + CaO2 conditions, whereas only a quarter were eradicated under hypoxia (Fig. 5C). Upon prolonging the exposure to 20 hours, TEM analysis revealed a significantly higher intracellular bacterial burden under hypoxia compared to normoxia and hypoxia + CaO2 (Figs. 5, D and E). The intracellular reactive oxygen species (ROS) levels in neutrophils (Fig. 5F), a crucial oxygen-dependent antibacterial component (14, 38), were significantly reduced under hypoxia. Interestingly, there was no significant difference in intracellular ROS levels between normoxia and hypoxia + CaO2, indicating that nano-CaO2 generated sufficient oxygen and subsequent ROS to sustain neutrophil intracellular bactericidal activities under hypoxia.

Apoptosis and nonphlogistic clearance of activated neutrophils by macrophages are crucial for maintaining local homeostasis and resolving acute inflammation (Fig. 5G) (39). To determine the impact of various factors on neutrophil apoptosis rates, we established a double-hit model by exposing lipopolysaccharide (LPS)-activated neutrophils to different oxygen supply conditions. Flow cytometry analysis demonstrated that hypoxia significantly reduced neutrophil apoptosis, particularly at the late stage characterized by nuclear condensation and PI+/Annexin V+ morphology (Figs. 5H and I). This decline in apoptosis was also observed in TEM images, with a lower percentage of apoptotic neutrophils under hypoxia compared to other groups (Fig. 5J). Additionally, hypoxia led to decreased caspase-3 activity, indicating inhibition of neutrophil apoptosis (Fig. 5K). However, the additional treatment with nano-CaO2 facilitated the apoptosis of neutrophils under hypoxia, similar to the pattern observed under normoxia (Figs. 5, H–K). Remarkably, we observed that intracellular infection antagonized neutrophil apoptosis, potentially prolonging the inflammatory phase (fig. S10).