软骨细胞来源外泌体靶向ATG4B激活巨噬细胞炎症小体的机制及在骨关节炎致病中的作用

The activated synovial macrophages are closely involved in the progress of osteoarthritis, but the mechanisms about the activation of synovial macrophages are still not clear. Our previous study found that chondrocytes can produce exosomes, and these exosomes could mediate the crosstalk between chondrocytes and synovial macrophages when the cartilage is injured. The chondrocytes-derived exosomes(ChC-Exo) significantly inhibits autophagy, but increase the IL-1β release and cellular mitoROS level in LPS-primed macrophages. The data from the experiments including autophagy cDNA array and exosomes RNA-seq reveal that the miR-449a-5p in chondrocytes-derived exosomes may regulate the expression of autophagy-related gene ATG4B，which may be involved in ChC-Exo-mediated autophagy inhibition of macrophages. Combined with literature report and our previous results, we propose that ChC-Exo could be a model of damage related molecule pattern that identified by synovial macrophages. The ChC-Exo could inhibits autophagy via ATG4B, activate inflammasomes and promote the release of IL-1β. In this study, we will investigate mechanisms of ChC-Exo-regulated ATG4B expression and inflammasomes activation in macrophages and reveal the role and significance of this signal pathway in the pathogenesis of OA , which may provide some novel clues for OA control in clinic.

活化的滑膜巨噬细胞与骨关节炎(OA)致病密切相关，但其激活机制不十分清楚。我们前期发现随着软骨基质丢失和软骨磨损，软骨细胞来源外泌体(ChC-Exo)可进入滑膜组织，促进致敏巨噬细胞IL-1β的释放，并抑制细胞自噬反应及升高mitoROS水平。ChC-Exo诱导的IL-1β释放可被mitoROS清除剂和NLRP3抑制剂所拮抗。自噬转录组芯片及外泌体RNA-seq等实验结果提示巨噬细胞自噬抑制可能与miR-449a-5p介导的自噬基因ATG4B表达下调有关。结合文献报道和前期实验结果，我们提出“ChC-Exo可作为滑膜巨噬细胞所识别的损伤相关分子模式，通过靶向ATG4B抑制巨噬细胞自噬反应，激活NLRP3炎症小体，促进IL-1β释放”这一假说。本项目拟通过体内和体外实验，阐明ChC-Exo调控巨噬细胞ATG4B表达及炎症小体激活的机制，揭示该通路在OA致病中的作用，为OA防治提供新的思路。

骨性关节炎（Osteoarthritis, OA）滑膜组织中主要的炎症浸润细胞是单核/巨噬细胞。活化的滑膜巨噬细胞及其产生的炎症因子IL-1β与骨关节炎的发生发展密切相关，但相关机制不十分清楚。在本项目资助下，我们发现：（1）分离得到OA患者来源ChC-EXOs具有典型的外泌体特征，包括双层膜结构、直径范围30~150nm及表达CD63等外泌体标志物；（2）与ChCNCpre-EXOs相比，ChCILpre-EXOs显著促进了细胞炎症小体的活化和产生IL-1β；（3）ChCILpre-EXOs抑制了LPS致敏巨噬细胞的自噬水平，阻断自噬后，ChCILpre-EXOs促炎症小体活化的作用明显减弱；（4）ChCILpre-EXOs显著下调了巨噬细胞中自噬关键分子ATG4B的表达；（5）Dio标记的ChC-EXOs与巨噬细胞共孵育后，可大量进入巨噬细胞，主要分布与细胞质部位，而关节腔注射的Dio-labeled ChC-EXOs明显富集于关节滑膜组织；（6）IL-1β预处理导致了软骨细胞及其外泌体中miR-449a-5p的显著升高，ChCILpre-EXOs对其靶细胞中ATG4B 3’UTR的抑制作用更为明显，其介导的ATG4B的mRNA和蛋白水平下降更为明显;（7）与ChCNCpre-EXOs相比，ChCILpre-EXOs介导了更明显的小鼠DMM术后关节滑膜炎症反应及软骨损伤；（8）与antagomir-NC相比，关节腔注射antagomir-449a-5p部分逆转了ChCILpre-EXOs介导的DMM小鼠软骨损伤。本研究提出了“在OA病程中，ChC-Exos可作为一种DAMPs激活滑膜巨噬细胞”的观点，为OA滑膜炎症的形成机制提供了新的理论依据；同时，揭示了自噬关键蛋白ATG4B通过自噬继而影响巨噬细胞炎症小体激活这一新的生物学效应，拓展了对ATG4B生物学功能的认知。