Extracellular CIRP induces macrophage endotoxin tolerance through IL-6R–mediated STAT3 activation
Mian Zhou, Monowar Aziz, Naomi-Liza Denning, Hao-Ting Yen, Gaifeng Ma, Ping Wang
Mian Zhou, Monowar Aziz, Naomi-Liza Denning, Hao-Ting Yen, Gaifeng Ma, Ping Wang
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Research Article Immunology Inflammation

Extracellular CIRP induces macrophage endotoxin tolerance through IL-6R–mediated STAT3 activation

Abstract

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern, whose effect on macrophages is not entirely elucidated. Here we identified that eCIRP promotes macrophage endotoxin tolerance. Septic mice had higher serum levels of eCIRP; this was associated with a reduced ex vivo immune response of their splenocytes to LPS. Pretreatment of macrophages with recombinant murine CIRP (rmCIRP) resulted in a tolerance to LPS stimulation as demonstrated by a reduction of TNF-α production. We found that eCIRP increased phosphorylated STAT3 (p-STAT3) in macrophages. A STAT3 inhibitor, Stattic, rescued macrophages from rmCIRP-induced tolerance by restoring the release of TNF-α in response to LPS stimulation. We discovered strong binding affinity between eCIRP and IL-6 receptor (IL-6R) as revealed by Biacore, fluorescence resonance energy transfer (FRET), and their colocalization in macrophages by immunostaining assays. Blockade of IL-6R with its neutralizing Ab inhibited eCIRP-induced p-STAT3 and restored LPS-stimulated TNF-α release in macrophages. Incubation of macrophages with rmCIRP skewed them toward an M2 phenotype, while treatment with anti–IL-6R Ab prevented rmCIRP-induced M2 polarization. Thus, we have demonstrated that eCIRP activates p-STAT3 via a novel receptor, IL-6R, to promote macrophage endotoxin tolerance. Targeting eCIRP appears to be a new therapeutic option to correct immune tolerance in sepsis.

Authors

Mian Zhou, Monowar Aziz, Naomi-Liza Denning, Hao-Ting Yen, Gaifeng Ma, Ping Wang

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Figure 5

Inhibition of IL-6R corrects eCIRP-induced endotoxin tolerance.

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Inhibition of IL-6R corrects eCIRP-induced endotoxin tolerance. (A) Sple...
(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 β-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-α 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 β-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-α 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-α 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 (+).