Zhang Zhi-Hong, Yang Hong-Xu, Yang Jun-Liang, Jiang Xue-Li, Wang Si-Qi, Wu Yan-Ling, Zhan Zi-Ying, Nan Ji-Xing, Lian Li-Hua
Key Laboratory of Traditional Chinese Korean Medicine Research of State Ethnic Affairs Commission, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China; Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
Longgang Central Hospital, 6082 longgang Road, Shenzhen, Guangdong Province 518116, China.
Int Immunopharmacol. 2025 Aug 28;161:115112. doi: 10.1016/j.intimp.2025.115112. Epub 2025 Jun 18.
Acute liver failure (ALF), characterized by inflammation-mediated hepatocyte dysfunction, poses a significant global health burden. Although neutrophils and macrophages are central to driving inflammatory responses in ALF, the molecular mechanisms governing IL-36-driven inflammation in toll-like receptor 3 (TLR3)-mediated NETosis and macrophage activation remain poorly understood. This study systematically delineates wherein TLR3 activation induces IL-36 hyperactivation and NETosis, which cooperatively trigger macrophage activation to accelerate ALF progression.
First, the role of IL-36 signaling was evaluated in the TLR3-provoked mouse peritoneal macrophages (MPMs) and murine bone-derived macrophages (BMDMs) in vitro. Further, the murine model of ALF was established with polyinosinic-polycytidylic acid (Poly(I:C))/D-GalN and acute ethanol gavage in Il1rl2-deficient mice generated by lentivirus-mediated shRNA, and the released NETs were evaluated with immunoblotting and immunofluorescence staining.
We observed Poly(I:C), the specific ligand of TLR3, when combined with extracellular ATP, provoked the IL-36 signaling and the release of IL-1β in macrophages. In contrast, Il36g deficiency reduced the secretion of proinflammatory cytokines, such as IL-1β and HMGB1, upon macrophage activation. While Poly(I:C)/D-GalN or binge ETOH-induced ALF in mice was accompanied by the enhanced level of IL-36γ and NET formation, IL-36R knockdown reversed the liver damage by decreasing the expressions of proinflammatory cytokines and chemokines, reducing the lipid accumulation and preventing the NET formation as well.
Our study demonstrates that TLR3-mediated NET formation was enhanced in ALF, and this process was tightly regulated by IL-36 signaling. These findings suggest that targeting IL-36 represents a potential therapeutic target for modulating inflammation in ALF.
急性肝衰竭（ALF）以炎症介导的肝细胞功能障碍为特征，给全球健康带来了重大负担。尽管中性粒细胞和巨噬细胞在驱动ALF炎症反应中起核心作用，但在Toll样受体3（TLR3）介导的中性粒细胞胞外陷阱形成（NETosis）和巨噬细胞激活过程中，调控白细胞介素-36（IL-36）驱动炎症的分子机制仍知之甚少。本研究系统地阐述了TLR3激活在何处诱导IL-36过度激活和NETosis，二者协同触发巨噬细胞激活以加速ALF进展。
首先，在体外评估IL-36信号通路在TLR3刺激的小鼠腹腔巨噬细胞（MPM）和小鼠骨髓来源巨噬细胞（BMDM）中的作用。此外，通过慢病毒介导的短发夹RNA（shRNA）构建Il1rl2基因缺陷小鼠，并用聚肌苷酸-聚胞苷酸（Poly(I:C)）/D-半乳糖胺（D-GalN）和急性乙醇灌胃建立ALF小鼠模型，通过免疫印迹和免疫荧光染色评估释放的NETs。
我们观察到，TLR3的特异性配体Poly(I:C)与细胞外ATP结合时，可激发巨噬细胞中的IL-36信号通路并释放白细胞介素-1β（IL-1β）。相反，Il36g基因缺陷会降低巨噬细胞激活后促炎细胞因子如IL-1β和高迁移率族蛋白B1（HMGB1）的分泌。虽然Poly(I:C)/D-GalN或暴饮乙醇诱导的小鼠ALF伴有IL-36γ水平升高和NET形成，但IL-36受体（IL-36R）基因敲低可通过降低促炎细胞因子和趋化因子的表达、减少脂质积累以及防止NET形成来逆转肝损伤。
我们的研究表明，ALF中TLR3介导的NET形成增强，且该过程受IL-36信号通路严格调控。这些发现表明，靶向IL-36是调节ALF炎症的一个潜在治疗靶点。
