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

描述(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，暗示有遗传倾向。人类TLR2基因座的遗传变异、环境因素及其相互作用与接触微生物产品的儿童哮喘易感性密切相关。然而，近亲繁殖群体中的遗传异质性阻碍了关联研究揭示哪些变异会影响疾病易感性。我们的目标是描述自然变异对人类过敏性炎症发生和易感性至关重要的基因的表达和功能的影响的潜在机制(S)。我们的模型基因是人类TLR2，它是先天性免疫的关键效应器，也是哮喘和过敏的调节器。我们发现：(1)人类TLR2突变体rs4696480与预防哮喘和过敏密切相关；(2)人类TLR2基因座包括其他与rs4696480连锁的多态。我们相信，最终，人类SNPs需要在生理基因组的背景下进行活体研究。在这里，我们希望测试一种假设，即人类TLR2基因变异和环境暴露之间的相互作用导致哮喘易感性可以在携带哮喘/过敏相关人TLR2单倍型的BAC转基因(TG)小鼠中有效地建模和解剖。更具体地说，我们提出：目标1：携带人类TLR2基因座单倍型的小鼠TLR2缺陷BAC TG系的产生、验证和分析，这些单倍型涉及影响哮喘易感性的基因-环境相互作用。这项工作将利用我们对TLR2变异的分析和我们在BAC重组工程方面的技能，并将导致产生表现出忠实的组织特异性和拷贝数依赖的人TLR2表达，但缺乏小鼠TLR2的TG系。目的2：确定hTLR2变异体与微生物产品暴露之间的基因-环境相互作用的机制，并评估其对哮喘/过敏易感性的影响。这项工作将依赖于体外和体外模型，以确定变异是否在转录和/或转录后水平上影响hTLR2的表达和功能。小鼠TLR2缺乏的背景将是表征人类TLR2依赖的表型的理想方法。通过提供受控的遗传背景，我们提出的TG模型将定义TLR2依赖表型所涉及的单倍型及其对TLR2表达和/或功能的修饰效应，为旨在中和遗传TLR2/环境影响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发生和易感性至关重要的基因的表达和功能产生影响的潜在机制。我们的模型是TLR2，一种参与先天性免疫的受体，其基因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，TLR2多态rs4696480与暴露在微生物分子中的儿童哮喘和过敏的易感性密切相关。我们对TLR2的变异进行了广泛的分析，并确定了与rs4696480连锁的多态。为了阐明人类TLR2基因变异与微生物制品接触导致哮喘/过敏易感性之间的相互作用机制，需要强有力的生理学方法。最终，多态现象需要在生理基因组环境中进行活体研究。在这一建议中，我们希望探索这样的假设：(1)与哮喘易感性表型相关的人类TLR2多态足以在定义和控制的环境条件下诱导TLR2表达和/或功能的显著变化，以及(2)自然遗传TLR2变异与环境之间的相互作用可以有效地在小鼠模型中进行建模和解剖，该模型携带定义的人类TLR2单倍型，其背景是TLR2缺乏。影响TLR2表达/功能的单倍型/环境模式是根据受控的遗传背景进行评估的，这一事实确保了这个体内模型将允许我们确定哪些多态性是必要的，并且足以诱导TLR2表达和/或功能活性的改变，从而导致TLR2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.