In vivo inflammatory challenge to elucidate the role of the toll-like receptor 4 pathway in depression

项目摘要 大约三分之一的严重抑郁障碍(MDD)患者表现出炎症，据信 在这种疾病中起着因果作用，但确切的机制尚不清楚。实验性内毒素血症 对健康个体的研究表明，行为、免疫和生理变化会导致 短暂的类抑郁状态，在注射脂多糖(LPS)后4-5小时消失。而当 这些研究不能揭示MDD中假定的、异常的炎症和调节机制。 因此，需要一种机械性的方法来确定哪些免疫调节机制存在缺陷。 MDD。这项提案将利用一项实验医学研究，该研究涉及急性注射脂多糖或 给MDD患者和健康对照组(HC)注射生理盐水以确定TLR4中与抑郁相关的变化 血液和大脑中的微核糖核酸(MiRNAs)及其调控途径。中环 假设TLR4信号在MDD中是通过暴露于外源性(例如病原体， 来自泄漏肠道的内毒素)和/或内源性(例如慢性应激)配体，因此，TLR4介导的炎症 与HC相比，MDD受试者的调节机制受损。TLR4检测病原体-和 产生炎性细胞因子的损伤相关分子模式(PAMP和DAMP)，如 白介素6(IL-6)和肿瘤坏死因子(TNF)在某些MDD患者中升高。同时， 与内毒素一样，PAMPS在MDD的发病机制中也发挥了作用，应激相关的湿气的产生也可能 激活TLR4。尽管TLR4途径在理论上很重要，但目前的证据基础很小， 现有的临床数据是横断面得出的，TLR4下游信号还没有被评估 尽管MDD中存在TLR4反应的miRNAs的异常表达。这些miRNAs可以从 细胞外小泡(EVS)穿过血脑屏障，因此，分离神经胶质来源的EVS将提供 MDD患者中枢神经系统相关TLR4调节的读数。我们的初步数据表明，炎症 细胞因子、IL-6和肿瘤坏死因子，以及TLR4在内毒素刺激下，MDD患者较HC患者升高。K99 阶段将识别外周和中枢TLR4介导的免疫信号机制在MDD中的使用 高灵敏度的免疫分析检测以确定细胞因子表达和TLR4途径的变化 并支持PI在流式细胞术、miRNA提取、星形细胞富集型EV(AEEV)方面的培训 分离，RNA-seq.R00阶段将确定外周和中枢TLR4介导的调节 利用下一代测序技术识别MDD中失调的miRNAs的MDD机制 并将为过渡到专门研究中枢神经系统依赖的TLR4的独立调查者提供支持- 抑郁症的炎症机制。识别TLR4介导的外周和中枢神经系统的能力 免疫调节反应与尖端技术，仍然是精神病学领域的新事物，将感动我们 离了解炎症相关抑郁症的机制又近了一步。

Project Summary Approximately 1/3 of individuals with major depressive disorder (MDD) display inflammation that is believed to play a causal role in the disorder, but the precise mechanisms are not understood. Experimental endotoxemia studies in healthy individuals have shown behavioral, immunological, and physiological changes leading to a transient depressive-like state that resolves 4-5 hours after administration of lipopolysaccharide (LPS). While informative, these studies cannot reveal putative, aberrant inflammatory and regulatory mechanisms in MDD. Hence, a mechanistic approach is required to pinpoint which immunoregulatory mechanisms are defective in MDD. This proposal will leverage an experimental medicine study involving acute administration of LPS or saline to individuals with MDD and healthy controls (HC) to identify depression-relevant changes in the TLR4 pathway and its regulation by microribonucleic acids (miRNAs) in both the blood and the brain. The central hypothesis is that TLR4 signaling is sensitized in MDD by a history of exposure to exogenous (e.g. pathogens, LPS from leaky gut) and/or endogenous (e.g. chronic stress) ligands, and thus, TLR4-mediated inflammatory and regulatory mechanisms are impaired in MDD subjects compared to HC. TLR4 detects pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) for production of inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor (TNF), which are elevated in some individuals with MDD. Whilst PAMPs, like LPS play a role in the pathogenesis of MDD, stress-associated production of DAMPs may also activate TLR4. Despite the theoretical importance of the TLR4 pathway, the evidence base is currently small, the extant clinical data are cross-sectionally derived, and TLR4 downstream signaling has not been assessed despite aberrant expression of TLR4-responsive miRNAs in MDD. These miRNAs can be isolated from extracellular vesicles (EVs) that cross the blood brain barrier, thus, isolating EVs of glial origin will provide a readout of CNS-associated TLR4 regulation in MDD. Our preliminary data suggest that the inflammatory cytokines, IL-6 and TNF, as well as TLR4 are increased in MDD compared to HC in response to LPS. The K99 phase will identify peripheral and central TLR4-mediated immune signaling mechanisms in MDD with the use of high-sensitivity, immunoassay detection to identify changes in cytokine expression and TLR4 pathway proteins and support the PI’s training in flow cytometry, miRNA extraction, astrocyte-enriched EV (AEEV) isolation, and RNA-seq. The R00 phase will identify peripheral and central TLR4-mediated regulatory mechanisms in MDD with the use of Next Generation Sequencing to identify miRNAs dysregulated in MDD and will provide support for transition to an independent investigator specializing in CNS-dependent TLR4- inflammatory mechanisms in depression. The ability to delineate TLR4-mediated peripheral and CNS immunoregulatory responses with cutting edge techniques, still novel to the field of psychiatry, will move us one step closer to understanding the mechanisms underlying inflammation-associated depression.