Defining the mechanisms of MSC extracellular vesicle modulation of microglia metabolism and bioenergetics in traumatic brain injury recovery

项目总结 神经炎症在创伤性脑损伤(TBI)的发生和发展中起关键作用； 然而，大多数治疗方法无法解决多方面的问题。颅脑损伤后小胶质细胞 被激活并产生炎性细胞因子(包括肿瘤坏死因子-α和白介素6)，从而损害血液 脑屏障(BBB)，紧密连接，并导致周围免疫细胞的渗透，如 中性粒细胞、单核细胞和T细胞。识别靶向的新免疫调节策略 小胶质细胞和促进神经保护环境是治疗这种毁灭性疾病的关键。 间充质基质细胞(MSCs)是一种很有前途的再生医学治疗方法 其免疫调节功能是由分泌的胞外小泡(MSC-Evs)介导的 具有独特的表面成分和泡囊内的货物。我们的团队已经展示了 MSC-EVS调节小胶质细胞、T细胞和小胶质细胞等神经炎症细胞类型的能力 周细胞。MSC-EVS是治疗脑外伤的一种很有前途的疗法，因为它们可以 对亲本MSCs的免疫调节功能，ii)跨越血脑屏障，以及iii)解决安全问题 与MSC交付相关(即肿瘤形成和血栓形成)。此外，目标定位能力 (由表面信号介导)和MSC-EV货物可以通过启动(预处理)进行工程设计 具有不同微环境信号的间充质干细胞，如细胞因子、干扰素-γ和肿瘤 肿瘤坏死因子α。然而，对于MSC-EV的作用和机制，人们的认识还存在差距 小胶质细胞在脑损伤中的调节，以及这种作用是否可以通过启动来增强。 这项拟议的工作试图阐明MSC-EVS调节小胶质细胞的机制。 特别关注MSC-EV线粒体转移。我们的中心假设是MSC-EVS产生了 从细胞因子启动的MSCs将通过调节小胶质细胞向 更多的神经保护表型(例如，炎性细胞因子和反应性细胞因子的产生减少 氧物种)，这一作用是通过MSC-EV来源的线粒体在体外和 在活体内。我们将从以下几个方面验证这一假说：1)确定线粒体的机制 MSC-EV转导对小胶质细胞代谢的影响；2)评价MSC-EV对小胶质细胞的治疗效果 猪脑外伤模型。拟议工作的成功完成将创造一种新颖的、可调整的方法 通过更好地了解MSC-EV，靶向大脑的免疫系统和治疗脑外伤 行动机制。

PROJECT SUMMARY Neuroinflammation plays a critical role in both the onset and progression of traumatic brain injury (TBI); however, most therapies are unable to address the multifaceted aspects. Following TBI, microglia become activated and produce inflammatory cytokines (including TNF-α and IL-6) that damage blood brain barrier (BBB), tight junctions, and lead to infiltration of peripheral immune cells such as neutrophils, monocytes, and T cells. Identification of new immunomodulatory strategies that target microglia and promote a neuroprotective environment is critical for treating this devastating disease. Mesenchymal stromal cells (MSCs) are a promising therapy for regenerative medicine applications due to their immunomodulatory function, which is mediated by secreted extracellular vesicles (MSC-EVs) that possess distinct surface composition and intravesicular cargo. Our group has demonstrated the ability of MSC-EVs to modulate cell-types involved in neuroinflammation such as microglia, T cells and pericytes. MSC-EVs are a promising therapeutic for TBI because they can i) have comparable immunomodulatory function to parent MSCs, ii) cross the BBB, and iii) address safety concerns associated with MSC delivery (i.e. tumorigenesis and thrombosis). Further, the targeting capabilities (mediated by surface signals) and MSC-EV cargo can be engineered through priming (preconditioning) of MSCs with different microenvironmental cues such as cytokines Interferon-gamma and Tumor necrosis factor alpha. However, there is a gap in knowledge over the role and mechanisms of MSC-EV modulation of microglia in the context of TBI and whether this effect can be enhanced through priming. The proposed work seeks to elucidate the mechanisms by which MSC-EVs modulate microglia with a specific focus on MSC-EV mitochondrial transfer. Our central hypothesis is that MSC-EVs produced from cytokine-primed MSCs will have greater functionality through modulation of microglia towards a more neuroprotective phenotype (e.g. reduced production of inflammatory cytokines and reactive oxygen species) and that this effect is mediated by MSC-EV derived mitochondrial transfer in vitro and in vivo. We will test this hypothesis in the following aims: 1) Define the mechanisms of mitochondrial transfer from MSC-EV on microglia metabolism, and 2) Assess MSC-EV therapeutic efficacy in a porcine TBI model. Successful completion of the proposed work will create a novel, tunable approach for targeting the brain’s immune system and treating TBI through a better understanding of MSC-EV mechanisms of action.