FIG 3.

The PI3K/Akt/mTOR pathway mediates the IL-10 response to nasal S. aureus isolates. (A) PBMCs were stimulated with two S. aureus isolates at the indicated MOIs for 30 min for Western blot experiments or for 18 h in experiments looking at IL-10 accumulation by ELISA. (B) Flow cytometric analysis of Akt phosphorylation (S473) in CD14⁺ gated human PBMCs stimulated with two representative S. aureus isolates inducing low-level (S5) or high-level (S8) IL-10 responses. (C) PBMCs were pretreated with wortmannin (1 μM) and then stimulated with S. aureus (MOI = 5) for 18 h. Quantification of IL-10 and TNF-α accumulation in the supernatants was done by ELISA. (D) Western blot of PBMCs pretreated with the pan-PI3K-p110 inhibitor wortmannin (Wort) or p110 isoform inhibitors PIK-75 (PIK; p110α inhibitor; 100 nM), TGX-221 (TGX; p110β inhibitor; 500 nM), AS-604580 (AS; p110γ inhibitor; 10 μM), and IC-87114 (IC; p110δ inhibitor; 5 μM) for 1 h and then stimulated with S. aureus S8 (MOI = 5) for 30 min. (E) PBMCs were pretreated with rapamycin (10 nM) and then stimulated with S. aureus (MOI = 5) for 18 h. Quantification of IL-10 and TNF-α accumulation in the supernatants was performed by ELISA. Data in panels A, B, and D are representative of three independent experiments from three different donors. Data in panels C and E are plotted as means ± SEMs from eight individual donors in experiments performed in triplicate. Statistical analysis was performed using theStudent t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.