High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils.

Abstract

High‑dose ionizing radiation induces multiple types of tissue injuries, including hematopoietic dysfunction characterized by neutropenia. Neutrophil extracellular traps (NETs) released during NETosis may contribute to the neutropenia, and subsequent infection and inflammation. Triggering receptor expressed on myeloid cells‑1 (TREM‑1) is one of receptors responsible for NET formation and extracellular cold‑inducible RNA‑binding protein (eCIRP) is a ligand for the TREM‑1 receptor. The present study aimed to investigate NET formation after exposure to high‑dose ionizing radiation and to explore the underlying role of the eCIRP/TREM‑1 axis as its mechanism. Bone marrow‑derived neutrophils (BMDNs) isolated from C57BL/6 mice were exposed to 5 to 15 Gy irradiation. C57BL/6 wild‑type (WT), CIRP^‑/‑ and TREM‑1^‑/‑ mice were exposed to 10 Gy total body irradiation (TBI). NET formation was analyzed 24 h after irradiation using flow cytometry and fluorescence microscopy, and also after treatment with eCIRP. TREM‑1 cell surface expression on neutrophils was assessed using flow cytometry. Peptidyl arginine deiminase 4 (PAD4) protein expression levels in BMDNs were evaluated using western blotting. TREM‑1 and PAD4 mRNA expression levels in BMDNs were assessed using reverse transcription‑quantitative PCR. In vitro irradiation of neutrophils resulted in a dose‑dependent increase in NET formation, as assessed using flow cytometry and validated using fluorescence microscopy, which demonstrated the characteristic long extracellular DNA structures of NETs in irradiated neutrophils. The in vivo mouse model of TBI exhibited similar results. Furthermore, TREM‑1 expression in BMDNs was significantly increased after irradiation. Protein and mRNA levels of PAD4 were significantly upregulated after irradiation. The addition of eCIRP to BMDNs further increased NET formation post‑irradiation in vitro. Conversely, knockout of CIRP and TREM‑1 in vivo significantly attenuated radiation‑induced NET formation compared with that of WT mice. High‑dose ionizing radiation induced NET formation through the eCIRP/TREM‑1 pathway and may contribute to early neutropenia post‑irradiation.