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 2

eCIRP induces STAT3 phosphorylation in macrophages.

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eCIRP induces STAT3 phosphorylation in macrophages. (A) RAW264.7 cells (...
(A) RAW264.7 cells (8 × 105 cells/mL) were stimulated with rmCIRP (2 μg/mL) for 1, 2, and 5 hours. Cells were harvested for protein extraction, followed by Western blot using Abs against p-STAT3, STAT3, and β-actin. Data are expressed as mean ± SEM (n = 3–5 samples/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 8. *P < 0.05 compared with PBS, #P < 0.05 compared with rmCIRP 1 hours, and †P < 0.05 compared with rmCIRP 2 hours. (B) RAW264.7 cells (8 × 105 cells/mL) were stimulated with 2 and 4 μg/mL rmCIRP for 5 hours. Cells were harvested for protein extraction, followed by Western blot assays using Abs against p-STAT3, STAT3, and β–actin. Data are expressed as mean ± SEM (n = 3–5 wells/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 8. *P < 0.05 compared with PBS, and #P < 0.05 compared with rmCIRP (2 μg/mL). (C) Splenocytes isolated from healthy mice (2 × 106 cells/mL) were stimulated with rmCIRP (4 μg/mL) for 10, 24, and 48 hours. Cells were harvested for protein extraction, followed by Western blot using Abs against p-STAT3, STAT3, and β-actin. Data are expressed as mean ± SEM (n = 3 samples/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 8. *P < 0.05 compared with PBS; #P < 0.05 compared with rmCIRP 10 hours. (D) Mice were injected with normal saline or rmCIRP (5 mg/kg BW) i.p. After 24 hours of PBS or rmCIRP injection, peritoneal macrophages were isolated for total protein extraction. Western blot was performed to determine p-STAT3, STAT3, and β-actin levels in each sample. Data are expressed as mean ± SEM (n = 3 mice/group). Experiments were repeated, and the repeated experimental data are shown in Supplemental Figure 8. *P < 0.05 compared with saline injection. Representative Western blots for p-STAT3, STAT3, and β-actin are shown. p-STAT3 expression in each sample was normalized to total STAT3 expression, and the mean values of PBS-treated groups were standardized as one for comparison. Data are expressed as mean ± SEM (n = 3 samples/group). The groups were compared by 1-way ANOVA and SNK method in multiple-group comparisons. Two groups were compared by 2-tailed Student’s t test.