Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea.
Research Institute for Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea.
Theranostics. 2020 Feb 10;10(7):3138-3150. doi: 10.7150/thno.43441. eCollection 2020.
Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS), which is a chronic progressive disease and is caused by uncontrolled activation of myelin antigen specific T cells. It has high unmet medical needs due to the difficulty of efficient drug delivery into the CNS to control tissue inflammation. In this study, we demonstrate that a fusion protein of NOD-like receptor family member X1 (NLRX1) and blood brain barrier (BBB)-permeable peptide, dNP2 ameliorates experimental autoimmune encephalomyelitis (EAE). : We purified recombinant LRR or NBD regions of NLRX1 protein conjugated with dNP2. To examine intracellular delivery efficiency of the recombinant protein, we incubated the proteins with Jurkat T cells or murine splenic T cells and their delivery efficiency was analyzed by flow cytometry. To investigate the therapeutic efficacy in an EAE model, we injected the recombinant protein into mice with 3 different treatment schemes e.g., prevention, semi-therapeutic, and therapeutic. To analyze their functional roles in T cells, we treated MACS-sorted naïve CD4 T cells with the proteins during their activation and differentiation into Th1, Th17, and Treg cells. : dNP2-LRR protein treatment showed significantly higher delivery efficiency than TAT-LRR or LRR alone in Jurkat T cells and mouse splenic T cells. In all three treatment schemes of EAE experiments, dNP2-LRR administration showed ameliorated tissue inflammation and disease severity with reduced number of infiltrating T cells producing inflammatory cytokines such as IFNγ. In addition, dNP2-LRR inhibited T cell activation, cytokine production, and Th1 differentiation. : These results suggest that dNP2-LRR is a novel agent, which regulates effector T cell functions and could be a promising molecule for the treatment of CNS autoimmune diseases such as multiple sclerosis.
多发性硬化症（MS）是一种中枢神经系统（CNS）脱髓鞘炎症性疾病，是一种慢性进行性疾病，由髓鞘抗原特异性 T 细胞的失控激活引起。由于难以将有效药物递送至 CNS 以控制组织炎症，因此存在高度未满足的医疗需求。在这项研究中，我们证明了 NOD 样受体家族成员 X1（NLRX1）和血脑屏障（BBB）可渗透肽的融合蛋白 dNP2 可改善实验性自身免疫性脑脊髓炎（EAE）。我们纯化了与 dNP2 缀合的 NLRX1 蛋白的 LRR 或 NBD 区域的重组蛋白。为了检查重组蛋白的细胞内递药效率，我们用重组蛋白孵育 Jurkat T 细胞或鼠脾 T 细胞，并通过流式细胞术分析其递药效率。为了研究 EAE 模型中的治疗功效，我们用 3 种不同的治疗方案（预防、半治疗和治疗）将重组蛋白注射到患有 EAE 的小鼠中。为了分析它们在 T 细胞中的功能作用，我们在 T 细胞的激活和分化为 Th1、Th17 和 Treg 细胞期间用蛋白质处理 MACS 分选的幼稚 CD4 T 细胞。dNP2-LRR 蛋白处理在 Jurkat T 细胞和鼠脾 T 细胞中显示出比 TAT-LRR 或 LRR 更高的递药效率。在 EAE 实验的所有 3 种治疗方案中，dNP2-LRR 给药均显示出改善的组织炎症和疾病严重程度，减少了产生炎性细胞因子（如 IFNγ）的浸润 T 细胞数量。此外，dNP2-LRR 抑制 T 细胞激活、细胞因子产生和 Th1 分化。这些结果表明，dNP2-LRR 是一种新型调节剂，可调节效应 T 细胞功能，可能是治疗多发性硬化症等中枢神经系统自身免疫性疾病的有前途的分子。
