髓源性抑制细胞来源的exosomes抑制小鼠胶原诱导性关节炎的实验研究

Myeloid-derived suppressor cells (MDSCs) are a phenotypically heterogeneous cell population that includes myeloid progenitor cells and immature myeloid cells. MDSCs are able to suppress T cells responses and play an essential role in immunosuppression. MDSCs accumulated in autoimmune diseases, but the roles of MDSCs in autoimmune diseases remain controversial. We found that MDSCs could suppress the onset of murine collagen-induced arthritis (CIA), whereas these cells couldn't reduce severity of established arthritis. Exosomes are small membrane-bound vesicles (30-100nm) that are secreted in extracellular medium by hemopoietic and nonhemopoietic cells. Exosomes deliver bioactive molecules including proteins and RNAs to the target cells by interacting with cell surface receptors or fusing with plasma membrane, they are unable to change their phenotypes following purification and are very stable. In the preliminary experiment, we found that G-MDSCs-derived exosomes (G-MDSCs-Ex) were able suppress the onset of CIA as well as reduce severity of established arthritis. In vitro, G-MDSCs-Ex inhibited the proliferation of CD4+T cells and their differentiation into Th17 cells, but they contribute to the generation of Treg cells. In this study, we will clarify that different subsets and sources of MDSCs-Ex play roles in murine CIA model. The functionally active molecules (including miRNA, mRNA and proteins) of MDSCs will be identified. We will examine the immunosuppressive effects of exosomes from MDSCs modified to overexpress functionally active molecules in murine CIA model. MDSCs-derived exosomes may represent a novel, cell-free therapy for the treatment of collagen-induced arthritis.

髓源性抑制细胞(MDSCs)是重要的免疫抑制细胞，但其在自身免疫性疾病中的作用存在争议。我们发现MDSCs在小鼠胶原诱导性关节炎（CIA）早期具有保护作用，而在疾病高峰期治疗效果不明显。Exosomes是细胞主动分泌到胞外的一种膜性囊泡，可以调控受体细胞发挥重要的信息传递作用，且性质稳定。我们发现来源于粒系MDSCs的exosomes（G-MDSCs-Ex）可有效治疗不同时期CIA，G-MDSCs-Ex在体外可抑制T细胞增殖，促进Treg细胞分化，抑制Th17细胞分化。由于MDSCs存在异质性，我们将研究不同来源、不同亚群MDSCs-Ex对CIA的治疗效果，选择效果最好的MDSCs-Ex；筛选鉴定MDSCs-Ex中功能性miRNA、mRNA和蛋白分子，验证其功能，并研究其作用机制；利用基因工程技术制备过量表达功能性分子的MDSCs-Ex，观察是否具有更好的疗效。本研究将为CIA提供新疗法。

类风湿关节炎(RA) 是一种慢性致残性疾病，目前仍缺乏有效治疗手段，需要寻找新的、更为有效的治疗方法。髓源性抑制细胞(MDSCs)是一群具有免疫抑制功能的未成熟髓系细胞，可分为G-MDSC和M-MDSC两个主要亚群，文献报道MDSCs 在自身免疫性疾病中的治疗作用并不明确，极易受到组织微环境的影响。Exosomes 是细胞分泌的膜性囊泡，可介导细胞间信息交流，性质稳定，具有较好的应用前景。我们制备不同亚群MDSC来源的exosomes，并进行性质鉴定。将MDSC不同亚群来源exosomes注射胶原诱导性关节炎（CIA）模型小鼠体内，结果发现G-MDSC来源exosomes可明显减缓CIA小鼠病程，减轻炎症反应，而M-MDSC来源exosomes并不具有这种作用；G-MDSC来源exosomes处理后，可明显降低小鼠体内Th1和Th17细胞比例。在体外，G-MDSC来源exosomes可抑制CD4+T 细胞增殖，以及Th1和Th17细胞的分化。通过芯片筛选和后续验证，发现G-MDSC来源exosomes包含多种microRNA，其中miR-93-5p可抑制 Th17细胞分化，miR-29a-3p可以抑制Th1细胞的分化。将携带miR-93-5p或（和）miR-29a-3p 外源性mimics的exosomes处理CIA小鼠，结果显示单独携带miR-93-5p或miR-29a-3p mimics的exosomes具有明显的抑制作用，携带miR-93-5p和miR-29a-3p mimics的exosomes的作用最为显著。将miR-29a-3p和miR-93-5p的mimics转染到人MDSC ，然后制备exosomes，体外结果显示携带miR-29a-sp和miR-93-5p的人MDSC-exosomes可以抑制人Th1和Th17细胞的分化。我们的研究还发现，G-MDSC来源exosomes处理CIA小鼠后，其体内Breg细胞比例升高升高，体外G-MDSC来源exosomes促进Breg细胞比例出现升高，并可能与促进细胞中GSK-3β、CREB磷酸化水平有关。此外，我们还发现G-MDSC来源exosomes可抑制小鼠炎性肠病（IBD），其作用与exosomes携带的Arg-1抑制Th1细胞有关。我们的研究结果为自身免疫性疾病的治疗提供了新的方法。