Integration of Leukotriene and Prostaglandin Receptor Signaling in Mast Cell Activation and Pulmonary Inflammation during Asthma

项目摘要 肥大细胞（MC）是哮喘的效应细胞，其活化引起半胱氨酰的分泌， 白三烯（cys-LTs）和白藜芦醇（PGs）。MC不仅分泌这些介质，而且它们还 拥有它们的感受器，来感知它们的信号。半胱氨酸-LT是有效的支气管收缩剂， 血管渗漏的诱导剂，气道高反应性的增强剂，并在 哮喘和其他炎症性疾病。Cys-LT主要通过两个G蛋白偶联受体发挥作用 （GPCR）、CysLT 1 R和CysLT 2 R。另一种GPCR，GPR 17由LTD 4激活。CysLT 2 R和 GPR 17负调控CysLT 1 R功能。LTE 4是cys-LT中最丰富和最稳定的，是一种 CysLT 1 R和CysLT 2 R的弱部分激动剂。然而，LTE 4在体内诱导独特的反应， 这是LTC 4或LTD 4无法概括的。在MC中，LTE 4比LTD 4更有效，并且中继信号 通过过氧化物酶体增殖物激活受体（PPAR-γ）和P2 Y12 R。最近，GPR 99 被鉴定为另一个CysLTR，其偏好LTE 4。了解所有这些cys-LT受体 （CysLTR）响应于多个配体而彼此相互作用是至关重要的，特别是考虑到它们的 在气道生理学中的作用。此外，cys-LT与PGE 2一起协同增强钙通量，c- Fos、考克斯-2、PGD 2和巨噬细胞炎性蛋白-1 β（MIP-1β; CCL 4））的产生。 有趣的是，LTD 4-PGE 2协同作用仅被CysLT 1 R拮抗剂联合治疗阻断。 （MK 571/ singulair）和EP 3拮抗剂（L-798），表明需要CysLT 1 R联合治疗 拮抗剂和EP 3R拮抗剂来治疗哮喘中的炎症。LTD 4 + PGE 2协同作用还 增强DF致敏小鼠的肺部炎症（免疫细胞、杯状细胞的募集 化生、炎性转录物的上调）。与LTD 4类似，LTE 4与PGE 2协同作用，但它 与LTD 4的不同之处在于涉及PPARγ的信号。基于这些观察，我们假设cys- LT诱导cys-LT-应答受体之间的复杂相互作用，以深刻地影响细胞的功能。 通过将抗炎性PGE 2信号传导转换为促炎性信号传导， MC中考克斯-2和PGD 2的产生影响Th 2炎症和哮喘。我们将检验这一假设 1）确定已知的（CysLT 1 R和CysLT 2 R）之间的相互作用， 和假定的（GPR 99、P2 Y12 R和PPAR γ）CysLTR响应cys-LT，影响MC功能，2） 揭示cys-LT-PGE 2协同作用诱导PGD 2产生和MC的机制， 3）确定cys-LT+ PGE 2相互作用和MC在Cys-LT+ PGE 2激活中的生理意义。 体内肺部炎症。我们将分析的病理，生理和免疫签名， 免疫反应和评估MC的贡献。这些研究将带来大量的 哮喘和过敏性疾病的致病和治疗意义，并提供了基础， 开发和翻译未来的治疗分子，调节炎症。

Project Summary Mast cells (MCs) are effector cells in asthma and their activation causes secretion of cysteinyl leukotrienes (cys-LTs) and prostaglandins (PGs). MCs not only secrete these mediators, but they also possess receptors for them, to perceive their signals. Cys-LTs are potent bronchoconstrictors, powerful inducers of vascular leakage, potentiators of airway hyper-responsiveness and play an important role in asthma and other inflammatory disorders. Cys-LTs mainly act through two G-protein-coupled receptors (GPCR), CysLT1R and CysLT2R. Another GPCR, GPR17 is activated by LTD4. Both CysLT2R and GPR17 negatively regulate CysLT1R function. LTE4, the most abundant and stable of the cys-LTs, is a weak, partial agonist for the CysLT1R and CysLT2R. However, LTE4 induces unique responses in vivo that cannot be recapitulated by LTC4 or LTD4. In MC, LTE4 is more potent than LTD4 and relays signals through peroxisome proliferator activating receptor (PPAR)-γ and P2Y12R. Recently, GPR99 was identified as another CysLTR with a preference for LTE4. Understanding how all these cys-LT receptors (CysLTR) interact with each other in response to multiple ligands is critical, especially considering their role in airway physiology. Further, cys-LTs together with PGE2 synergistically potentiate calcium flux, c- Fos, COX-2, PGD2 and Macrophage Inflammatory Protein-1β (MIP-1β; CCL4)) generation in MCs. Interestingly, LTD4-PGE2 synergism is blocked only by combined treatment of CysLT1R antagonist (MK571/ singulair) and EP3 antagonist (L-798), suggesting the need for a combination of CysLT1R antagonists and EP3R antagonists to treat inflammation in asthma. LTD4+PGE2 synergism also potentiates pulmonary inflammation in der f sensitized mice (recruitment of immune cells, goblet cell metaplasia, up-regulation of inflammatory transcripts). Similar to LTD4, LTE4 synergizes with PGE2 but it differs from LTD4 via signals involving PPARγ. Based on these observations, we hypothesize that cys- LTs induce complex interactions between cys-LT-responsive receptors to profoundly influence the downstream signaling by switching anti-inflammatory PGE2 signaling to pro-inflammatory, upregulating COX-2 and PGD2 production in MC impacting Th2 inflammation and asthma. We will test this hypothesis in the following specific aims: 1) To determine the interplay between the known (CysLT1R and CysLT2R) and putative (GPR99, P2Y12R and PPARɣ) CysLTRs in response to cys-LTs, influencing MC function, 2) To uncover the mechanism by which cys-LT-PGE2 synergism induces PGD2 production and MC activation and 3) To determine the physiological significance of cys-LT+PGE2 interactions and MC in pulmonary inflammation in vivo. We will analyze pathologic, physiologic, and immunologic signatures of the immune response and evaluate the contribution of MCs. These studies will carry substantial pathogenic and therapeutic implications for asthma and allergic diseases as well as provide the basis for development and translation of future therapeutic molecules that regulate inflammation.