Frontline Science: Extracellular CIRP generates a proinflammatory Ly6G+ CD11bhi subset 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+ CD11bhi and Ly6G+ CD11blo 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+ CD11bhi and Ly6G+ CD11blo LDNs in the blood compared to WT mice. In sepsis mice or rmCIRP-injected mice, compared to Ly6G+ CD11blo LDNs, the expression of CXCR4, ICAM-1, and iNOS and formation of reactive oxygen species, and neutrophil extracellular traps in Ly6G+ CD11bhi LDNs in the blood were significantly increased. Treatment of WT bone marrow-derived neutrophils (BMDNs) with rmCIRP increased Ly6G+ CD11bhi LDN frequency, whereas treatment of TLR4-/- BMDNs with rmCIRP significantly decreased the frequency of Ly6G+ CD11bhi LDNs. BMDNs' stimulation with rmCIRP increased the expression of transcription factors in LDNs. eCIRP induces the formation of a proinflammatory phenotype Ly6G+ CD11bhi of LDNs through TLR4. Targeting eCIRP may provide beneficial outcomes in sepsis by decreasing proinflammatory Ly6G+ CD11bhi LDNs.