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 6

eCIRP induces mitochondrial DNA fragmentation in macrophages.

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eCIRP induces mitochondrial DNA fragmentation in macrophages. After rmCI...
After rmCIRP (1 μg/mL) stimulation of peritoneal macrophages for 4 hours, the cell culture media was discarded. MitoTracker Red was added to the culture medium. After 1 hour of culture, the cells were fixed with 2% paraformaldehyde solution for 20 minutes and then washed with PBS and blocked with normal 5% BSA for 1 hour. The cells were stained with TUNEL. The cell nucleus was stained with Hoechst 33258. The macrophages were then examined by confocal microscopy. (A) Representative images of the macrophages stained with TUNEL (green fluorescence), MitoTracker Red (red fluorescence), and nuclear counterstaining with Hoechst (blue fluorescence). Scale bar: 10 μM. Total original magnification, 400×. (B) Cells were counted in 3 random fields in each independent experiment. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by Student’s t test (*P < 0.05 versus PBS-treated macrophages). The data passed the normality test (Shapiro-Wilk), with a subsequent 2-tailed P < 0.05. The experiments were performed 2 times, and all data were used for analysis.