Frontline Science: Extracellular CIRP generates a proinflammatory Ly6G+CD11bhisubset of low-density neutrophils in sepsis

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern. Neutrophils present in the mononuclear cell fraction of Ficoll gradient separation are called low-density neutrophils (LDNs). Here we report the novel role of eCIRP on LDNs' heterogeneity in sepsis. Sepsis was induced in male C57BL/6 wild-type (WT) and CIRP(-/-)mice by cecal ligation and puncture (CLP). At 20 h after CLP, LDNs in the blood were isolated by Ficoll gradient separation, followed by staining the cells with anti-Ly6G and anti-CD11b Abs and detection by flow cytometry. Sepsis or recombinant murine CIRP (rmCIRP) injection in mice resulted in significant increase in the frequency (%) and number of Ly6G(+)CD11b(hi)and Ly6G(+)CD11b(lo)LDNs in the blood compared to sham- or vehicle-treated mice. At 20 h of CLP, CIRP(-/-)mice had significantly lower frequency and number of Ly6G(+)CD11b(hi)and Ly6G(+)CD11b(lo)LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP-injected mice, compared to Ly6G(+)CD11b(lo)LDNs, the expression of CXCR4, ICAM-1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G(+)CD11b(hi)LDNs in the blood were significantly increased. Treatment of WT bone marrow-derived neutrophils (BMDNs) with rmCIRP increased Ly6G(+)CD11b(hi)LDN frequency, whereas treatment of TLR4(-/-)BMDNs with rmCIRP significantly decreased the frequency of Ly6G(+)CD11b(hi)LDNs. BMDNs' stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G(+)CD11b(hi)of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G(+)CD11b(hi)LDNs.

Automated summary

The study reveals the important role of eCIRP in the heterogeneity of LDNs in sepsis, and targeting it may help reduce inflammatory responses.