Extracellular CIRP induces macrophage endotoxin tolerance through IL-6R–mediated STAT3 activation
Mian Zhou, … , Gaifeng Ma, Ping Wang
Mian Zhou, … , Gaifeng Ma, Ping Wang
Published February 6, 2020
Citation Information: JCI Insight. 2020;5(5):e133715. https://doi.org/10.1172/jci.insight.133715.
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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 1

eCIRP induces macrophage tolerance.

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eCIRP induces macrophage tolerance. (A) Sepsis was induced in mice by CL...
(A) Sepsis was induced in mice by CLP. Blood and spleen were collected 72 hours after CLP. eCIRP levels in the serum were assessed. Splenocytes were isolated from the septic mouse and stimulated with LPS (100 ng/mL) for 5 hours ex vivo and assessed for TNF-α in the culture supernatants. Data show the correlation between serum level of eCIRP and culture supernatant level of TNF-α from splenocytes treated with ex vivo LPS for each mouse. Green circle, eCIRP; orange circle, TNF-α. n = 7 mice/group. (B) TNF-α levels in the serum were assessed and presented with serum levels of eCIRP. Data are expressed as mean ± SEM (n = 7 mice/group). (C and D) A total of 7 × 105/mL peritoneal macrophages isolated from healthy mice were prestimulated with PBS or rmCIRP (1 μg/mL) for 24 hours, and the cells were washed with medium. Macrophages were restimulated with LPS (50 ng/mL) for 5 hours and assessed for (C) TNF-α and (D) IL-6 in the culture supernatants. Data are expressed as mean ± SEM (n = 5–6 wells/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 7. *P < 0.05 vs. PBS control; #P < 0.05 vs. pre-rmCIRP (–), LPS (+). (E and F) RAW264.7 macrophages (3 × 105/mL) were pretreated with PBS or rmCIRP at 0.5 and 1.0 μg/mL for 24 hours. Cells were washed with medium, restimulated with LPS (10 ng/mL) for 5 hours and assessed for (E) TNF-α and (F) IL-6 in the culture supernatants. Data are expressed as mean ± SEM (n = 4 wells/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 7. *P < 0.05 vs. PBS control; #P < 0.05 vs. pre-rmCIRP (–), LPS (+); †P < 0.05 vs. rmCIRP (0.5 μg/mL). (G) Mice were injected with normal saline or rmCIRP (5 mg/kg BW) intraperitoneally (i.p.); 24 hours after injection, peritoneal macrophages were isolated. A total of 2 × 105 peritoneal macrophages were stimulated with 25 and 50 ng/mL LPS for 5 hours ex vivo and assessed for IL-6 in the culture supernatants. Data are expressed as mean ± SEM (n = 6–12 samples/group). Experiments were performed 2 times, and all data were used for analysis. The groups were compared by 1-way ANOVA and Student-Newman-Keuls (SNK) method. *P < 0.05 vs. PBS in respective injection group, #P < 0.05 vs. LPS (25 ng/mL) in respective injection group, and †P < 0.05 vs. saline injection in respective LPS dose. CLP, cecal ligation and puncture.