Supplementary Material for: Increased bacterial load per neutrophil reduces intracellular killing capacity

Introduction: Neutrophils are the most abundant innate immune cells in the peripheral blood and eliminate bacteria through phagocytosis and antimicrobial mechanisms. Early during infection, they often encounter high bacterial loads before full recruitment. Individual neutrophils can ingest many bacteria, but it remains unclear how high bacterial loads per neutrophil affect intracellular killing. Methods: Neutrophils were isolated from healthy donor blood by fluorescence-activated cell sorting (FACS). Intracellular bacterial load was quantified using imaging flow cytometry to measure spot counts and green fluorescent protein (GFP) intensity after exposure to GFP-expressing Staphylococcus aureus. A single-cell killing assay assessed intracellular killing across bacterial-load categories by sorting individual GFP+ neutrophils into 384-well plates and counting wells with outgrowth after 100 hours. Phagolysosomal acidification was measured using dual-labeled (pH-sensitive pHrodo and pH-insensitive PromoFluor 520 LSS NHS ester [PF520]) S. aureus bioparticles. Results: Bacterial uptake by neutrophils was highly heterogeneous in vivo and in vitro. GFP spot counts strongly correlated with GFP intensity (R² = 0.66), allowing stratification into GFP fluorescence intensity categories. In the single-cell killing assay, higher bacterial loads per neutrophil were associated with reduced intracellular killing (χ²(4) = 11.72, p = 0.0003). Higher bacterial loads per neutrophil corresponded with diminished phagolysosomal acidification capacity (χ²(4) = 24.00, p < 0.0001). Conclusion: Neutrophils ingesting higher bacterial loads exhibit reduced intracellular killing, likely due to decreased phagolysosomal acidification. These findings highlight how bacterial load per neutrophil shapes antimicrobial capacity and early infection control.