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 6

eCIRP induces M2 polarization through IL-6R.

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eCIRP induces M2 polarization through IL-6R. (A) RAW264.7 cells (1 × 106...
(A) RAW264.7 cells (1 × 106/mL) were treated with rmCIRP (1 μg/mL) for 5, 24, or 48 hours. Arg-1 expression at the mRNA level was assessed by quantitative (qPCR). Expression of Arg-1 was normalized to β-actin expression and represented as fold induction compared with the normalized values of PBS control–treated cells. Data are expressed as mean ± SEM (n = 4–6 samples/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 11. The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 vs. PBS (control); #P < 0.05 vs. rmCIRP (5 hours); †P < 0.05 vs. rmCIRP (24 hours). (B and C) RAW264.7 cells (1 × 106/mL) were treated with rmCIRP at doses of 0.625 and 1.25 μg/mL for 48 hours; the expression of Arg-1 and CD206 mRNAs was assessed by qPCR and normalized to β-actin expression. Results are represented as fold induction compared with the normalized values of PBS control–treated cells. Data are expressed as mean ± SEM (n = 4 samples/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 11. The groups were compared by 1-way ANOVA and SNK method. *P < 0.05 vs. rmCIRP (0 μg/mL or PBS); #P < 0.05 vs. rmCIRP (0.625 μg/mL). (D and E) RAW264.7 cells (1 × 106/mL) were pretreated with IgG (3 μg/mL) or anti–IL-6R Ab (3 μg/mL) for 30 minutes. These cells were then stimulated with PBS or rmCIRP (1 μg/mL) for 24 hours, and then Arg-1 and CD206 were assessed by qPCR and flow cytometry, respectively. Arg-1 mRNA was normalized to β–actin, and data expressed in fold induction were compared with the PBS-treated condition. Data are expressed as mean ± SEM (n = 4–6 samples/group). Experiments were repeated 2 times, and all data were used for analysis. The groups were compared by 1-way ANOVA and SNK method (*P < 0.05 vs. PBS; #P < 0.05 vs. IgG + rmCIRP).