Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4^-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4^-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) α_vβ₃ or α_vβ₅ integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins α_vβ₃ and α_vβ₅ Using a preclinical model of sepsis, we found that CIRP^-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of α_vβ₃/α_vβ₅ integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.