Neuroinflammatory Regulation of Colonic Mechanosensory Activity

项目摘要 结肠超敏反应和胃肠道（GI）疼痛中的结肠直肠机械感觉增强， 由背根神经节（DRG）中的初级传入神经元启动和介导，可能由Piezo 2介导 结肠伤害性传入神经元亚群中的机械转导。巨噬细胞集落- 刺激因子（CSF 1）和脑源性神经营养因子（BDNF）是两种有效的介质， 然而，DRG伤害性神经元不表达CSF 1受体Csf 1 r， 神经元有BDNF受体TrkB。巨噬细胞和神经胶质细胞，包括卫星神经胶质细胞（SGCs）表达 Csf 1 r和/或TrkB亚型，因此它们可能是CSF 1和BDNF的主要接受者。在DRG中， 巨噬细胞和神经胶质细胞包围感觉神经元以形成神经元-神经胶质-免疫细胞三联体。后 激活后，巨噬细胞和神经胶质细胞释放多种炎性因子，如原伤害感受因子， 细胞因子肿瘤坏死因子α（TNFα）可作用于附近的感觉神经元。在我们的初步研究中， 巨噬细胞和胶质细胞在雌雄小鼠结肠背根节产生TNFα中的作用差异 超敏反应模型TNFα和CSF 1条件化的人巨噬细胞培养基激活一个亚群， 表达Piezo 2的结肠传入神经元。巨噬细胞也被BDNF激活产生TNFα。我们 假设巨噬细胞和神经胶质细胞被DRG中的CSF 1和BDNF激活，产生TNFα， 激活表达Piezo 2的结肠传入神经元，导致结肠机械感觉敏化。我们将 实现三个相互关联的具体目标。在AIM 1中，我们将区分巨噬细胞和神经胶质细胞在 产生TNFα，在雄性和雌性小鼠的DRG中产生神经炎症，以调节结肠 超敏反应我们将实施遗传工具，用于巨噬细胞或神经胶质细胞的条件性消融/抑制， 并通过体内DRG成像检查结肠直肠扩张（CRD）诱发的机械感觉活动。在AIM 2中， 我们将确定DRG伤害感受神经元中的Piezo 2介导结肠炎性神经炎性调节， 超敏反应我们将使用我们的Piezo 2条件性敲除小鼠系进行直接神经化学 测量和研究Piezo 2在神经炎症（TNFα）调节的介导中的功能作用。 结肠机械感觉活动。在AIM 3中，我们将描述巨噬细胞和神经胶质细胞的调节 CSF 1和BDNF在雄性和雌性小鼠产生TNFα调节Piezo 2表达的结肠传入中的作用 神经元在许多物种和系统中，TrkB受体的表达与性别有关。我们将研究 TrkB受体在巨噬细胞和神经胶质细胞中是否具有性二态性，以解释TrkB受体在巨噬细胞和神经胶质细胞中的不同作用。 巨噬细胞和神经胶质细胞在结肠超敏反应的雄性和雌性小鼠，并提出机制， 患者中与性别相关的胃肠道疼痛。我们的研究使用体内，体外，遗传工具和成像技术， 描述神经炎症对结肠机械感觉活动的影响将为研究结肠机械感觉活动开辟新的途径。 设计治疗胃肠道疼痛和功能性肠病的特定靶点。

PROJECT SUMMARY The enhanced colorectal mechanosensation in colonic hypersensitivity and gastrointestinal (GI) pain is initiated and mediated by primary afferent neurons in dorsal root ganglia (DRG), perhaps by Piezo2-mediated mechanotransduction in a subpopulation of colonic nociceptive afferent neurons. Macrophage colony- stimulating factor (CSF1) and brain-derived neurotrophic factor (BDNF) are two potent mediators in the genesis of mechanical pain, however, DRG nociceptive neurons do not express CSF1 receptor Csf1r and few neurons have BDNF receptor TrkB. Macrophages and glial cells including satellite glial cells (SGCs) express Csf1r and/or TrkB isoforms, therefore they are likely primary recipients of CSF1 and BDNF. In DRG, macrophages and glial cells surround sensory neurons to form a neuronal-glial-immune cell triad. Upon activation, macrophages and glial cells release a variety of inflammatory factors such as the pronociceptive cytokine tumor necrosis factor alpha (TNFα) that can act on nearby sensory neurons. In our preliminary study, macrophages and glial cells act differentially in producing TNFα in DRG of male and female mice in colonic hypersensitivity models. TNFα and CSF1-conditioned human macrophage culture medium activate a subset of Piezo2 expressing colonic afferent neurons. Macrophages are also activated by BDNF to produce TNFα. We postulate that macrophages and glial cells are activated by CSF1 and BDNF in DRG to generate TNFα that activates Piezo2 expressing colonic afferent neurons, leading to colonic mechanosensory sensitization. We will pursue three interrelated Specific Aims. In AIM 1, we will distinguish the roles of macrophages and glial cells in producing TNFα to generate neuroinflammation in DRG in male and female mice to regulate colonic hypersensitivity. We will implement genetic tools for conditional ablation/inhibition of macrophages or glial cells, and examine colorectal distension (CRD)-evoked mechanosensory activity by in vivo DRG imaging. In AIM 2, we will determine Piezo2 in DRG nociceptive neurons in mediation of neuroinflammatory regulation of colonic hypersensitivity. We will use our Piezo2 conditional knockout mouse line to perform direct neurochemical measurement and study the functional roles of Piezo2 in mediation of neuroinflammation (TNFα)-regulated colonic mechanosensory activity. In AIM 3, we will characterize the regulation of macrophages and glial cells by CSF1 and BDNF in male and female mice in producing TNFα to regulate Piezo2 expressing colonic afferent neurons. In many species and systems, the expression of TrkB receptors is sex-related. We will examine whether TrkB receptors are sexually dimorphic in macrophages and glial cells to interpret the distinct roles of macrophages and glial cells in colonic hypersensitivity in male and female mice, and suggest mechanism of gender-related GI pain in patients. Our studies using in vivo, in vitro, genetic tools, and imaging techniques in characterizing the impact of neuroinflammation on colonic mechanosensory activity will open new avenues in designing specific target(s) in the treatment of GI pain and functional bowel disorders.