Regulation of TRPV1 and Nociceptor Sensitization by the Complement System

描述（由申请人提供）：免疫系统在损伤或疾病引发的疼痛的发病机制中起关键作用。精确理解特定免疫介质促进伤害性神经元通路致敏的机制对于开发更有效的治疗策略至关重要。补体系统是先天免疫的主要组成部分。该提议集中于补体系统的两种高活性分裂产物C3 a和C5 a。据报道，在与疼痛相关的各种病理状态中，包括关节炎、胰腺炎、炎性肠病、烧伤和手术创伤，C3 a和C5 a的产生增加。阻断C3 a和C5 a的合成或拮抗它们的受体在炎性、神经性和术后疼痛的动物模型中产生镇痛作用。此外，在动物模型中，补体片段的直接施用增加了对热和机械刺激的伤害感受敏感性。尽管有强有力的证据表明补体系统参与疼痛超敏反应的发生，但其潜在机制尚不清楚。该项目的目标是研究在受影响组织中产生C3 a和C5 a而增强疼痛敏感性的机制。我们假设，C3 a和C5 a受体的表达和功能耦合与TRPV 1在一个子集的主要伤害感受器，这些受体的激活敏感的伤害感受器通过蛋白激酶C依赖性调节的TRPV 1通道，这是已知的疼痛产生刺激的分子整合器的功能。我们将使用多学科的方法，包括免疫组织化学，单细胞RT-PCR，[Ca 2 +]i成像，膜片钳分析，单纤维记录和伤害性行为的测量来测试我们的中心假设。在目的1中，我们将描述C3 a和C5 a受体（分别为C3 aR和C5 aR）和TRPV 1在通过特定分子标记物鉴定的各种感觉神经元中的表达和亚细胞分布。我们还将使用Ca 2+成像和膜片钳记录来测试TRPV 1与C3 aR和C5 aR的功能偶联。在目标2中，我们将研究将C3 aR和C5 aR的激活与TRPV 1致敏联系起来的细胞内信号级联。在目标3中，我们将使用单纤维记录和行为研究来检查PKC依赖性调节C3 aR和C5 aR激活下游的TRPV 1在调节伤害感受器兴奋性以及伤害感受器的热和化学敏化中的作用。拟议的研究将有助于表征C3 a和C5 a受体在调节伤害感受器功能中的新作用，并可能导致开发针对补体系统的新治疗策略以缓解疼痛。

DESCRIPTION (provided by applicant): The immune system plays a critical role in the pathogenesis of pain triggered by injury or illness. A precise understanding of the mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for the development of more efficacious treatment strategies. The complement system is a principal component of innate immunity. This proposal focuses on two highly active split products of the complement system, C3a and C5a. Increased production of C3a and C5a has been reported in various pathological states associated with pain, including arthritis, pancreatitis, inflammatory bowel disease, burns and surgical trauma. Blocking the synthesis of C3a and C5a or antagonizing their receptors produces analgesic effects in animal models of inflammatory, neuropathic and postoperative pain. Moreover, direct administration of the complement fragments increases nociceptive sensitivity to heat and mechanical stimuli in animal models. In spite of this strong evidence implicating the complement system in the development of pain hypersensitivity, the underlying mechanisms are not understood. The goal of this project is to investigate mechanisms that are responsible for the enhanced pain sensitivity produced by the generation of C3a and C5a in the affected tissues. We hypothesize that C3a and C5a receptors are expressed and functionally coupled with TRPV1 in a subset of primary nociceptors; activation of these receptors sensitize nociceptors via protein kinase C-dependent modulation of the TRPV1 channel, which is known to function as a molecular integrator of pain producing stimuli. We will use a multidisciplinary approach involving immunohistochemistry, single-cell RT-PCR, [Ca2+]i imaging, patch-clamp analysis, single-fiber recordings and measurement of nociceptive behavior to test our central hypothesis. In Aim 1, we will characterize the expression and subcellular distribution of C3a and C5a receptors (C3aR and C5aR, respectively) and TRPV1 in various classes of sensory neurons identified by specific molecular markers. We will also use Ca2+ imaging and patch-clamp recordings to test functional coupling of TRPV1 with C3aR and C5aR. In Aim 2, we will investigate intracellular signaling cascades that link the activation of C3aR and C5aR with TRPV1 sensitization. In Aim 3, we will use single-fiber recordings and behavioral studies to examine the role of PKC- dependent modulation of TRPV1 downstream of C3aR and C5aR activation in regulating nociceptor excitability as well as heat and chemical sensitization of nociceptors. The proposed studies will help to characterize the novel roles of C3a and C5a receptors in the regulation of nociceptor function, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain.