Figure 3.

Training for 9 weeks promotes whole blood antimicrobial responses. Blood was collected from control mice and mice trained 9 weeks before collection (n = 4-5 mice per group). (A, B) Blood was incubated for 6 h with 10 ng/ml LPS, 1 μg/ml PHA, 10 μM CpG and 10⁸ heat-killed L. monocytogenes and C. albicans. The concentration of IL-6 in blood supernatants was quantified by ELISA. Each dot of the violin plots represents data from one individual mouse, and the dashed horizontal lines the median. *P < 0.05 (A). The concentrations of G-CSF, IFNγ, IL-1α, IL-1β, IL-6, IL-10, IL-12p40, IL-18, TNF, CCL2, CCL3, CXCL2, CXCL5 and CXCL10 in blood supernatants were quantified by multiplex immunoassay (Luminex). Heatmap and hierarchical clustering of cytokine levels was performed using Z-score normalization (B). (C–E) Blood (C), PMNs (D) and monocytes (E) were incubated for 2 h with 6 x 10⁴, 6 x 10³ and 1.8 x 10³ CFU L. monocytogenes, respectively. Serial dilutions of blood or samples of lysed cells were plated on blood agar plates. After and incubation for 24 h, CFUs were enumerated (F, G) PMNs were culture 1 h with or without 10⁸ heat-killed L. monocytogenes. Myeloperoxidase (Mpo) relative mRNA levels were measured by RT-PCR and normalized to actin housekeeping gene expression (F). MPO concentrations in cell culture supernatants were quantified by ELISA (G). Each dot in the violin plots represents data from one individual mouse and the dashed horizontal lines the quartiles. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control (A, B).