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 3

TBK1 and IRF3 in lungs in HS and rmCIRP-treated mice.

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TBK1 and IRF3 in lungs in HS and rmCIRP-treated mice. (A–C) After 4 hour...
(A–C) After 4 hours of HS, lungs were harvested and assessed for (A and B) pTBK1 and TBK1 and (A and C) pIRF3 and IRF3. Representative Western blots are shown. pTBK1 and pIRF3 expression in each sample was normalized to total TBK1 and IRF3 expression, and the mean values of the WT-sham group were standardized as 1 for comparison. Data are expressed as mean ± SEM (n = 6 mice/group) and compared by ANOVA and SNK tests (*P < 0.05 versus WT-sham; #P < 0.05 versus WT-HS mice). (D) eCIRP levels in serum of WT and STING–/– mice following sham or 4 hours after HS were assessed by ELISA. Data are expressed as mean ± SEM (n = 6 mice/group) and compared by ANOVA and SNK tests (*P < 0.05 versus WT-sham). (E–G) After 4 hours of injection with rmCIRP (5 mg/kg body weight) or equivalent volume PBS (vehicle), lungs were harvested from each group of mice and assessed for (E and F) pTBK1 and TBK1 and (E and G) pIRF3 and IRF3 proteins. Representative Western blots are shown. pTBK1 and pIRF3 expression in each sample was normalized to total TBK1 and IRF3 expression, and the mean values of the WT PBS-treated group were standardized as 1 for comparison. (H–K) Lung tissues of PBS or rmCIRP-injected WT and STING–/– mice were analyzed for the expression of (H and I) IFN-α and (J and K) IFN-β at mRNA and protein levels by real-time PCR and ELISA, respectively. Data are expressed as mean ± SEM (n = 6 mice/group) and compared by ANOVA and SNK tests (*P < 0.05 versus PBS-injected and #P < 0.05 versus rmCIRP-injected mice). The experiments were performed 3 times, and all data were used for analysis.