Extracellular CIRP activates STING to exacerbate hemorrhagic shock
Kehong Chen, Joaquin Cagliani, Monowar Aziz, Chuyi Tan, Max Brenner, Ping Wang
Kehong Chen, Joaquin Cagliani, Monowar Aziz, Chuyi Tan, Max Brenner, Ping Wang
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Research Article Inflammation

Extracellular CIRP activates STING to exacerbate hemorrhagic shock

Abstract

Stimulator of IFN genes (STING) activates TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) to produce type I IFNs. Extracellular cold-inducible RNA-binding protein (eCIRP) is released from cells during hemorrhagic shock (HS). We hypothesized that eCIRP activates STING to induce inflammation and acute lung injury (ALI) after HS. WT and STING–/– mice underwent controlled hemorrhage by bleeding, followed by fluid resuscitation. Blood and lungs were collected at 4 hours after resuscitation. Serum ALT, AST, LDH, IL-6, and IFN-β were significantly decreased in STING–/– mice compared with WT mice after HS. In STING–/– mice, the levels of pTBK1 and pIRF3, and expression of TNF-α, IL-6, and IL-1β mRNAs and proteins in the lungs, were significantly decreased compared with WT HS mice. The 10-day mortality rate in STING–/– mice was significantly reduced. I.v. injection of recombinant mouse CIRP (rmCIRP) in STING–/– mice showed a significant decrease in pTBK1 and pIRF3 and in IFN-α and IFN-β mRNAs and proteins in the lungs compared with rmCIRP-treated WT mice. Treatment of TLR4–/–, MyD88–/–, and TRIF–/– macrophages with rmCIRP significantly decreased pTBK1 and pIRF3 levels and IFN-α and IFN-β mRNAs and proteins compared with WT macrophages. HS increases eCIRP levels, which activate STING through TLR4/MyD88/TRIF pathways to exacerbate inflammation.

Authors

Kehong Chen, Joaquin Cagliani, Monowar Aziz, Chuyi Tan, Max Brenner, Ping Wang

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Figure 4

Stimulation of macrophages with rmCIRP induces type I IFN expression via STING-TBK1-IRF3 pathway.

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Stimulation of macrophages with rmCIRP induces type I IFN expression via...
Peritoneal macrophages were isolated from WT and STING–/– mice 4 days after i.p. injection of a single dose of 4% thioglycolate. A total of 2 × 106 cells/mL were stimulated with 5 μg/mL of rmCIRP or an equal volume of PBS as vehicle control. (A–C) After 4 hours of stimulation with rmCIRP, total protein was extracted from each sample and assessed for (A and B) pTBK1 and TBK1 and (A and C) pIRF3 and IRF3 proteins by Western blot. The blot was stripped and incubated with anti–β-actin Abs to serve as the loading control. Representative Western blots for pTBK1, TBK1, pIRF3, IRF3, and β-actin are shown. Each blot was quantified by densitometry analysis. pTBK1 and pIRF3 expression in each sample was normalized to total TBK1 and IRF3 expression, respectively, and the mean values of the PBS-treated group were standardized as 1 for comparison. (D–G) Assessment of type I IFNs. After treatment of a total of 2 × 106 cells/mL with 5 μg/mL of rmCIRP or an equal volume of PBS for 4 hours, mRNA and protein were extracted from each sample and assessed for (D and E) IFN-α and (F and G) IFN-β at mRNA and protein levels by real-time PCR and ELISA, respectively. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by ANOVA and SNK tests (*P < 0.05 versus PBS-treated and #P < 0.05 versus rmCIRP-treated macrophages). The experiments were performed 3 times, and all data were used for analysis.