Figure 5. IL-33 and STAT6 promote M2 differentiation after sepsis.

(a) Peritoneal cells were collected from C57BL/6 J or Rag1^−/− mice 15 days after CLP and antibiotic treatment and the frequency of F4/80⁺CD206⁺ macrophages determined by FACS (n≥3 mice per group). (b) Peritoneal cells of BALB/c, Il1rl1^−/− and Stat6^−/− mice were harvested in ‘a' above and the frequency of F4/80⁺CD206⁺ macrophages determined by FACS (n≥5 mice per group). (c) Representative FACS plots of IL-4Rα⁺F4/80⁺ peritoneal macrophages of C57BL/6 J mice at day 15 after CLP (n=5 per group). (d,e) Peritoneal macrophages harvested in b above were stimulated with IL-4 in vitro. (d) STAT6 phosphorylation in the cells was determined by FACS (n=4 per group). (e) Concentrations of CCL22 in the 18 h culture supernatants determined by ELISA (n=4 mice per group). (f) Peritoneal cells of BALB/c, Il1rl1^−/− and Stat6^−/− mice were harvested in a above. The number of viable bacteria recovered from lysates of peritoneal macrophages exposed, in vitro, to L. pneumophila for 72 h in the presence or absence of arginase inhibitor BEC. (g) BALB/c macrophages from BMDM were cultured for 2 days with M-CSF (M0) or IL-4, IL-13 and IL-33 (M2). The number of viable bacteria recovered from lysates of M2-polarized macrophages exposed, in vitro, to L. pneumophila for 72 h in the presence or absence of BEC. (h) M0 or M2 macrophages were adoptively transferred (4 × 10⁶ cells, i.v.) into naive BALB/c mice, which were challenged 12 days later with L. pneumophila. Survival of mice was recorded (n=10 mice for M0 group and n=11 for M2 group). ND, not detected. *P<0.05, **P<0.01 and ***P<0.001 (two-tailed unpaired Student's t-test in a,b, one-way ANOVA result with Bonferroni's posthoc tests in d, Mantel-Cox log-rank test in e). Data are representative of two (a,c–g) independent experiments; or pooled from two (b,h) experiments (mean±s.e.m. in a,b,e–h).