Fig. 1: mTOR inhibition potentiates the inflammatory phenotype of primary uninfected human monocytes following TLR4 stimulation.

a Freshly isolated human monocytes were pretreated as indicated with rapamycin (100 nM) or one of two structurally distinct mTORi (AZD2014, mTORi #1; INK128, mTORi #2; each 5 μM) or DMSO for 6 h prior to stimulation with LPS (1 μg, 30 m). Representative western blot series from one independent donor of three quantified, with adjusted density representing fold change of phosphorylated target staining, normalized to GAPDH staining, relative to the stimulated control (LPS). b Monocytes from four donors were pretreated as in a, stimulated with LPS (1 μg, 24 h), and supernatant cytokine content was quantified on the MSD platform. c–e PBMCs from three donors were pretreated with AZD2014 for 6 h and stimulated with LPS (1 ng, 18 h) prior to staining. Representative flow plot (c) and aggregates (d and e) represent gating on leukocyte/singlet/live/CD14⁺. Boolean gating (e) facilitated identification of subpopulations producing different combinations of cytokines. e Illustrates that some monocytes produced no cytokines at all (turquoise), whether unstimulated, only stimulated with LPS, or stimulated with LPS after mTORi pretreatment. In contrast, the color coding described in the rightmost panel in e indicates that others produced one cytokine or different combinations of two or three cytokines, with “polyfunctionality” being defined as production of 2 or 3 cytokines in different combinations. Enahnced polyfunctionality was seen among the mTORi pretreated, LPS-stimulated monocytes. Statistical analyses: for the blot quantification (a), significance was determined using an ANOVA with Dunnett’s multiple comparisons, and asterisks (*) indicate a comparison to the stimulated control. For b, significance was determined via one-way ANOVA Tukey’s multiple comparisons, except in cases where Friedman tests and Dunn’s multiple comparisons were appropriate. For d, asterisks (*) indicate comparisons between US and LPS/mTORi (AZD2014) and hashes (#) indicate comparisons between LPS and LPS/mTORi (AZD2014), and significance was computed via two-way ANOVA and Tukey’s multiple comparisons. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001. Error bars represent mean ± SD. For e, cytokine profiles were compared using Chi-square tests.