Fig 2: Hypothesis schema.eCIRP promotes macrophage endotoxin tolerance. eCIRP is increased during sepsis or other disease conditions and recognizes its novel receptor, IL-6R, expressed in macrophages. This leads to the activation of downstream transcription factor STAT3, which results in immune tolerance as depicted by decreased levels of TNF-a and IL-6 following LPS stimulation to these macrophages. eCIRP treatment of macrophages also induces regulatory phenotype M2 polarization in macrophages through IL-6R–dependent STAT3 activation. Inhibition of IL-6R by using its neutralizing Ab decreases eCIRP-induced STAT3 activation in macrophages and corrects immune tolerance and M2 polarization.

Fig 3: Inhibition of STAT3 by Stattic rescues macrophages from eCIRP-induced endotoxin tolerance.RAW264.7 cells (5 × 105/mL) were pretreated with PBS or rmCIRP (1 µg/mL) in the absence or presence of Stattic (3 µM) for 24 hours. Cells were then washed with medium to remove rmCIRP in the supernatants, and they were further stimulated with LPS (10 ng/mL). After 5 hours, culture supernatants were collected and assessed for (A) TNF-a and (B) IL-6. Data are expressed as mean ± SEM (n = 7 samples/group). The experiments were performed 3 times, and all data were used for analysis. 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 (+).

Fig 4: Stat3 and mTOR regulate IL-22 production by CD4+ T cells.a–d WT CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM) (n = 3/group). Phosphorylated Stat3 (6 h) (a, b) and phosphorylated mTOR (24 h) (c, d) were assessed by western blot and flow cytometry. Phosphorylated S6K was analyzed by flow cytometry (e). f–i CBir1 Tg CD4+ T cells were activated with APCs and CBir1 peptide under Th1 conditions with butyrate (0.5 mM) ± rapamycin (1 µM) or HJC0152 (1 µM). IL-22 mRNA (f) and protein (g) were assessed by qRT-PCR and ELISA at 60 h (n = 3/group). Expression of Hif1a (h) and Ahr (i) was analyzed at 48 h by qRT-PCR. j WT and Stat3-/- CD4+ T cells were treated with or without butyrate (0.5 mM) for 5 days (n = 3/group). IL-22 production was measured by flow cytometry. One representative of three independent experiments was shown. Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test (a–e, j) or two-tailed one-way ANOVA (f–i). a *p = 0.0134; b ***p = 0.0002; c **p = 0.0059; d ***p = 0.0002; e **p = 0.0010; f, ****p < 0.0001; g **p = 0.0019 (butyrate vs control) and 0.0069 (butyrate + rapamycin vs butyrate), *p = 0.0141; h ***p = 0.0004, **p = 0.0012; i, ***p = 0.0004 (butyrate vs control) and 0.009 (butyrate + HJC0152 vs butyrate), **p = 0.0030; j **p = 0.0017, *p = 0.0338.

Fig 5: 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 (+).