Abstract
Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation and tissue injury in sepsis. Neutrophil extracellular traps (NETs) are released by activated neutrophils during sepsis. NETs contribute to pathogen clearance, but excessive NET formation (NETosis) causes inflammation and tissue damage. Peptidylarginine deiminase 4 (PAD4) is associated with NETosis by increasing histone citrullination and chromatin decondensation. We hypothesized that CIRP induces NETosis in the lungs during sepsis via upregulating PAD4 expression. Sepsis was induced in C57BL/6 wild-type (WT) and CIRP−/− mice by cecal ligation and puncture (CLP). After 20 h of CLP induction, NETs in the lungs of WT and CIRP−/− mice were quantified by flow cytometry by staining the single cell suspensions with MPO and CitH3 Abs. PAD4 expression in the lungs of WT and CIRP−/− mice after sepsis was assessed by Western blotting. In vitro effects of recombinant mouse (rm) CIRP for NETosis and PAD4 expression in the bone marrow-derived neutrophils (BMDN) were assessed by flow cytometry and Western blotting, respectively. After 20 h of CLP, NETosis in the lungs was significantly decreased in CIRP−/− mice compared to WT mice, which also correlated with the decreased PAD4 expression. Intratracheal administration of rmCIRP into WT mice significantly increased NETosis and PAD4 expression in the lungs compared to vehicle-injected mice. In vitro culture of BMDN with rmCIRP significantly increased NETosis and PAD4 expression compared to PBS-treated control. Fluorescence microscopy revealed typical web-like structures consistent with NETs in rmCIRP-treated BMDN. Thus, CIRP serves as a novel inducer of NETosis via PAD4 during sepsis.
Highlights
In inflammation, cold-inducible RNA-binding protein (CIRP) is released into the extracellular space[4]
We found that at 10 h after cecal ligation and puncture (CLP) the frequencies of neutrophil extracellular traps (NETs)-forming neutrophils were increased as compared to sham mice
We found that the total numbers of NET-forming neutrophils in the lungs were increased in the WT mice following sepsis, while the numbers of NET-forming neutrophils in the lungs of CIRP−/− mice were significantly decreased by 64% as compared to WT septic mice (Fig. 1C)
Summary
Cold-inducible RNA-binding protein (CIRP) is released into the extracellular space[4]. We found that CIRP-neutralizing antibodies (Ab) ameliorate sepsis[4], CIRP−/− mice are protected from sepsis and ALI9, and healthy mice injected with recombinant mouse (rm) CIRP develop lung injury[10]. In order to do this, we first induced sepsis in WT and CIRP−/− mice and compared the status of NETosis and PAD4 expression in their lungs. To determine the direct effect of CIRP, we administered rmCIRP into the mice and assessed NETosis and PAD4 expression in the lungs. Our data showed that CIRP could induce NET formation and PAD4 expression in the lungs in mice during sepsis, thereby revealing a new drug candidate by targeting CIRP to control sepsis and ALI through the downregulation of PAD4-dependent NETosis in the lungs during sepsis
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