Reilly Bridgette, Tan Chuyi, Murao Atsushi, Nofi Colleen, Jha Alok, Aziz Monowar, Wang Ping
Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, Manhasset, NY, USA.
Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Manhasset, NY, USA.
J Inflamm Res. 2022 Jul 17;15:4047-4059. doi: 10.2147/JIR.S370615. eCollection 2022.
Extracellular cold-inducible RNA-binding protein (eCIRP) is an endogenous pro-inflammatory mediator that exacerbates injury in inflammation and sepsis. The mechanisms in which eCIRP is released have yet to be fully explored. Necroptosis is a programmed cell death that is dependent on the activation of mixed lineage kinase domain-like pseudo kinase (MLKL) which causes the release of damage-associated molecular patterns. We hypothesize that eCIRP is released through necroptosis and intensifies inflammation in sepsis.
RAW264.7 cells were treated with pan-caspase inhibitor z-VAD (15 μM) 1 h before stimulation with LPS (1 μg/mL). Necroptosis inhibitor, Necrostatin-1 (Nec-1) (10 μM) was added to the cells with LPS simultaneously. After 24 h of LPS stimulation, cytotoxicity was determined by LDH assay. eCIRP levels in the culture supernatants and phospho-MLKL (p-MLKL) from cell lysates were assessed by Western blot. p-MLKL interaction with the cell membrane was visualized by immunofluorescence. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Mice were treated with Nec-1 (1 mg/kg) or DMSO. 20 h post-surgery, serum and peritoneal fluid levels of eCIRP, TNF-α and IL-6 were determined by ELISA. H&E staining of lung tissue sections was performed.
We found that in RAW264.7 cells, LPS+z-VAD induces necroptosis as evidenced by an increase in p-MLKL levels and causes eCIRP release. Nec-1 reduces both p-MLKL activation and eCIRP release in LPS+z-VAD-treated RAW264.7 cells. Nec-1 also inhibits the release of eCIRP, TNF-α and IL-6 in the serum and peritoneal fluid in CLP-induced septic mice. We predicted a transient interaction between eCIRP and MLKL using a computational model, suggesting that eCIRP may exit the cell via the pores formed by p-MLKL.
Necroptosis is a novel mechanism of eCIRP release in sepsis. Targeting necroptosis may ameliorate inflammation and injury in sepsis by inhibiting eCIRP release.
细胞外冷诱导RNA结合蛋白（eCIRP）是一种内源性促炎介质，可加重炎症和脓毒症中的损伤。eCIRP释放的机制尚未完全阐明。坏死性凋亡是一种程序性细胞死亡，依赖于混合谱系激酶结构域样假激酶（MLKL）的激活，后者会导致损伤相关分子模式的释放。我们假设eCIRP通过坏死性凋亡释放，并加剧脓毒症中的炎症反应。
在用脂多糖（LPS，1μg/mL）刺激前1小时，用泛半胱天冬酶抑制剂z-VAD（15μM）处理RAW264.7细胞。将坏死性凋亡抑制剂Necrostatin-1（Nec-1，10μM）与LPS同时加入细胞中。LPS刺激24小时后，通过乳酸脱氢酶（LDH）测定法测定细胞毒性。通过蛋白质免疫印迹法评估培养上清液中的eCIRP水平以及细胞裂解物中的磷酸化MLKL（p-MLKL）。通过免疫荧光观察p-MLKL与细胞膜的相互作用。通过盲肠结扎和穿刺（CLP）在C57BL/6小鼠中诱导脓毒症。小鼠用Nec-1（1mg/kg）或二甲基亚砜（DMSO）处理。手术后20小时，通过酶联免疫吸附测定法（ELISA）测定血清和腹腔液中eCIRP、肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的水平。对肺组织切片进行苏木精-伊红（H&E）染色。
我们发现，在RAW264.7细胞中，LPS+z-VAD诱导坏死性凋亡，p-MLKL水平升高证明了这一点，并导致eCIRP释放。Nec-1降低了LPS+z-VAD处理的RAW264.7细胞中p-MLKL的激活和eCIRP的释放。Nec-1还抑制了CLP诱导的脓毒症小鼠血清和腹腔液中eCIRP、TNF-α和IL-6 的释放。我们使用计算模型预测了eCIRP与MLKL之间的瞬时相互作用，表明eCIRP可能通过p-MLKL形成的孔离开细胞。
坏死性凋亡是脓毒症中eCIRP释放的新机制。靶向坏死性凋亡可能通过抑制eCIRP释放来改善脓毒症中的炎症和损伤。
