Yamaga Satoshi, Murao Atsushi, Aziz Monowar, Wang Ping, Brenner Max
Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA.
Int J Mol Med. 2025 Oct;56(4). doi: 10.3892/ijmm.2025.5598. Epub 2025 Aug 1.
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 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 . Conversely, knockout of CIRP and TREM‑1 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.
高剂量电离辐射会引发多种类型的组织损伤，包括以中性粒细胞减少为特征的造血功能障碍。NETosis过程中释放的中性粒细胞胞外陷阱（NETs）可能导致中性粒细胞减少，以及随后的感染和炎症。髓系细胞表达的触发受体-1（TREM-1）是负责NET形成的受体之一，细胞外冷诱导RNA结合蛋白（eCIRP）是TREM-1受体的配体。本研究旨在调查暴露于高剂量电离辐射后NET的形成情况，并探索eCIRP/TREM-1轴作为其机制的潜在作用。从C57BL/6小鼠分离的骨髓来源中性粒细胞（BMDNs）接受5至15 Gy的辐射。C57BL/6野生型（WT）、CIRP和TREM-1小鼠接受10 Gy全身照射（TBI）。照射后24小时使用流式细胞术和荧光显微镜分析NET的形成情况，以及用eCIRP处理后的情况。使用流式细胞术评估中性粒细胞上TREM-1的细胞表面表达。使用蛋白质印迹法评估BMDNs中肽基精氨酸脱亚氨酶4（PAD4）蛋白表达水平。使用逆转录定量PCR评估BMDNs中TREM-1和PAD4 mRNA表达水平。使用流式细胞术评估中性粒细胞的体外照射导致NET形成呈剂量依赖性增加，并通过荧光显微镜进行验证，荧光显微镜显示了照射后中性粒细胞中NETs典型的长细胞外DNA结构。TBI小鼠模型显示出类似的结果。此外，照射后BMDNs中TREM-1的表达显著增加。照射后PAD4的蛋白质和mRNA水平显著上调。向BMDNs中添加eCIRP进一步增加了照射后NET的形成。相反，与WT小鼠相比，CIRP和TREM-1基因敲除显著减弱了辐射诱导的NET形成。高剂量电离辐射通过eCIRP/TREM-1途径诱导NET形成，并可能导致照射后早期中性粒细胞减少。
