Impact of TLR2/environment interactions on asthma susceptibility: mouse models

描述（由申请人提供）：哮喘和过敏是一种复杂的疾病，具有强烈的家族性成分，提示有遗传倾向。人类TLR 2基因座的遗传变异、环境因素及其相互作用与暴露于微生物制品的儿童哮喘易感性密切相关。然而，远交群体内的遗传异质性阻止了关联研究揭示哪些变体影响疾病易感性。我们的目标是表征自然变异体对人类过敏性炎症发展和易感性关键基因表达和功能影响的潜在机制。我们的模型基因是人TLR 2，它是先天免疫中的关键效应子，也是哮喘和过敏的调节剂。我们证明：（1）人TLR 2变体rs 4696480与针对哮喘和过敏的保护强烈相关;（2）人TLR 2基因座包括与rs 4696480连锁的其他多态性。我们确信，最终，人类SNP需要在生理基因组背景下进行体内研究。在这里，我们希望验证这样的假设：人类TLR 2遗传变异和导致哮喘易感性的环境暴露之间的相互作用可以在携带哮喘/过敏相关人类TLR 2单倍型的BAC转基因（TG）小鼠中有效地建模和解剖。具体而言，我们建议：目标1：小鼠Tlr 2缺陷BAC TG系的产生、验证和分析，其携带参与影响哮喘易感性的基因-环境相互作用的人TLR 2基因座单倍型。这项工作将利用我们对TLR 2变异的分析和我们在BAC重组工程中的技能，并将导致产生表现出忠实的组织特异性和拷贝数依赖性表达人TLR 2，但缺乏鼠TLR 2的TG系。目标二：确定hTLR 2变异体与微生物制品暴露之间基因-环境相互作用的潜在机制，并评估其对哮喘/过敏易感性的影响。本工作将依赖于体外和离体模型，以确定变异是否在转录和/或转录后水平上对hTLR 2的表达和功能起作用。鼠TLR 2缺陷背景将是人TLR 2依赖性表型表征的理想选择。通过提供受控的遗传背景，我们提出的TG模型将定义参与TLR 2依赖性表型的单倍型及其对TLR 2表达和/或功能的修饰作用，为旨在中和遗传TLR 2/环境对哮喘和过敏的影响的策略铺平道路。 公共卫生相关性：我们工作的总体目标是表征自然遗传变异和环境因素对人类过敏性炎症的发展和易感性至关重要的基因的表达和功能的影响的机制。我们的模型是TLR 2，一种参与先天免疫的受体，其遗传变异与微生物产物相互作用，影响哮喘和过敏的易感性。我们发现TLR 2基因多态性rs 4696480与接触微生物分子的儿童对哮喘和过敏的易感性密切相关。我们对TLR 2变异进行了广泛的分析，并确定了与rs 4696480连锁的多态性。为了阐明人类TLR 2基因变异和暴露于导致哮喘/过敏易感性的微生物产物之间的相互作用所涉及的机制，需要强有力的生理方法。最终，多态性需要在生理基因组背景下进行体内研究。在这个提议中，我们希望探索以下假设：（1）与哮喘易感性表型相关的人类TLR 2多态性足以在限定和受控的环境条件下诱导TLR 2表达和/或功能的明显改变，和（2）天然遗传TLR 2变异与环境之间的相互作用，影响人类TLR 2调节。在鼠Tlr 2缺陷背景上携带确定的人TLR 2单倍型的小鼠模型中，可以有效地建模和解剖功能。根据受控的遗传背景评估影响TLR 2表达/功能的单倍型/环境模式的事实确保了该体内模型将允许我们确定哪些多态性是诱导改变的TLR 2表达和/或功能活性所必需的和足够的，并因此导致负责TLR 2的分子机制。

DESCRIPTION (provided by applicant): Asthma and allergies are complex diseases with a strong familial component suggestive of a genetic predisposition. Genetic variants in the human TLR2 locus, environmental factors and their interactions are strongly associated with susceptibility to asthma in children exposed to microbial products. However, genetic heterogeneity within outbred populations prevents association studies from revealing which variants affect disease susceptibility. Our goal is to characterize the mechanism(s) underlying the impact of natural variants on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model gene is human TLR2, a key effector in innate immunity and modulator of asthma and allergy. We showed that: (1) human TLR2 variant rs4696480 is strongly associated with protection against asthma and allergy; (2) the human TLR2 locus includes other polymorphisms linked to rs4696480. We are convinced that, ultimately, human SNPs need to be studied in vivo within a physiologic genomic context. Here we wish to test the hypothesis that the interplay between human TLR2 genetic variation and environmental exposures that results in asthma susceptibility can be effectively modeled and dissected in BAC transgenic (TG) mice carrying asthma/allergy-associated human TLR2 haplotypes. More specifically, we propose: Aim 1: Generation, validation and analysis of murine Tlr2-deficient BAC TG lines carrying human TLR2 locus haplotypes involved in gene-environment interactions that affect asthma susceptibility. This work will capitalize on our analysis of variation in TLR2 and our skills in BAC recombineering, and will lead the generation of TG lines exhibiting faithful tissue-specific and copy number-dependent expression of human TLR2, but lacking murine Tlr2. Aim 2: Identification of the mechanisms underlying gene-environment interactions between hTLR2 variants and exposure to microbial products, and assessment of their impact on asthma/allergy susceptibility. This work will rely on in vitro and ex vivo models in order to determine whether variation acts on expression and functions of hTLR2 at transcriptional and/or post-transcriptional level. The murine tlr2-deficient background will be ideal for the characterization of human TLR2-dependent phenotypes. By providing a controlled genetic background, the TG model we propose will define the haplotypes involved in TLR2-dependent phenotypes and their modifying effects on TLR2 expression and/or function, paving the way for strategies aimed at neutralizing the effects of genetic TLR2/environment impact on asthma and allergies. PUBLIC HEALTH RELEVANCE: The overall goal of our work is to characterize the mechanisms underlying the impact of natural genetic variation and environmental factors on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model is TLR2, a receptor involved in innate immunity whose genetic variation interacting with microbial products affects the susceptibility to asthma and allergy. We have found that a TLR2 polymorphism, rs4696480, is strongly associated with susceptibility to asthma and allergy in children exposed to microbial molecules. We have performed an extensive analysis of TLR2 variation and identified polymorphisms in linkage with rs4696480. To elucidate the mechanisms involved in the interaction between human TLR2 genetic variation and exposure to microbial products that result in asthma/allergy susceptibility powerful and physiologic approaches are required. Ultimately, polymorphisms need to be studied in vivo within the physiologic genomic context. In this proposal we wish to explore the hypothesis that (1) human TLR2 polymorphisms associated with asthma susceptibility phenotypes are sufficient to induce appreciable alterations of TLR2 expression and/or function in defined and controlled environmental conditions, and (2) the interactions between natural genetic TLR2 variation and environment that affect human TLR2 regulation/function can be effectively modeled and dissected in mouse models carrying defined human TLR2 haplotypes on a murine Tlr2-deficient background. The fact that haplotype/environment patterns affecting TLR2 expression/function are assessed against controlled genetic background, ensures that this in vivo model will allow us to determine which polymorphisms are necessary and sufficient to induce altered TLR2 expression and/or function activity and therefore leading to the molecular mechanisms responsible for TLR2.