Tissue repair, extracellular vesicular biogenesis, and the control of immune responses

摘要 皮肤伤口愈合中的解决方案是通过细胞间通讯过程协调的， 包括含有生物活性蛋白质和核酸的胞外小泡(EV)。而当 EV的生物发生已经在体外进行了研究，系统地确定了它们在体内的相关性 是有限的。已经确定TBC/RabGAPs的表达控制EV的生物发生和 RE-Program的EV内容为了在炎症中解偶联EV的促修复活性，我们 建议使用遗传方法来针对EV生物发生所涉及的分泌机制 (即RAB 5、11和27A)，以确定TBC/RabGAP的这些目标如何调节有效载荷 电动汽车。我们将确定EVS以及EVS的变化如何影响炎症的消退， 并深入了解电动汽车在伤口愈合中的作用。在目标1中，我们将使用囊泡流 用细胞术确定EV亚群，然后过继转移EVS以确定其容量 激活糖尿病和免疫缺陷基因敲除小鼠的特异性免疫细胞群 调解皮肤伤口闭合的模型。目标2将研究特定的RAB以确定如何 这些RAB影响EV的生物发生和内容。此外，利用改装后的电动汽车 RAB环境下，我们将建立这些电动汽车对 特异性免疫反应的分解动力学。总之，这些研究将确立 电动汽车作为潜在疗法的相关性，并解决系统地 创造专门针对皮肤修复的特定阶段进行优化的治疗性电动汽车。

SUMMARY Resolution in skin wound healing is coordinated by intercellular communication processes that include extracellular vesicles (EVs) containing biologically active protein and nucleic acid. While EV biogenesis has been studied in vitro, systematic approaches to identify their in vivo relevance are limited. Having established that the expression of TBC/RabGAPs controls EV biogenesis and ‘re-programs’ EV content to uncouple the pro-reparative activities of EVs in inflammation, we propose to use a genetic approach to target the secretory machinery implicated in EV biogenesis (i.e. Rabs 5, 11, and 27A) to determine how these targets of TBC/RabGAPs regulate the payload of EVs. We will determine how EVs, and changes in EVs, impact the resolution of inflammation, and gain insight into the role of EVs in wound healing. In Aim 1, we will use vesicle flow cytometry to define EV subsets, followed by adoptive transfer of EVs to determine their capacity to activate specific immune cell populations in diabetic and immunodeficient knockout mouse models that mediate cutaneous wound closure. Aim 2 will study specific Rabs to determine how these Rabs impact EV biogenesis and content. In addition, using the EVs formed in the altered Rab environment, we will establish the mechanism of action and effect of these EVs on the kinetics of resolution of specific immune responses. Together, these studies will establish the relevance of EVs as potential therapeutics and address the over-arching goal of systematically creating therapeutic EVs that are specifically optimized for specific phases of skin repair.