负载caspase-1抑制剂的树突状细胞外泌体对实验性自身免疫性重症肌无力大鼠的靶向治疗作用及机制研究

Caspase-1 activation is key factors of the production of interleukin (IL)-1beta, and plays an important role in the development of autoimmune and inflammatory disease. We previously demonstrated that caspase-1 inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response via suppressing DC IL-1 β, CD4+ T and γδT cells IL-17 pathways, and regulates humoral responses via IL-6-dependent inhibiton of STAT3. However, long-term administration of caspase-1 inhibitor is associated with dose-dependent innocent bystander or off-target side effects, and the treatment has no antigen specificity. As a result, a targeted caspase-1 inhibitor delivery is urgently needed to address this severe toxicity. Here the targeting exosomes are generated by engineering dendritic cells to express Lamp2b fused with AChR alpha subunit, and exosomes used to encapsulate caspase-1 inhibitor are delivered to dendritic cells in EAMG rats. Then the therapeutic potential and mechanisms of targeting exosomes on EAMG rats are further explored. Furthermore, the interaction between exogenous EXOs and endogenous DCs derived from different immune organs is detected in vivo and in vitro. We attempt to explore the mechanisms of induction of immune tolerance and provide a novel theoretical basis for treatment of EAMG and even autoimmune diseases.

Caspase-1的激活是白介素（IL）-1β产生的关键因素，在自身免疫及炎症疾病的发生发展中起重要作用。我们前期的研究发现caspase-1抑制剂可以通过抑制树突状细胞（DC）-IL-1-IL-17和IL-6-STAT3-Bcl-6途径，从而抑制实验性自身免疫性重症肌无力（EAMG）大鼠疾病进展。但是长期大量应用caspase-1抑制剂造成免疫抑制剂的组织毒性作用，而且其对EAMG的治疗不具有抗原特异性。基于此，本课题利用AchR亚单位胞外段-Lamp2b的融合蛋白质粒转染大鼠DCs，提取外泌体（exosome）并负载caspase-1抑制剂，完成对EAMG大鼠AchR反应性DCs的靶向投递，评价其对EAMG大鼠的治疗作用，从细胞和体液免疫方面深入探讨其作用机制，并阐明外泌体与内源性不同免疫器官中DCs的相互作用及诱导免疫耐受机制，为重症肌无力甚至其他免疫性疾病治疗提供新的思路和理论依据

Caspase-1 是治疗重症肌无力的重要药物靶点，我们前期的研究发现caspase-1抑制剂可以通过抑制树突状细胞（DC）-IL-1 -IL-17和IL-6-STAT3-Bcl-6途径，从而抑制实验性自身免疫性重症肌无力（EAMG）大鼠疾病进展，但是长期大剂量的 caspase-1 抑制剂应用产生严重的组织毒性作用。外泌体（exosome）作为新型药物载体，具有免疫原性低、在血液中的稳定性高、向细胞运输药物的效率高等优势，在自身免疫疾病治疗中起到重要作用。我们成功提取大鼠骨髓树突状细胞（BMDC）外泌体，通过电镜、纳米颗粒追踪分析、zeta电位检测进行鉴定；反复进行优化方法及重复验证，保证了外泌体载药效率及稳定性，获得exo-VX-765备用并质谱分析外泌体载药浓度；体外验证exo-VX-765对BMDCs表面分子及IL-1β影响，进一步证实exo-VX-765能够减少骨髓巨噬细胞 IL-1β水平，明显降低CD4+IL-17+ T细胞、CD4+IFN-γ+ T细胞比例，体外探索exo-VX765治疗EAMG作用及相关机制，为重症肌无力甚至其他免疫性疾病治疗提供新的思路和理论依据。