Mechanisms of Initiation and Persistence of Allergic Asthma

描述（由申请人提供）：这个哮喘和过敏性疾病中心补助金申请是一个努力，以继续在过敏性气道炎症和哮喘的潜在机制的长期利益之间的4 UCSF教职员工长期富有成效的合作。本申请的中心目标是确定过敏性气道炎症和气道高反应性的起始和持续的关键机制。该应用程序包括两个项目，将利用小鼠模型和第三个项目，将检查这些模型中确定的分子靶点和途径与人类哮喘患病率，严重程度和药物反应性的相关性。该中心的主要研究者和项目1的领导人，院长谢泼德，已确定的关键作用，激活转化生长因子？(TGF??)由两种不同的整合素（？v？6、？v？8)在过敏性哮喘慢性模型气道高反应性和同源免疫应答调节中的作用。这项建议将利用一系列的转基因小鼠，直接检查TGF？？在这些影响中，以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Richard Locksley的最新数据，即几丁质是真菌，寄生虫和甲壳类动物的重要结构成分，可激活巨噬细胞并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细胞系来研究关键的几丁质反应细胞以及该途径引发过敏性炎症的机制和相关性。项目3，由Esteban Burchard和John Fahy共同领导，将跟进几丁质降解酶序列变异与TGF？和过敏性致敏和哮喘，更深入地询问协会和基因-基因相互作用的序列变异几丁质酶和多种成分的TGF？激活和信号通路，包括项目1中检查的整合素亚基。本项目还将使用来自哮喘患者和健康对照受试者的支气管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个项目提供广泛的支持，并得到集中管理核心的支持。本中心将评估哮喘发生和持续的潜在机制。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通常具有破坏性的疾病的新疗法提供线索。 项目1：？v同源免疫和气道高反应性中的整合素（SHEPPARD，D） 项目1描述（由申请人提供）：缺乏上皮整合素的小鼠，？v？6，我们已经证明激活潜在的TGF-β，在慢性过敏原攻击后，可防止持续气道高反应性（AHR）。令人惊讶的是，这种保护是不相关的任何减少上皮下气道纤维化，中央TGF-？此模型的依赖特性。白细胞特异性敲除相关整合素的小鼠，？v？8，这也激活TGF-？显示出增强的适应性免疫力在这项建议中，我们将确定是否这些改变的反应？v？6亚基基因敲除小鼠的直接后果是丧失？v？6整合素和/或TGF-？激活进行气道上皮细胞使用“救援”小鼠表达野生型整合素或活性TGF-？呼吸道上皮细胞。我们将评估的影响？v？6抗体和TGF-？RII-Ig嵌合体对这些相同终点的作用，以进一步证实该途径的重要性，并评估靶向该途径进行治疗干预的可行性。以确定的机制，其中损失？v？6保护AHR的诱导，我们将评估气道反应性和表达之间的关系，在初步实验中，利用表达微阵列鉴定为与这种表型相关的少量候选基因。我们还将通过免疫染色和/或原位杂交确定表达的细胞分布，并将使用表达特定候选物的无效突变的市售小鼠系评估功能意义。因为细胞因子IL-13是已知的AHR在多种模型中的诱导中发挥核心作用，并且因为通过微阵列鉴定的两个最有希望的候选者，白三烯C4合酶和白细胞介素-18已被建议在气道中的IL-13诱导的上游，我们还将研究IL-13在慢性挑战的野生型和？v？6只敲除小鼠。最后，我们将确定如何损失白细胞？v？8导致适应性免疫增强，并检查该途径与过敏性气道炎症及其后果的相关性。奠定总结-本项目将研究如何一个单一的生长因子，转化生长因子？可以促进慢性哮喘的发展或抑制过敏性致敏及其后果，这取决于这种生长因子在何处以及如何被激活。

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.