Fig 2: Role of CD11b+ Macrophages in Regulating Pseudomonas aeruginosa-Induced Lung Inflammatory Injury and Lethality(A–C) Lungs receiving 1 × 104 CFU of P. aeruginosa (PA) i.t. were harvested at the indicated times and homogenized, and supernatants were used to quantify IL-6 (A), IL-1ß (B), and IFN-ß (C) levels using an ELISA kit (n = 4 mice/group).(D) Lung edema determined as described in Figure 1G. Plot shows individual values (n = 5 mice/group).(E) Mouse survival was assessed every 6–12 h after 1 × 106 CFU of PA instillation (n = 10 mice/group).(A–E) are shown as mean ± SD from experiments that were repeated three times independently. *p < 0.05, ***p < 0.001 relative to unexposed macrophages (Mf) or macrophagedep mice; ###p < 0.001 relative to macrophage group after PA challenge at indicated times. Analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig 3: Co-infection Leads to Elevated Levels of Plasma Heme and Favors Bacterial Persistence Dependent on Uptake of Extracellular Iron(A) C57BL/6 mice were infected with P. chabaudi-RBCs and/or with C. rodentium. (A) Heme levels were measured in plasma samples collected between 9 and 14 days post-infection. Data represent pooled results from two independent experiments (n = 3–9 mice/group/time point). Each symbol represents an individual mouse and bars denote group means. Statistical significance was determined by the Mann-Whitney test. **p < 0.01 and ****p < 0.0001.(B) Spleen cells were collected between 9 and 14 dpi, and mitochondrial ROS was assessed in CD11b+ cells by measuring MitoSox by flow cytometry after culture with medium or PMA. Data shown are representative of one experiment (n = 4–14 mice per group). Each symbol represents an individual mouse and bars denote group means. Statistical significance was determined by the Mann-Whitney test (*p < 0.05).(C and D) Additional cohorts of uninfected or C. rodentium-infected C57BL/6 mice received hemin intraperitoneally (50 µmol kg/body weight per dose) once per day, starting at 3 dpi until 11 dpi and then body weight and survival were monitored. Symbols represent mean ± SEM as a percentage of the initial body weight. Data shown are representative of two independent experiments (n = 4–10 mice per group).(E and F) Additional cohorts of C57BL/6 mice were infected orally with 1 × 109 CFU of the isogenic C. rodentium fepA mutant strain or with its control strain DBS120 C. rodentium or co-infected with these strains and P. chabaudi at day 0. Body weights and survival were monitored. Symbols represent mean ± SEM as a percentage of the initial body weight. Data shown were pooled from two independent experiments (n = 10–33 mice/group).

Fig 4: Eomes Is Required for Normal NK Cell Maturation(A) ILC-Eomes?/? and Eomes WT mice were treated with the Tam-3d regimen. Splenic NK cells were sorted to YFP+CD27+ (stage II/III) or YFP+CD27-CD11b+ (stage IV) and transferred into CD45.2 WT recipients. 2 weeks later, YFP+ cell maturation was assessed.(B) Stage distribution of ILC-Eomes?/? or Eomes WT sorted stage II/III or IV NK cells before (light blue) or 2 weeks after transfer (dark blue).(C) Representative flow cytometry data showing maturation of stage II/III ILC-Eomes?/? or Eomes WT NK cells after 2 weeks in vivo. N = 3 pooled mice per group, three independent experiments. Data were compared using t tests.*p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: TNF inhibits M2 gene expression.(A) RNA from wild-type BMDMs left untreated (Ctrl), stimulated with IL4/IL13 or IL4/IL13 + TNF was used for qRT-PCR. Data shown are the mean fold-increase compared with the Ctrl group. (B) Wild-type BMDMs, which were treated with solvent (Ctrl), IL4/IL13 or IL4/IL13 + TNF for 5, 15, 30, 60, or 120 min, were used to isolate whole-cell lysates and analyzed by Western blotting for the indicated proteins. (C) Representative plots of wild-type BMDMs stimulated with IL4/IL13 or IL4/IL13 + TNF and percentages of RELMa+ of F4/80+ macrophages were determined by flow cytometry analysis. (D) Wild-type BMDMs were stimulated with IL4/IL13, IL4/IL13 + TNF and/or Etanercept and percentages of RELMa+ macrophages were determined by flow cytometry analysis. Data represent three independent experiments. (E) CD45+CD11b+F4/80+ macrophages from skin excisional wounds were sorted, RNA was extracted and analyzed by qRT-PCR. Data are representative for two independent experiments. (F) Small peritoneal macrophages were analyzed after in vivo injection of solvent or Etanercept. Percentages of Arg1+ and RELMa+ cells were analyzed by flow cytometry analysis. See also Fig S1. All values are means ± SEM; *P < 0.05; ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Tukey’s correction or by paired t test. If not indicated otherwise superscripts show statistical significance compared with the control group. n = 3 biological replicates.