Pharmacologic and genetic inhibition of mTOR and HIF-1α signaling inhibit NET-mediated extracellular bacterial killing. (A) Total, phagocytotic, and extracellular NET-mediated bacterial killings by LPS-stimulated human PMNs (100 ng/mL) for 1 hour were determined with or without pretreatment with rapamycin (200nM; gray bars) or FK-506 (200nM; black bars). The bars indicate mean percentage of bacterial killing ± SEM in 10 separate experiments. *Statistically significant difference (P < .05) between the rapamycin-pretreated PMNs and both vehicle and FK-506 control preincubated PMNs in the both the Total and Extracellular NET Killing groups. We used the Student t test and Mann-Whitney statistical tests. (B) Phagocytotic and extracellular NET-mediated bacterial killings by LPS-stimulated human PMNs (100 ng/mL) for 1 hour were determined with or without 2-ME2 (2μM; gray bars) pretreatment. The bars indicate mean percentage of bacterial killing ± SEM in 4 separate experiments. *Statistically significant difference (P < .05) among the 2-ME2 pretreated, LPS-stimulated PMNs and LPS-stimulated PMNs. We used the Student t test for statistical analysis. (C) We determined phagocytotic and NET-mediated extracellular bacterial killing in LPS-stimulated (100 ng/mL; 1 hour) HIF-1αKD and SC Control surrogate PMNs as described for panel A. The bars indicate mean percentage of bacterial killing ± SEM in 4 separate experiments. **P < .001. We used 1-way ANOVA with Bonferroni multiple comparisons posttesting.