Tian Jing, Liu Ying, Gao Wei, Shi Xiuyun, Cheng Feng, Xie Bing
Department of Orthopedics, General Hospital of Northern Theater, Shenyang City, Liaoning Province, China.
Department of Emergency, General Hospital of Northern Theater, Shenyang City, Liaoning Province, China.
Immunol Lett. 2025 Oct;275:107007. doi: 10.1016/j.imlet.2025.107007. Epub 2025 Apr 22.
Acute gouty arthritis is a metabolic disease characterized by hyperuricemia, with acute attacks involving neutrophil-released NETs activating immune responses through their major component, DNA, as danger-associated molecular patterns (DAMPs).
To investigate whether DNA from NETs activates the AIM2 inflammasome in synovial fibroblasts during acute gouty arthritis attacks, inducing pyroptosis and exacerbating inflammation.
The AIM2 gene knockdown mouse model of acute gouty arthritis was constructed, the joint pathological changes were observed by H&E staining, the synovium fibroblasts and neutrophils were sorted by flow cytometry, and the expressions of AIM2, Caspase-1 and GSDMD related proteins were detected by Western blot. The levels of TNF-α, IL-6, IL-1β and IL-18 in serum and cell supernatant were detected by ELISA. Neutrophils were induced to release NETs by urate, and NETs markers (dsDNA, MPO-DNA, NE-DNA) were detected by immunofluorescence (Cit-H3, PAD4) and ELISA. NETs media were co-cultured with synovial fibroblasts, cell activity and migration were evaluated by CCK8 and scrape assay, markers of synovitis (Thy1, VCAM-1, PDPN) were detected by immunofluorescence, and pyroptosis was evaluated by TUNEL and LDH release. By silencing or overexpression of AIM2 gene, Western blot and ELISA, the role of AIM2 in NETs induced pyrodeath and inflammatory response was investigated.
AIM2 gene knockdown significantly alleviated the symptoms of MSU-induced acute gouty arthritis in mice, reducing joint swelling and pathological damage. Expression levels of inflammatory factors (TNF-α, IL-6, IL-1β, IL-18) and cleaved Caspase-1/Caspase-1, GSDMD-NT/GSDMD) were decreased. It was found that neutrophils released NETs in response to sodium urate stimulation, manifested by significant upregulation of Cit-H3 and PAD4, as well as increased dsDNA, MPO-DNA, and NE-DNA complexes. NETs can induce inflammatory response in synovial fibroblasts, which is manifested as decreased cell activity and migration ability, increased release of inflammatory factors, and significantly increased markers of synovitis (Thy1, VCAM-1, PDPN). In addition, NETs induce scorch death of synovium fibroblasts by activating AIM2 inflammatories, which aggravates the inflammatory response, and AIM2 gene knockdown can effectively inhibit the scorch death and inflammatory response induced by NETs, indicating that NETs play a key role in the occurrence and development of gout arthritis through AIM2-mediated scorch death of synovium fibroblasts.
NETs-activated AIM2-mediated synovial fibroblast pyroptosis plays a crucial role in acute gouty arthritis, providing a new therapeutic target.
急性痛风性关节炎是一种以高尿酸血症为特征的代谢性疾病，急性发作时中性粒细胞释放的中性粒细胞胞外陷阱（NETs）通过其主要成分DNA作为危险相关分子模式（DAMPs）激活免疫反应。
探讨急性痛风性关节炎发作时NETs的DNA是否激活滑膜成纤维细胞中的AIM2炎性小体，诱导细胞焦亡并加剧炎症。
构建急性痛风性关节炎的AIM2基因敲低小鼠模型，通过苏木精-伊红（H&E）染色观察关节病理变化，采用流式细胞术分选滑膜成纤维细胞和中性粒细胞，通过蛋白质免疫印迹法检测AIM2、半胱天冬酶-1（Caspase-1）和Gasdermin D（GSDMD）相关蛋白的表达。采用酶联免疫吸附测定（ELISA）检测血清和细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的水平。用尿酸盐诱导中性粒细胞释放NETs，通过免疫荧光（瓜氨酸化组蛋白H3（Cit-H3）、肽基精氨酸脱亚氨酶4（PAD4））和ELISA检测NETs标志物（双链DNA（dsDNA）、髓过氧化物酶-DNA（MPO-DNA）、中性粒细胞弹性蛋白酶-DNA（NE-DNA））。将NETs培养基与滑膜成纤维细胞共培养，通过CCK8法和划痕试验评估细胞活性和迁移能力用免疫荧光检测滑膜炎标志物（Thy1、血管细胞黏附分子-1（VCAM-1）、血小板源性蛋白聚糖（PDPN）），通过末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）法和乳酸脱氢酶（LDH）释放评估细胞焦亡。通过沉默或过表达AIM2基因，采用蛋白质免疫印迹法和ELISA，研究AIM2在NETs诱导的细胞焦亡和炎症反应中的作用。
AIM2基因敲低显著减轻了小鼠由单钠尿酸盐（MSU）诱导的急性痛风性关节炎症状，减少了关节肿胀和病理损伤。炎症因子（TNF-αIL-6、IL-1β、IL-18）和裂解的Caspase-1/Caspase-1、GSDMD-NT/GSDMD的表达水平降低。发现中性粒细胞在尿酸盐刺激下释放NETs，表现为Cit-H3和PAD4显著上调，以及dsDNA、MPO-DNA和NE-DNA复合物增加。NETs可诱导滑膜成纤维细胞产生炎症反应，表现为细胞活性和迁移能力降低、炎症因子释放增加以及滑膜炎标志物（Thy1、VCAM-1、PDPN）显著增加。此外，NETs通过激活AIM2炎性小体诱导滑膜成纤维细胞焦亡，从而加剧炎症反应，而AIM2基因敲低可有效抑制NETs诱导的焦亡和炎症反应，表明NETs通过AIM2介导的滑膜成纤维细胞焦亡在痛风性关节炎的发生发展中起关键作用。
NETs激活的AIM2介导的滑膜成纤维细胞焦亡在急性痛风性关节炎中起关键作用，提供了一个新的治疗靶点。
