Regulation of TRPV1 and Nociceptor Sensitization by the Complement System

描述(申请人提供)：免疫系统在受伤或疾病引发的疼痛的发病机制中起着关键作用。准确了解特定免疫介质对伤害性神经通路的敏感化机制对于开发更有效的治疗策略是至关重要的。补体系统是先天免疫的主要组成部分。这项建议侧重于补体系统的两个高活性分裂产物C3a和C5a。据报道，在与疼痛相关的各种病理状态下，C3a和C5a的产生增加，包括关节炎、胰腺炎、炎症性肠病、烧伤和手术创伤。阻断C3a和C5a的合成或拮抗它们的受体在炎症、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，直接给予补体片段增加了对热和机械刺激的伤害性敏感性。尽管有这些强有力的证据表明补体系统与疼痛过敏症的发生有关，但其潜在的机制尚不清楚。这个项目的目标是研究在受影响的组织中产生C3a和C5a导致疼痛敏感性增强的机制。我们假设C3a和C5a受体在初级伤害性感受器中表达并与TRPV1在功能上偶联；这些受体的激活通过蛋白激酶C依赖的TRPV1通道的调节而使伤害性感受器敏感，TRPV1通道被认为是痛觉产生刺激的分子积分器。我们将使用免疫组织化学、单细胞RT-PCR、[Ca~(2+)]i成像、膜片钳分析、单纤维记录和伤害行为测量等多学科方法来验证我们的中心假设。在目标1中，我们将研究C3a和C5a受体(分别为C3aR和C5aR)和TRPV1在由特定分子标记鉴定的各类感觉神经元中的表达和亚细胞分布。我们还将使用钙离子成像和膜片钳记录来测试TRPV1与C3aR和C5aR的功能偶联。在目标2中，我们将研究将C3aR和C5aR的激活与TRPV1敏化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学研究来研究依赖PKC的调节C3aR下游TRPV1和C5aR激活在调节伤害性感受器兴奋性以及伤害性感受器的热和化学敏化中的作用。这些研究将有助于确定C3a和C5a受体在伤害性感受器功能调节中的新作用，并可能导致针对补体系统的新的治疗策略的开发以减轻疼痛。 公共卫生相关性：疼痛管理仍然是最严重的公共卫生问题之一。拟议的研究将有助于更好地了解补体系统如何在手术创伤或关节炎、胰腺炎和炎症性肠道综合征等疾病引起的疼痛的发病机制中发挥作用，并可能导致针对补体系统的新治疗策略的发展，以减轻疼痛。

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. PUBLIC HEALTH RELEVANCE: Pain management remains one of the most serious public health problems. The proposed studies will help to better understand how the complement system contributes to the pathogenesis of pain caused by surgical trauma or illnesses such as arthritis, pancreatitis and inflammatory bowel syndrome, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain.