Department of Anesthesiology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China.
Department of Anesthesiology and Perioperative Medicine, Shanghai Fourth People's Hospital Affiliated to Tongji University, Shanghai, China.
Front Immunol. 2022 Jun 29;13:854202. doi: 10.3389/fimmu.2022.854202. eCollection 2022.
Damage-associated molecular patterns (DAMPs) are the primary promoter of progressive neuroinflammation and are associated with chronic stress-related emotional disorders. The present study investigated the role and mechanism of extracellular nucleosomes and histones, the newly defined DAMPs, in mice with chronic stress. C57BL/6 mice were exposed to chronic unpredictable mild stress (CUMS) and corticosterone drinking, respectively, for 4 weeks. Negative emotional behaviors were comprehensively investigated. Microglial morphology, oxidative stress, and inflammation, as well as C-type lectin receptor 2D (Clec2d) and Toll-like receptor 9 (TLR9) expression in medial prefrontal cortex (mPFC) were assessed with flow cytometer and cell sorting. Specifically, microglial pro-inflammatory activation and inflammation were further investigated with stereotactic injection of recombinant nucleosomes and histones in mPFC and further evaluated with AAV-Clec2d knocking-down, DNase I, and activated protein C (APC) pretreatment. Moreover, the rescue effect by AAV-Clec2d knocking-down was observed in mice with chronic stress. Mice with chronic stress were presented as obviously depressive- and anxiety-like behaviors and accompanied with significant microglial oxidative stress and inflammation, indicating by reactive oxygen species (ROS) production, primed nuclear factor-κB (NF-κB) signaling pathway, activated NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, and upregulated Clec2d and TLR9 in mPFC, together with histones dictation in cerebrospinal fluid and extracellular trap formation. Stereotactic injection of nucleosomes was contributed to promote microglial inflammation rather than histones in mPFC, indicating that the pro-inflammatory role was derived from extracellular histones-bound DNA but not freely histones. AAV-Clec2d knocking-down, DNase I, and APC were all effective to inhibit nucleosome-induced microglial oxidative stress and inflammation. Moreover, AAV-Clec2d knocking-down in mice with chronic stress exhibited reduced microglial inflammation and improved negative emotional behaviors. Our findings reveal a novel mechanism of DAMP-associated inflammation that extracellular nucleosomes accelerate microglial inflammation Clec2d and TLR9, and then contribute to chronic stress-induced emotional disorders.
损伤相关分子模式（DAMPs）是进行性神经炎症的主要启动子，与慢性应激相关的情绪障碍有关。本研究探讨了细胞外核小体和组蛋白（新定义的 DAMPs）在慢性应激小鼠中的作用和机制。C57BL/6 小鼠分别接受慢性不可预测轻度应激（CUMS）和皮质酮饮水处理，持续 4 周。综合评估负性情绪行为。使用流式细胞仪和细胞分选评估内侧前额叶皮质（mPFC）中小胶质细胞形态、氧化应激和炎症，以及 C 型凝集素受体 2D（Clec2d）和 Toll 样受体 9（TLR9）的表达。具体而言，使用立体定向注射重组核小体和组蛋白进一步研究小胶质细胞的促炎激活和炎症，并进一步用 AAV-Clec2d 敲低、DNase I 和活化蛋白 C（APC）预处理进行评估。此外，在慢性应激的小鼠中观察到 AAV-Clec2d 敲低的挽救作用。慢性应激的小鼠表现出明显的抑郁和焦虑样行为，并伴有明显的小胶质细胞氧化应激和炎症，表现为活性氧（ROS）产生、启动核因子-κB（NF-κB）信号通路、激活 NACHT、LRR 和富含 PYD 结构域蛋白 3（NLRP3）炎症小体，以及 mPFC 中 Clec2d 和 TLR9 的上调，以及脑脊液中组蛋白的规定和细胞外陷阱的形成。核小体的立体定向注射有助于促进 mPFC 中小胶质细胞的炎症，而不是组蛋白，表明促炎作用来自细胞外组蛋白结合的 DNA，而不是游离组蛋白。AAV-Clec2d 敲低、DNase I 和 APC 均能有效抑制核小体诱导的小胶质细胞氧化应激和炎症。此外，在慢性应激的小鼠中 AAV-Clec2d 敲低可减少小胶质细胞炎症并改善负性情绪行为。我们的研究结果揭示了 DAMPs 相关炎症的一种新机制，即细胞外核小体加速小胶质细胞炎症反应，激活 Clec2d 和 TLR9，进而导致慢性应激引起的情绪障碍。
