MicroRNA-155 potentiates the inflammatory response in hypothermia by suppressing IL-10 production

Abstract

Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS-induced production of TNF and IL-6, while blunting IL-10 production. This dysregulation was associated with increased expression of microRNA-155 (miR-155), which potentiates Toll-like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR-155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold-exposed monocytes. In contrast, miR-155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL-10 production. In a murine model of LPS-induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR-155, suppression of Ship1 and Socs1, and alterations in TNF and IL-10. Importantly, miR-155-deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL-10. These results establish a causal role of miR-155 in the dysregulation of the inflammatory response to hypothermia.

Keywords: cold exposure; peritonitis; sepsis.

Figure 1.

Cold exposure alters the balance of proinflammatory and anti-inflammatory cytokines in primary human monocytes stimulated with LPS. Levels of cytokines in supernatants of isolated primary human monocytes incubated at 32 or 37°C and stimulated with LPS (100 ng/ml); n = 7 healthy human donors consisting of 4 independent experiments. A) TNF. B) IL-6. C) IL-12. D) IL-10. Wilcoxon ranked-sign test was used to compare 32 to 37°C. *P < 0.05.

Figure 2.

Role of miR-155 on the inflammatory response in cold-exposed primary human monocytes. A–C) SHIP1 (A), SOCS1 (B), and miR-155 (C) expression in monocytes incubated at 32 and 37°C and stimulated with LPS; n = 7 healthy human donors consisting of 4 independent experiments. Wilcoxon ranked-sign test was used to compare 32 to 37°C. *P < 0.05. D, E) Expression of miR-155 targets, SHIP1 (D) and SOCS1 (E), in monocytes transfected with scramble RNA [negative control (NC)] or miR-155 mimics or antagomirs at 32 and 37°C as indicated; n = 7 healthy human donors consisting of 4 independent experiments. *P < 0.05 comparing negative control with miR-155 mimics or antagomirs. **P < 0.05 comparing 32°C vs. 37°C negative controls. F) Effect of miR-155 overexpression at 37°C on TNF, IL-6, IL-12, and IL-10 protein levels. G) Effect of miR-155 knockdown at 32°C on TNF, IL-6, IL-12, and IL-10 protein levels; n = 6 healthy human donors consisting of 3 independent experiments. Wilcoxon ranked-sign test was used to compare miR-155 mimic/antagomir-treated samples with negative control samples. *P < 0.05.

Figure 3.

Role of SHIP1 and SOCS1 in cytokine production in cold-exposed monocytes. A–D) Production of cytokines in monocytes transfected with scramble RNA (NC) or SHIP1 siRNA and stimulated with LPS and kept at 37°C; n = 7 healthy human donors consisting of 4 independent experiments. Wilcoxon ranked-sign test was used to compare SHIP1 siRNA-treated samples with negative control samples. *P < 0.05. E–H) Production of cytokines in monocytes transfected without or with SOCS1 siRNA and stimulated as described above; n = 7 healthy human donors consisting of 4 independent experiments. Wilcoxon ranked-sign test was used to compare SOCS1 siRNA-treated samples with negative control samples. *P < 0.05.

Figure 4.

Role of IL-10 in the dysregulation of inflammatory cytokine production in cold-exposed monocytes. A, B) Effect of IL-10 receptor blockade on TNF (A) and miR-155 (B) expression in monocytes stimulated with LPS and maintained at 37°C. C, D) Effect of addition of rhIL-10 on TNF (C) and miR-155 (D) in monocytes stimulated with LPS and maintained at 32°C. Data are mean of 6 different donors consisting of 3 independent experiments. Wilcoxon ranked-sign test was used to compare between treatment groups. *P < 0.05.

Figure 5.

Hypothermia potentiates the inflammatory response in mice undergoing peritonitis. A) Experimental design. B) Body temperatures of cold-exposed mice and mice kept at room temperature for 48 h after treatment with LPS (4 mg/kg i.p.); n = 11–34/measurement and group; results comprise data from 2–3 independent experiments depending on time point. C–E) Cytokine expression in cold-exposed mice and mice kept at room temperature at 6, 12, and 48 h after LPS administration. Top panels show changes in mRNA-expression in PECs, and the bottom panels show peritoneal exudate cytokine concentrations for TNF (C), IL-6 (D), and IL-10 (E). F–H) Expression of miR-155 (F), Ship1 (G), and Socs1 (H) in PECs of cold-exposed mice and mice kept at room temperature at 6, 12, and 48 h after LPS treatment. 6 h: n = 3 mice/group, 12 h: n = 9 mice/group, 48 h: n = 11 mice/group; each time point represents 2–3 independent experiments. Student's t test was used to compare cold-exposed mice with mice kept at room temperature at each time point.

Figure 6.

Effect of hypothermia on peritoneal macrophage subsets and cytokine expression. A, B) Representative flow scatter plots of mice kept at room temperature or cold-exposed mice 48 h after LPS (4 mg/kg i.p.). C) Changes in F4/80⁺ GR1⁻ peritoneal macrophages in LPS-treated cold-exposed mice and mice kept at room temperature. D–L) Gating for F4/80^hiCD11b^hi macrophages and F4/80^loCD11b^lo macrophages in unstimulated as well as LPS-stimulated cold-exposed mice and mice kept at room temperature based on F4/80 and CD11b expression (32). Changes in F4/80^hiCD11b^hi (M) and F4/80^loCD11b^lo (N) peritoneal macrophage populations over 72 h after LPS treatment in cold-exposed mice and mice kept at room temperature. 6 h: n = 3 mice/group, 12 h: n = 9 mice/group, 48 h: n = 11 mice/group, 72 h: n = 10 mice/group. O) Expression of Tnf, Il6, Il12, Il10, and miR-155 in sorted F4/80^hiCD11b^hi peritoneal macrophages of cold-exposed mice, mice kept at room temperature after 48 h, and unstimulated naive mice; n = 3 in unstimulated mice, n = 8 in cold-exposed mice, and n = 7 in mice kept at room temperature. Student's t test was used to compare expression changes in F4/80^hiCD11b^hi macrophages in cold-exposed mice with mice kept at room temperature at the indicated time point. *P < 0.05.

Figure 7.

Hypothermia decreases survival in mice undergoing peritonitis. A) Body temperatures of cold-exposed mice and mice kept at room temperature for 72 h after treatment with LPS (20 mg/kg ip). B) Survival curve of LPS-treated cold-exposed mice and mice kept at room temperature; n = 10 for cold-exposed mice and n = 16 for mice kept at room temperature from 2 independent experiments. The log-rank test was used for the analysis of the survival data. *P < 0.05.

Figure 8.

Effects of miR-155 deficiency on body temperature, the inflammatory response and survival in cold-exposed mice. A) Experimental design: WT and miR-155-KO mice were exposed to hypothermia, both groups received LPS. B) Body temperatures of cold-exposed WT and miR-155-KO mice after treatment with LPS (4 mg/kg i.p.); n = 10/measurement and group, data are from 2 independent experiments. C) IL-10 expression in cold-exposed WT and miR-155-KO mice 48 h after LPS treatment. The left panel shows changes in mRNA-expression, the right panel shows peritoneal cytokine concentrations. D–F) Expression of miR-155 (D), Ship1 (E), and Socs1 (F) in cold-exposed WT and miR-155-KO mice 48h after LPS treatment; n = 10 mice per group, data are from 2 independent experiments. G) Body temperatures of cold-exposed WT and miR-155 KO mice after treatment with LPS (20 mg/kg i.p.). H) Survival curve of LPS-treated/cold-exposed WT and miR-155-KO mice; n = 9 for WT mice and n = 10 for miR-155-KO mice; data are from 2 independent experiments. Student's t test was used to compare cold-exposed mice with mice kept at room temperature at each time point, the log-rank test was used for the analysis of the survival data. *P < 0.05.