Lead compound discovery from proprietary mycobacterial strains for treatment of PTSD

项目摘要 免疫调节，即调节性和效应性T细胞的平衡表达，被认为在 现代高收入环境，部分原因是与共生和环境衍生的接触减少 细菌，也被称为“老朋友”。失败的免疫调节被认为是导致最近 与应激相关的慢性炎症性疾病以及与应激相关的精神障碍在 炎症是危险因素，如抑郁、焦虑和创伤后应激障碍(PTSD)，在 高收入国家。其中一位老朋友是母牛分枝杆菌11659，它是一种非致病性、 具有抗炎和免疫调节作用的环境腐生植物。以下列形式进行的免疫 一种热灭活的母牛分枝杆菌制剂已被证明可以提高小鼠的应激恢复能力，通过测量 防止应激引起的焦虑增加，防止应激引起的自发性夸张 结肠炎，以及预防炎症模型中化学诱导的结肠炎的应激诱导的夸大 肠病。在恐惧条件作用之前或之后进行的母牛分枝杆菌免疫接种，也 在一种恐惧增强的惊吓大鼠模型中，被证明可以增强恐惧的消退。在其他研究中，免疫接种 研究表明，使用母牛分枝杆菌可以防止压力引起的焦虑样防御行为的夸大 与应激诱导的小胶质细胞抑制相关的获得性无助大鼠模型的反应 引爆和神经炎症。然而，这些压力弹性和压力保护的程度 对其他分枝杆菌菌株的影响尚不清楚。在这项建议中，我们建议 用小鼠骨髓来源的树突状细胞(BMDCs)、人单核细胞来源的巨噬细胞进行筛选 (THP-1细胞)和一株小胶质细胞(BV2细胞)，20株环境分枝杆菌专利菌株 用于抗炎和免疫调节特性。在筛选方面，我们会针对符合 以下标准：它们可以很容易地分离培养；它们与已知的分枝杆菌没有密切联系 病原体；它们代表着跨越已知分枝杆菌系统发育广度的不同谱系 多样性(其中有150株已描述的分枝杆菌菌株)。然后我们将筛选两名领先的候选人， 利用雄性和雌性大鼠，鉴定具有应激保护作用的新的分枝杆菌菌株，如 通过防止不可避免的压力(IS)导致的焦虑、条件性恐惧和逃避的增加来衡量 应激诱导的海马区小胶质细胞启动和神经炎症的缺陷和预防。我们会 在雄性和雌性大鼠的恐惧强化惊吓和暗示恐惧的模型中，也评估这些菌株。在 完成这些研究后，我们预计我们将发现一种单一的铅毒株 具有进一步临床前试验和临床开发的最佳特性的分枝杆菌。这些结果 有望对创伤和应激源相关疾病的治疗产生重要的积极影响 因为目前的药物治疗有很大的局限性。

Project Summary Immunoregulation, i.e., balanced expression of regulatory and effector T cells, is thought to be compromised in modern high-income settings due in part to reduced contact with commensal and environmentally derived bacteria, also known as “old friends”. Failed immunoregulation is thought to be one factor contributing to recent increases in stress-related and chronic inflammatory disorders as well stress-related psychiatric disorders in which inflammation is a risk factor, such as depression, anxiety, and posttraumatic stress disorder (PTSD), in high-income countries. One of these “old friends” is Mycobacterium vaccae NCTC 11659, a nonpathogenic, environmental saprophyte with anti-inflammatory and immunoregulatory properties. Immunization in the form of a heat-killed preparation of M. vaccae has been shown to increase stress resilience in mice, as measured by prevention of stress-induced increases in anxiety, prevention of stress-induced exaggeration of spontaneous colitis, and prevention of stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease. Immunization with M. vaccae, when conducted either before or after fear conditioning, has also been shown to enhance fear extinction in a rat model of fear-potentiated startle. In other studies, immunization with M. vaccae has been shown to prevent stress-induced exaggeration of anxiety-like defensive behavioral responses in a rat model of learned helplessness in association with suppression of stress-induced microglial priming and neuroinflammation. However, the extent to which these stress resilience and stress-protective effects generalize to other strains of mycobacteria are not known. In this STTR proposal, we propose to screen, using murine bone marrow-derived dendritic cells (BMDCs), human monocyte-derived macrophages (THP-1 cells), and a microglial cell line (BV2 cells), twenty proprietary strains of environmental mycobacteria for anti-inflammatory and immunoregulatory properties. For screening, we will target strains that meet the following criteria: they can readily be cultivated in isolation; they are not closely related to known mycobacterial pathogens; and they represent distinct lineages spanning the phylogenetic breadth of known mycobacterial diversity (of which there are >150 described mycobacterial strains). We will then screen two lead candidates, using male and female rats, to identify novel strains of mycobacteria with stress-protective effects, as measured by prevention of inescapable stress (IS)-induced increases in anxiety, conditioned fear, and escape deficits, and prevention of stress-induced priming of hippocampal microglia and neuroinflammation. We will also evaluate these strains in models of fear-potentiated startle and cued fear in male and female rats. At the completion of these studies, it is our expectation that we will have identified a single lead strain of mycobacteria that has the best properties for further preclinical testing and clinical development. These results are expected to have an important positive impact for treatment of trauma- and stressor-related disorders because current pharmacological treatments have significant limitations.