EXTRACELLULAR CIRP INHIBITS NEUTROPHIL APOPTOSIS TO PROMOTE ITS AGING BY UPREGULATING SERPINB2 IN SEPSIS

Abstract

ABSTRACT Background: Sepsis reduces neutrophil apoptosis. As the result, neutrophils may become aged, exacerbating inflammation and tissue injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern to promote inflammation and tissue injury in sepsis. SerpinB2, a serine protease inhibitor, has been shown to inhibit apoptosis. We hypothesize that eCIRP upregulates SerpinB2 to promote aged neutrophil subset by inhibiting apoptosis in sepsis. Methods: We stimulated bone marrow–derived neutrophils (BMDNs) of wild-type (WT) mice with 1 μg/mL of recombinant mouse CIRP (i.e., eCIRP) and assessed cleaved caspase-3 and SerpinB2 by western blotting. Apoptotic neutrophils were assessed by Annexin V/PI. Bone marrow–derived neutrophils were stimulated with 1 μg/mL eCIRP and treated with or without PAC-1 (caspase-3 activator) and aged neutrophils (CXCR4hiCD62Llo) were assessed by flow cytometry. To induce sepsis, we performed cecal ligation and puncture in WT or CIRP−/− mice. We determined the percentage of aged neutrophils and SerpinB2+ neutrophils in blood and spleen by flow cytometry. Results: We found that cleaved caspase-3 levels were increased at 4 h of PBS treatment compared with 0 h but decreased by eCIRP treatment. Extracellular cold-inducible RNA-binding protein reduced apoptotic cells after 20 h of treatment. Extracellular cold-inducible RNA-binding protein also increased the frequencies of aged neutrophils compared with PBS after 20 h, while PAC-1 treatment reduced aging in eCIRP-treated BMDNs. Extracellular cold-inducible RNA-binding protein significantly increased the expression of SerpinB2 at protein levels in BMDNs at 20 h. In WT mice, the frequencies of aged and SerpinB2+ neutrophils in blood and spleen were increased after 20 h of cecal ligation and puncture, while in CIRP−/− mice, aged and SerpinB2+ neutrophils were significantly decreased compared with WT mice. We also found that aged neutrophils expressed significantly higher levels of SerpinB2 compared with non-aged neutrophils. Conclusions: eCIRP inhibits neutrophil apoptosis to increase aged phenotype by increasing SerpinB2 expression in sepsis. Thus, targeting eCIRP could be a new therapeutic strategy to ameliorate inflammation caused by neutrophil aging in sepsis.