Department of Anatomy and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea.
Cell Commun Signal. 2024 Feb 2;22(1):96. doi: 10.1186/s12964-023-01447-z.
Neutrophil extracellular trap (NET) has been implicated in the pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the specific contributions of NLRP3, a NET-associated molecule, to EAE pathogenesis and its regulatory role in NET formation remain unknown.
To investigate the detrimental effect of NETs supported by NLRP3 in MS pathogenesis, we induced EAE in WT and NLRP3 KO mice and monitored the disease severity. At the peak of the disease, NET formation was assessed by flow cytometry, immunoblotting, and immunofluorescence staining. To further identify the propensity of infiltrated neutrophils, NET-related chemokine receptors, degranulation, ROS production, and PAD4 expression levels were evaluated by flow cytometry. In some experiments, mice were injected with DNase-1 to eliminate the formed NETs.
Our data revealed that neutrophils significantly infiltrate the brain and spinal cord and form NETs during EAE pathogenesis. NLRP3 significantly elevates NET formation, primarily in the brain. NLRP3 also modulated the phenotypes of brain-infiltrated and circulating neutrophils, augmenting CXCR2 and CXCR4 expression, thereby potentially enhancing NET formation. NLRP3 facilitates NET formation in a ROS-dependent and PAD4-independent manner in brain-infiltrated neutrophils. Finally, NLRP3-supported NET formation exacerbates disease severity, triggering Th1 and Th17 cells recruitment.
Collectively, our findings suggest that NLRP3-supported NETs may be an etiological factor in EAE pathogenesis, primarily in the brain. This study provides evidence that targeting NLRP3 could be a potential therapeutic strategy for MS, specifically by attenuating NET formation.
中性粒细胞胞外诱捕网（NET）已被牵涉到多种多发性硬化症（MS）和实验性自身免疫性脑脊髓炎（EAE）的病理学中。然而，NET 相关分子 NLRP3 对 EAE 发病机制的具体贡献及其对 NET 形成的调控作用尚不清楚。
为了研究 NET 支持的 NLRP3 在 MS 发病机制中的有害作用，我们在 WT 和 NLRP3 KO 小鼠中诱导 EAE，并监测疾病严重程度。在疾病高峰期，通过流式细胞术、免疫印迹和免疫荧光染色评估 NET 形成。为了进一步确定浸润中性粒细胞的倾向，通过流式细胞术评估 NET 相关趋化因子受体、脱颗粒、ROS 产生和 PAD4 表达水平。在一些实验中，用 DNase-1 注射小鼠以消除形成的 NET。
我们的数据显示，中性粒细胞在 EAE 发病过程中显著浸润大脑和脊髓并形成 NET。NLRP3 显著增加 NET 的形成，主要在大脑中。NLRP3 还调节了脑浸润和循环中性粒细胞的表型，增加了 CXCR2 和 CXCR4 的表达，从而可能增强 NET 的形成。NLRP3 以 ROS 依赖和 PAD4 独立的方式促进脑浸润中性粒细胞中的 NET 形成。最后，NLRP3 支持的 NET 形成加重了疾病严重程度，引发 Th1 和 Th17 细胞募集。
总之，我们的研究结果表明，NLRP3 支持的 NET 可能是 EAE 发病机制中的一个病因因素，主要在大脑中。本研究提供的证据表明，靶向 NLRP3 可能是 MS 的一种潜在治疗策略，特别是通过减轻 NET 的形成。
