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

总结 皮肤伤口愈合的解决是由细胞间通讯过程协调的， 包括含有生物活性蛋白质和核酸细胞外囊泡（EV）。而 EV生物发生已经在体外进行了研究，系统的方法来确定它们在体内的相关性 是有限的。已经确定TBC/RabGAP的表达控制EV生物发生， “重新编程”EV内容以解除EV在炎症中的促修复活性， 我建议使用遗传学方法来靶向与EV生物发生有关的分泌机制 (i.e. Rabs 5、11和27 A）以确定TBC/RabGAP的这些靶标如何调节有效载荷 的EV。我们将确定EV以及EV的变化如何影响炎症的消退， 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 细胞术以确定EV亚群，然后过继转移EV以确定其能力 在糖尿病和免疫缺陷敲除小鼠中激活特异性免疫细胞群 介导皮肤伤口闭合的模型。目标2将研究特定的Rabs，以确定如何 这些Rabs影响EV生物发生和含量。此外，使用在改变的环境中形成的EV， 拉布环境，我们将建立这些电动汽车的作用机制和影响， 特异性免疫应答的消退动力学。总之，这些研究将建立 EV作为潜在治疗剂的相关性，并解决系统性 创造治疗性EV，其针对皮肤修复的特定阶段进行了特别优化。

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.