Neutrophils contain bacteria inside the phagolysosome for more than 2 days. (A) Coculture of neutrophils and SA-GFP in fibrin gels. Increasing concentrations of neutrophils from healthy controls were incubated with a fixed amount of bacteria (5 × 106 CFUs per mL) (representative data; n = 20). Symbols represent the mean of duplicates. Bacterial proliferation is expressed in GFP-relative fluorescence units (RFU). (B) TUO of SA-GFP (5 × 106 CFUs per mL) cocultured in fibrin gels with 3 × 106 neutrophils per mL (n = 15), 5 × 106 neutrophils per mL (n = 22), and 10 × 106 neutrophils per mL (n = 17). (C) Propidium iodide was added to cocultures of SA-GFP (5 × 106 CFUs per mL) and 10 × 106 neutrophils per mL in fibrin gel to determine viability and containment capacity of neutrophils simultaneously. Lines represent median fluorescence intensity (n = 4). (D) Effect of addition of the phagocytosis inhibitor cytochalasin D (20 μM end concentration) on TUO during coculture of 10 × 106 polymorphonuclear (PMN) cells per mL and 5 × 106 CFUs per mL SA-GFP in fibrin gel at different (early) time points (n = 4). (E) Effect of neutrophil lysis by Triton X-100 (0.5% end concentration) on TUO during coculture of 10 × 106 PMN cells per mL and 5 × 106 CFUs per mL SA-GFP in fibrin gel at different (late) time points (n = 4). (F) Correlation between TUO and amount of bacteria present in fibrin gels. Various amounts of SA-GFP were added to fibrin gels, and TUO was determined (representative experiment; n = 5). (G) TUO after lysis of 10 × 106 PMN cells per mL at the start vs lysis at 16 hours when cocultured with 5 × 106 CFUs per mL SA-GFP in fibrin gel (n = 9). Data in panels A-B are mean + SEM. Data in panels E-G are mean ± SEM. *P < .05; **P < .01; ***P < .001 as determined by 1-way analysis of variance (ANOVA) or repeated measures (RM) 1-way ANOVA with the Greenhouse-Geisser correction followed by Tukey’s multiple comparison test or Student t test for paired samples. ns, not significant.
