树突细胞源性exosomes对实验性自身免疫性脑脊髓炎小鼠免疫耐受诱导的潜能研究

Our previous studies showed that atorvastatin-induced immature bone marrow dendritic cells (BMDCs) were able to secrete tolerogenic exosomes (EXOs). These tolerogenic EXOs could induce immune tolerance in experimental autoimmune myasthenia gravis (EAMG). The therapeutic effects of tolerogenic EXOs on EAMG rats were associated with up-regulated expression of indoleamine 2,3-dioxygenase (IDO) on these tolerogenic EXOs. Up to now, the exact mechanisms of EXOs-derived atorvastatin-modified BMDCs (s-EXOs) on experimental autoimmune encephalomyelitis (EAE) mice are not entirely clear. In present study, EXOs are purified from the culture supernatants of BMDCs after cultured with atorvastatin or DMSO respectively. Then the mechanisms of s-EXOs on EAE mice are further explored, in which IDO/Treg are considered as key markers. Meanwhile, the interaction between exogenous EXOs and endogenous DCs derived from different immune organs is detected in vivo and in vitro. Furthermore, the mechanisms of induction of Treg are explored in vitro. In conclusions, our study focuses on s-EXOs, which provides a novel cell-free approach for treatment of MS and even autoimmune diseases.

我们前期研究发现，抗原特异性髓源树突状细胞（BMDCs）在体外经阿托伐他汀修饰后能够分泌耐受性exosomes（EXOs），这些耐受性EXOs可以诱导实验性自身免疫性重症肌无力大鼠的免疫耐受，其作用机制与EXOs上高表达的吲哚胺2,3-双加氧酶（IDO）密切相关。迄今为止，阿托伐他汀修饰的小鼠BMDCs源性EXOs（s-EXOs）在实验性自身免疫性脑脊髓炎（EAE）小鼠免疫耐受中的作用机理未见相关报道。本课题通过诱导耐受性s-EXOs的产生，评价s-EXOs是否能够诱导EAE的免疫耐受，并围绕IDO、Treg为中心深入探讨其作用机制；通过体内外实验阐明外源性EXOs与内源性不同免疫器官中DCs的相互作用机制；体外进一步探讨不同免疫器官源性DCs与EXOs共同诱导Treg产生的作用机理。本课题专注于s-EXOs这一新视角，有望为治疗多发性硬化（MS）甚至其他免疫性疾病提供新的思路和方法。

树突状细胞（DC）具有重要的抗原提呈作用，在自身免疫性疾病中发挥着重要的免疫调控作用。在实际应用中DC不易保存，而其来源的外泌体（exosome）可以携带DC的细胞成份，具有与DC相似的调控作用，且具有易保存性。我们在体外通过他汀类药物修饰小鼠髓源树突状细胞（BMDC），成功提取了其分泌的exosome（statin-mDex），通过不同技术对提取的exosome进行了鉴定，进一步研究了statin-mDex对实验性自身免疫性脑脊髓炎（EAE）小鼠的外周和中枢免疫调节机制。发现statin-mDex治疗后可以改善EAE小鼠的临床症状，这种作用与其抑制中枢神经系统中炎症细胞的浸润密切相关。实验进一步验证了statin-mDex可以分布到EAE小鼠的脾脏、淋巴结和脑组织中发挥调控作用。在体外，statin-mDex可以抑制脾脏单个核细胞CD80、CD86和MHC-II分子的表达、上调脾脏CD4⁺T细胞中CD25+Foxp3+T细胞的水平、抑制脾脏单个核细胞中NK细胞的水平。重要的是我们发现，mDex可以被中枢的小胶质细胞吞噬，通过调控小胶质细胞的功能进一步发挥其对EAE的免疫调控作用。通过本课题的研究，阐明了statin-mDex诱导EAE中枢免疫耐受的作用机制，为药物干预DC源性exosome治疗EAE甚至MS提供了新的思路和理论依据。