Fig 2: Inhibition of IL-6R corrects eCIRP-induced endotoxin tolerance.(A) Splenocytes were pretreated with IgG or anti–IL-6R Ab and stimulated with rmCIRP for 5 hours. Cells were fixed, permeabilized, and stained with anti–p-STAT3 and F4/80 Abs and analyzed. Data are expressed as mean ± SEM (n = 3 mice/group). The groups were compared by 1-way ANOVA and SNK method (*P < 0.05 vs. PBS; #P < 0.05 vs. IgG + rmCIRP). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 9. (B) Peritoneal macrophages were pretreated with IgG or anti–IL-6R Abs for 30 minutes and stimulated with PBS or rmCIRP for 24 hours. Total proteins were subjected to Western blotting using anti–p-STAT3, STAT3, and ß-actin Abs. Data are expressed as mean ± SEM (n = 4 samples/group). The groups were compared by 1-way ANOVA and SNK method (*P < 0.05 vs. PBS; #P < 0.05 vs. IgG + rmCIRP). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 10. (C) Peritoneal macrophages were pretreated with PBS or rmCIRP with IgG or anti–IL-6R Ab for 24 hours. Cells were washed with medium and restimulated with LPS for 5 hours. TNF-a levels in the supernatants were assessed. Data are expressed as mean ± SEM (n = 9–11 samples/group). Results were pooled from 2 independent experiments. The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 vs. pre-rmCIRP (–), LPS (–); #P < 0.05 vs. pre-rmCIRP (–), LPS (+); †P < 0.05 vs. pre-rmCIRP (+), LPS (+). (D) RAW264.7 cells were treated with rmIL-6 for 1 and 5 hours. Total protein was extracted and subjected to Western blotting using p-STAT3, STAT3, and ß-actin Abs. Data are expressed as mean ± SEM (n = 3 samples/group). The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 compared with PBS-treated cells; #P < 0.05 compared with rmIL-6 at 1 hour. (E) RAW264.7 cells were treated with rmIL-6 or rmCIRP for 20 hours and were restimulated with LPS for 5 hours, and TNF-a levels in the medium were assessed. Data are expressed as mean ± SEM (n = 6 samples/group). The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 vs. PBS (+), LPS (–); #P < 0.05 vs. PBS (+), LPS (+); †P<0.05 vs. LPS (+), rmIL-6 (+). (F and G) RAW264.7 cells were transfected with mock, IL-6R siRNA, or negative control (NC) siRNA and treated with rmCIRP for 20 hours. Cells were restimulated with LPS for 5 hours and (F) TNF-a and (G) IL-6 levels in the culture medium were assessed. Data are expressed as mean ± SEM (n = 4 samples/group). Experiments were performed twice, and all data were used for analysis. The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 vs. mock (+), LPS (–); #P < 0.05 vs. mock (+), rmCIRP (–), LPS (+); †P < 0.05 vs. NC (+), rmCIRP (+), LPS (+).

Fig 3: Arg1 controls the bioenergetic state of inflammation-induced regulatory macrophages.(A) Representative blots of wild-type BMDMs stimulated with LPS or IL4/IL13 for the indicated time and percentages of Arg1+, PD-L1+ and PD-L2+ or F4/80+ macrophages were determined by flow cytometry analysis. Data shown are representative of three independent experiments. SSC-A, side scatter area; APC, allophycocyanin; PE, phycoerythrin. (B) Wild-type BMDMs were stimulated with solvent (Ctrl) or lactate for the indicated time, and the percentages of Arg1+, PD-L1+, and PD-L2+ or F4/80+ macrophages were determined by flow cytometry analysis. Data represent two independent experiments. (C) Wild-type and Nos2-/- BMDMs were left untreated or were treated with LPS*. The cells were sequentially treated with oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin A (Rot/AA), and the oxygen consumption rate (OCR) was determined. (D) Wild-type or Arg1-deficient BMDMs were left untreated or were treated with LPS*, and mitochondrial respiration was measured. All values are means ± SEM; *P < 0.05; **P < 0.01; and ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Tukey correction. If not indicated otherwise, superscripts show statistical significance compared to the control group. n = 3 biological replicates.

Fig 4: Gating strategy for identification of respiratory leukocytes, neutrophils, eosinophils and dendritic cells. Cells were gated based on scatter light (FSC, SSC) characteristics and respiratory leukocytes were identified by CD45 expression (A, B). Out of the CD45+ cells, neutrophils were identified by Gr1brightCD11bbright expression. Subsequently, out of the neutrophil negative fraction, eosinophils were identified by SiglecF+MHC-class-IIneg (I-A/I-Eneg) expression. Out of the neutrophil and eosinophil negative fraction macrophages were identified as SiglecF++F4/80+ double positive cells (A). Total dendritic cells were identified as CD11c+ SiglecFneg CD49bneg respiratory leukocytes (B).