Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern (DAMP). Intercellular adhesion molecule-1 (ICAM-1) expressing neutrophils produce excessive amounts of neutrophil extracellular traps (NETs). We reveal that eCIRP generates ICAM-1⁺ neutrophils through triggering receptor expressed on myeloid cells-1 (TREM-1) and the ICAM-1⁺ neutrophils involve Rho GTPase to promote NETosis. Treatment of BMDN with rmCIRP increased the frequency of ICAM-1⁺ BMDN, while rmCIRP-treated TREM-1^-/- BMDN or pretreatment of BMDN with TREM-1 inhibitor LP17 significantly decreased the frequency of ICAM-1⁺ neutrophils. The frequencies of ICAM-1⁺ neutrophils in blood and lungs were markedly decreased in rmCIRP-injected mice or septic mice treated with LP17. Coculture of ICAM-1^-/- neutrophils or wild-type (WT) neutrophils with WT macrophages in the presence of a peptidylarginine deiminase 4 (PAD4) inhibitor reduced TNF-α and IL-6 compared to WT neutrophils treated with rmCIRP. Treatment of ICAM-1^-/- neutrophils with rmCIRP resulted in reduced quantities of NETs compared to WT rmCIRP-treated neutrophils. Treatment of BMDN with rmCIRP-induced Rho activation, while blockade of ICAM-1 significantly decreased Rho activation. Inhibition of Rho significantly decreased rmCIRP-induced NET formation in BMDN. TREM-1 plays a critical role in the eCIRP-mediated increase of ICAM-1 expression in neutrophils, leading to the increased NET formation via Rho activation to exaggerate inflammation.