Small extracellular vesicles mediated signaling and pain

摘要 慢性疼痛是美国最普遍、最致残、最昂贵的公共卫生状况 各州。这个项目的目标是阐明如何利用人体自身的止痛机制 来提供止痛药。我们建议研究30-150 nm的细胞外小泡(SEV) 通过循环将mRNAs、miRNAs、蛋白质和脂质介体输送到受体细胞。 SEV的摄取诱导受体细胞中基因表达的变化，因此，SEV发挥着 在细胞间通讯中发挥重要作用。我们观察到RAW 264.7的SEV 巨噬细胞在完全弗洛伊德佐剂(CFA)中显示治疗和预防效果 小鼠炎症性疼痛模型。我们的初步研究表明，小鼠血清来源的SEV 在幼稚的受体小鼠鞘内注射时，也提供了预防，两周 随后，接受后爪注射CFA。因此，接受SEV的小鼠可以记住这一点 刺激至少2周，对CFA的反应减弱。这种长期的 内存开发未知。尽管慢性疼痛很普遍，但免疫策略并没有 我们的研究将提供这样一个理论和机制基础 策略。在这里，我们建议检验单核/巨噬细胞来源的SEV亚群的假设 对于减轻炎性疼痛、过敏性反应和 预防措施。我们还将调查单核/巨噬细胞SEV是否招募或促进抗- 大鼠背根神经节和脊髓免疫细胞炎性表型转换 CFA治疗前后原位定量免疫表型分析。最近的研究表明 小胶质细胞，即驻留在中枢神经系统的巨噬细胞，可以增强或抑制 通过表观遗传修饰对延迟的二次侮辱的反应。我们假设 单核/巨噬细胞来源的SEV在小鼠脊髓小胶质细胞中传递表观遗传免疫记忆 受体小鼠，赋予其减轻未来侮辱带来的疼痛的能力，并有助于 SEV的预防作用。提出的研究将阐明SEV在免疫中的作用 调节和记忆。

Abstract Chronic pain is the most prevalent, disabling, and expensive public health condition in the United States. The goal of this project is to elucidate how to harness body’s own analgesic mechanisms to provide pain relief. We propose to investigate 30-150 nm small extracellular vesicles (sEVs) that transport mRNAs, miRNAs, proteins, and lipid mediators to recipient cells via circulation. Uptake of sEVs induce gene expression changes in recipient cells and thus, sEVs play an important role in intercellular communication. We observed that sEVs from RAW 264.7 macrophage cells show therapeutic and prophylactic efficacy in a complete Freud adjuvant (CFA) mouse model of inflammatory pain. Our preliminary studies show that mouse serum derived sEVs also conferred prophylaxis when injected intrathecally in naïve recipient mice that, two weeks later, received a hind paw injection of CFA. Thus, mice that received sEVs can remember this stimulation for at least 2 weeks and show an attenuated response to CFA. How this long-term memory develop is unknown. Though chronic pain is prevalent, an immunization strategy has not yet been tested and our studies will provide the rationale and mechanistic basis for such a strategy. Here we propose to test the hypothesis that monocyte/macrophage-derived sEV subsets in serum are necessary and sufficient to attenuate inflammatory pain hypersensitivity and confer prophylaxis. We will also investigate if monocyte/macrophage sEVs recruit, or promote anti- inflammatory phenotype switching of immune cells in dorsal root ganglion and spinal cord by quantitative immunophenotyping in situ, before and after CFA treatment. Recent studies show that microglia, the resident macrophages of the central nervous system can enhance or suppress responses to a delayed secondary insult through epigenetic modifications. We hypothesize that monocyte/macrophage-derived sEVs impart epigenetic immune memory in spinal microglia of recipient mice, granting the capacity to attenuate pain from a future insult and contribute to the prophylactic effect of sEVs. The studies proposed will elucidate the role of sEVs in immune regulation and memory.