Mechanisms of Initiation and Persistence of Allergic Asthma

描述(由申请人提供)：这份哮喘和过敏性疾病中心的拨款申请是为了继续加州大学旧金山分校4名教职员工之间长期卓有成效的合作，他们对过敏性呼吸道炎症和哮喘的机制长期感兴趣。这项应用的中心目标是确定过敏性呼吸道炎症和气道高反应性启动和持续的关键机制。该应用包括两个将利用小鼠模型的项目和第三个将检查在这些模型中确定的分子靶点和途径与人类哮喘患病率、严重性和药物反应性的相关性的项目。项目1的中心首席研究员兼负责人谢泼德院长已经确定了转化生长因子激活的关键作用？(转化生长因子？？)通过两种不同的整合素(？v？6和？v？8)在慢性过敏性哮喘模型的气道高反应性和同源免疫反应的调节中的作用。这项建议将利用一系列转基因小鼠的品系来直接检测转化生长因子的作用？？在这些效应中，确定负责的关键细胞类型，并确定这些反应背后的分子机制。项目2基于项目负责人理查德·洛克斯利的最新数据，甲壳素是真菌、寄生虫和甲壳类动物的主要结构成分，它激活巨噬细胞和启动先天免疫细胞，以启动2型免疫反应。本项目中提出的工作将利用一系列新的报告线来检查关键的甲壳素反应细胞以及这一途径启动变态反应性炎症的机制和相关性。由Esteban Burchard和John Fahy共同领导的项目3将对甲壳素降解酶序列变异与转化生长因子之间的遗传关联进行初步观察。以及过敏性致敏和哮喘，以更深入地询问几丁质酶和转化生长因子多种成分的序列变异的关联和基因-基因相互作用？激活和信号通路，包括项目1中检测的整合素亚单位。该项目还将使用哮喘患者和健康对照组的肺泡灌洗样本和组织来评估相关基因的功能意义。这些项目将得到一个生理学和组织分析核心的支持，该核心将为所有3个项目提供广泛支持，并将得到一个集中管理核心的支持。LAY概要-该中心将评估哮喘启动和持续的潜在机制。通过确定新的途径和分子靶点，并测试它们与人类哮喘、哮喘严重程度和药物反应的相关性，该中心的工作应该为开发这种常见且往往具有破坏性的疾病的新疗法提供线索。 项目1：同源免疫和呼吸道高反应性中的V整合素(谢泼德，D) 项目1描述(由申请人提供)：缺乏上皮整合素？V？6的小鼠，我们已经证明激活了潜在的转化生长因子？，可以保护小鼠免受慢性过敏原攻击后的持续性呼吸道高反应性(AHR)。令人惊讶的是，这种保护与上皮下层气道纤维化的任何减少无关，这是该模型依赖于转化生长因子的中心特征。白细胞特异性敲除了相关整合素？V？8的小鼠，也激活了转化生长因子？显示出增强的适应性免疫的证据。在这个方案中，我们将确定V？6亚单位敲除小鼠的这些改变的反应是否是V？6整合素和/或转化生长因子？用表达野生型整合素或活性转化生长因子的“拯救”小鼠激活传导呼吸道上皮细胞？在呼吸道上皮细胞中。我们将评估？V？6抗体和转化生长因子？RII-LG嵌合体在这些相同的终点上，以进一步证实这一途径的重要性，并评估针对这一途径进行治疗干预的可行性。为了确定？V？6基因缺失防止AHR诱导的机制，我们将在初步实验中利用表达微阵列评估与该表型相关的少数候选基因的表达与气道反应性之间的关系。我们还将通过免疫染色和/或原位杂交来确定表达的细胞分布，并将使用商业上可获得的表达特定候选基因零突变的小鼠品系来评估功能意义。由于已知的细胞因子IL-13在多种模型的AHR的诱导中起核心作用，并且由于基因芯片确定的两个最有希望的候选者，白三烯C4合成酶和IL-18被认为是IL-13在呼吸道诱导的上游，我们也将在慢性挑战野生型和？v？6基因敲除小鼠中研究IL-13的细胞来源。最后，我们将确定白细胞V8的丢失如何导致获得性免疫的增强，并检查这一途径与过敏性呼吸道炎症及其后果的相关性。本项目将研究一种单一的生长因子，转化生长因子？，如何促进慢性哮喘的发展或抑制过敏性过敏及其后果，这取决于这种生长因子在哪里被激活以及如何被激活。

DESCRIPTION (provided by applicant): This Asthma and Allergic Diseases Center Grant application is an effort to continue a long-standing fruitful collaboration among 4 UCSF faculty members with a long-standing interest in the mechanisms underlying allergic airway inflammation and asthma. The central goals of this application are to determine critical mechanisms underlying the initiation and persistence of allergic airway inflammation and airway hyperresponsiveness. The application includes two projects that will utilize murine models and a third that will examine the relevance of molecular targets and pathways identified in these models to asthma prevalence, severity and drug responsiveness in humans. The Center Principal Investigator and leader of Project 1, Dean Sheppard, has identified critical roles for activation of transforming growth factor ? (TGF??) by two different integrins (?v?6 and ?v?8) in airway hyperresponsiveness in a chronic model of allergic asthma and in modulation of cognate immune responses. This proposal will take advantage of a series of lines of genetically modified mice to directly examine the role of TGF?? in these effects, to determine the critical cells types responsible, and to identify the molecular mechanisms underlying these responses. Project 2 is based on recent data from Richard Locksley, the project leader, that chitin, a prominent structural component of fungi, parasites and crustaceans, activates macrophages and primes innate immune cells for initiation of type 2 immune responses. Work proposed in this project will utilize a series of novel reporter lines to examine the critical chitin-responsive cells and the mechanisms and relevance of this pathway for initiation of allergic inflammation. Project 3, co-led by Esteban Burchard and John Fahy will follow-up on preliminary observations about genetic associations between sequence variants in chitin-degrading enzymes and TGF?? and allergic sensitization and asthma to more deeply interrogate associations and gene-gene interactions for sequence variants in chitinases and multiple components of the TGF? activation and signaling pathways, including the integrin subunits examined in Project 1. This project will also evaluate the functional significance of associated genes using bronchoalveolar lavage samples and tissue from asthmatic patients and healthy control subjects. These projects will be supported by a Physiology and Tissue Analysis core that will provide extensive support for all 3 projects, and by a centralized Administrative core. Lay summary - This Center will evaluate the mechanisms underlying initiation and persistence of asthma. By identifying novel pathways and molecular targets and testing their relevance to asthma, asthma severity and drug response in humans, the work in the Center should provide clues for the development of new treatments for this common and often devastating disease. PROJECT 1: ?v Integrins in Cognate Immunity and Airway Hyperresponsiveness (SHEPPARD, D) PROJECT 1 DESCRIPTION (provided by applicant): Mice lacking the epithelial integrin, ?v?6, that we have shown activates latent TGF-?, are protected from the persistent airway hyperresponsiveness (AHR) that follows chronic allergen challenge. Surprisingly, this protection is not associated with any decrease in sub-epithelial airway fibrosis, a central TGF-? -dependent feature of this model. Mice with leukocyte specific knockout of the related integrin, ?v?8, which also activates TGF-? show evidence of enhanced adaptive immunity. In this proposal, we will determine whether these altered responses in ?v?6 subunit knockout mice are a direct consequence of loss of the ?v?6 integrin and/or of TGF-? activation from conducting airway epithelial cells using "rescue" mice expressing either the wild type integrin or active TGF-? in airway epithelial cells. We will evaluate the effects of ?v?6 antibodies and a TGF-? RII-lg chimera on these same endpoints to further confirm the importance of this pathway and evaluate the feasibility of targeting this pathway for therapeutic intervention. To determine the mechanisms by which loss of ?v?6 protects from induction of AHR, we will evaluate the relationship between airway responsiveness and expression of a small number of candidate genes identified as linked to this phenotype in preliminary experiments utilizing expression microarrays. We will also determine the cellular distribution of expression by immunostaining and/or in situ hybridization, and will evaluate functional significance using commercially available lines of mice expressing null mutations of specific candidates. Because the cytokine IL-13 is known to play a central role in induction of AHR in multiple models, and because two of the most promising candidates identified by microarrays, leukotrienes C4 synthase and interleukin-18 have been suggested to be upstream of IL-13 induction in the airways, we will also examine the cellular sources of IL-13 in chronically challenged wild type and ?v?6 knockout mice. Finally, we will determine how loss of leukocyte ?v?8 leads to enhancement of adaptive immunity and examine the relevance of this pathway to allergic airway inflammation and its consequences. Lay summary - This project will examine how a single growth factor, transforming growth factor ?, can either contribute to development of chronic asthma or inhibit allergic sensitization and its consequences, depending on where and how this growth factor is activated.