Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1 neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1 neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found increased expression of CIRP and higher frequencies and numbers of ICAM-1 neutrophils in the lungs. Conversely, the CIRP mice showed significant inhibition in the frequencies and numbers of ICAM-1 neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1 phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1 neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared, respectively, with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1 BMDN were significantly higher than those in ICAM-1 BMDN. Following sepsis the ICAM-1 neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1 neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus, CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1 neutrophils in the lungs during sepsis.

BackgroundSepsis morbidity and mortality are aggravated by acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Mouse B-1a cells are a phenotypically and functionally unique sub-population of B cells, providing immediate protection against infection by releasing natural antibodies and immunomodulatory molecules. We hypothesize that B-1a cells ameliorate sepsis-induced ALI.MethodsSepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). PBS or B-1a cells were adoptively transferred into the septic mice intraperitoneally. After 20 h of CLP, lungs were harvested and assessed by PCR and ELISA for pro-inflammatory cytokines (IL-6, IL-1β) and chemokine (MIP-2) expression, by histology for injury, by TUNEL and cleaved caspase-3 for apoptosis, and by myeloperoxidase (MPO) assay for neutrophil infiltration.ResultsWe found that septic mice adoptively transferred with B-1a cells significantly decreased the mRNA and protein levels of IL-6, IL-1β and MIP-2 in the lungs compared to PBS-treated mice. Mice treated with B-1a cells showed dramatic improvement in lung injury compared to PBS-treated mice after sepsis. We found apoptosis in the lungs was significantly inhibited in B-1a cell injected mice compared to PBS-treated mice after sepsis. B-1a cell treatment significantly down-regulated MPO levels in the lungs compared to PBS-treated mice in sepsis. The protective outcomes of B-1a cells in ALI was further confirmed by using B-1a cell deficient CD19−/− mice, which showed significant increase in the lung injury scores following sepsis as compared to WT mice.ConclusionsOur results demonstrate a novel therapeutic potential of B-1a cells to treat sepsis-induced ALI.

No abstract

Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation in sepsis. Neutrophil reverse transendothelial migration (rTEM) allows neutrophils to migrate from tissues into the circulation. The phenotype of neutrophils following reverse migration is CD54CXCR1. We hypothesize that CIRP induces neutrophil rTEM in sepsis. Sepsis was induced in male C57BL/6 mice by cecal ligation and puncture (CLP), and at 5, 10, or 20 h after CLP, the frequencies of reversely migrated (RM) neutrophils were assessed in the blood by flow cytometry. Since 20 h of CLP showed highest increase in the frequency of RM neutrophils, we further assessed RM neutrophils in the blood of WT and CIRP mice at this time point. The effect of CIRP on neutrophil rTEM was determined by injecting mice with recombinant mouse CIRP (rmCIRP) intratracheally (i.t.) and assessed the frequencies of RM neutrophils. The expression of neutrophil elastase (NE) and junctional adhesion molecule-C (JAM-C) in the lungs were measured by Western blot. The mean frequency of RM neutrophils in sham mice was 0.4%, while the frequencies were significantly increased to 1%, 3%, and 7% at 5, 10, and 20 h of CLP, respectively. The mean frequency of RM neutrophils in the blood of CIRP mice was significantly lower than that of WT mice at 20 h of CLP. The RM neutrophils in the blood was significantly increased after administration of rmCIRP i.t. into mice in a time- and dose-dependent manners. NE expression was upregulated while JAM-C expression was downregulated in the lungs after CLP or rmCIRP administration. For the first time, we have showed that CIRP induces neutrophil rTEM in sepsis by increasing NE and decreasing JAM-C.

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.