Date of download: 6/28/2016 Copyright © 2016 American Society of Anesthesiologists. All rights reserved. Increased interleukin (IL)-10 release induced by the activation of the heat-shock response is associated with a decreased intracellular killing by alveolar macrophages, decreased bacterial phagocytosis by neutrophils, and increased mortality in a mouse model of Pseudomonas aeruginosa pneumonia. (A) MH-S cells, a mouse alveolar macrophage cell line (1 × 10 6 cells), were left untreated or heat shocked with heat for 30 min at 43°C, then exposed 1 h later to P. aeruginosa PAK (2 × 10 7 colony-forming units [CFU]/ml; multiplicity of infection [MOI] = 10:1) or its vehicle for 4 h. Enzyme-linked immunosorbent assay of IL-10 was carried out according to the manufacturer’s protocol. Results are shown as mean ± SD (n = 5 experiments repeated in triplicate); *P ≤ 0.05 from control cells; **P ≤ 0.05 from cells treated with PAK alone. (B) MH-S cells were heat shocked at 43°C for 30 min or kept at 37°C, then recovered at 37°C for 1 h before adding 10 7 CFU/ml of P. aeruginosa PAK for 45 min at 37°C. Gentamicin (150 μg/ml) was added and cells were incubated for 1 or 3 h at 37°C. After cell lysis, percent of intracellular bacterial killing was calculated, as described in the Materials and Methods section. In some experiments, MH-S cells were pretreated with a mouse IL-10-blocking antibody or its isotype control antibody 1 h before undergoing the heat-shock response and subsequent exposure to P. aeruginosa bacteria. Results are shown as mean ± SD (n = 5 experiments repeated in triplicate); *P ≤ 0.05 from non–heat-shocked cells; **P ≤ 0.05 from heat-shocked cells pretreated with an isotype control antibody. (C) Primary mouse neutrophils were isolated, exposed to increasing concentrations of mouse IL-10 (0.1–10 ng/ml), then exposed 60 min later to P. aeruginosa expressing green fluorescent protein (PAK-GFP) for 30 min. After cell fixation, permeabilization, and staining with phalloidin, the number of intracellular PAK-GFP was counted, as described in the Materials and Methods section. Results are shown as mean ± SD (n = 3 experiments repeated in triplicate); *P ≤ 0.05 from neutrophils exposed to IL-10 vehicle. (D) Primary mouse neutrophils were isolated as described in the Materials and Method section. Cells were heat shocked at 43°C for 30 min or kept at 37°C, then exposed 60 min later to P. aeruginosa PAK-GFP for 30 min. After cell fixation, permeabilization, and staining with phalloidin, the number of intracellular PAK- GFP was counted, as described in the Materials and Methods section. Results are shown as mean ± SD (n = 3 experiments repeated in triplicate); *P ≤ 0.05 from non heat-shocked cells; **P ≤ 0.05 from heat-shocked cells pretreated with an isotype control antibody. (E and F) C57BL/6 wild-type (WT) or IL-10-null mice underwent a heat-shock response with whole-body hyperthermia that was followed 24 h later by airspace instillation of P. aeruginosa (PAK strain, 1 × 10 7 CFU) or its vehicle, as described in the Materials and Method section. Number of live bacteria in the lung homogenates and mortality at 24 h are reported. Results are shown as mean ± SD (n = 6 mice per group for lung bacterial clearance and n = 8 mice per group for mortality); *P ≤ 0.05 from non–heat- shocked mice, **P ≤ 0.05 from wild-type mice. Figure Legend: From: Heat-shock Response Increases Lung Injury Caused by Pseudomonas aeruginosa via an Interleukin-10-dependent Mechanism in Mice Anesthesiology. 2014;120(6): doi: /ALN From: Heat-shock Response Increases Lung Injury Caused by Pseudomonas aeruginosa via an Interleukin-10-dependent Mechanism in Mice Anesthesiology. 2014;120(6): doi: /ALN