THE MICROBIOTA AND MUCOSAL IMMUNE RESPONSES IN THE DEVELOPMENT OF ASTHMA

 描述（由申请人提供）：哮喘是一种常见的过敏性疾病，其特征为气道炎症和阻塞。尽管哮喘等过敏性疾病的患病率正在增加，但其原因尚不完全清楚。环境因素而不是遗传因素被认为是主要原因，部分原因是过敏与西方生活方式有关。该项目将检验呼吸道和肠道微生物群是赋予过敏性疾病易感性或保护的关键因素的假设， 包括哮喘。将使用常规饲养的哮喘小鼠模型和致菌性哮喘小鼠模型来检验以下假设：由免疫系统识别的细菌亚群负责调节对哮喘的易感性。微生物群先前已被证明在小鼠哮喘发病机制中发挥作用，但对哪些微生物负责，甚至这些生物体居住在哪里（身体栖息地）知之甚少。在这项资助的第一个目标中，来自不同供应商的具有不同微生物群的小鼠将被筛选出易患气道过敏性炎症的微生物群落。哮喘将通过卵清蛋白致敏随后抗原激发来诱导。这些动物中哮喘的严重程度将通过其肺部的气道阻塞和组织炎症程度来确定。然后，他们的呼吸道和肠道微生物群将通过细菌16S rRNA测序和BugFACS进行表征，BugFACS是一种允许基于其与免疫球蛋白A（IgA）结合来分离和鉴定活细菌的技术，免疫球蛋白A是粘膜表面产生的主要抗体。我将确定影响哮喘表型的候选细菌菌株，最初是通过共同饲养两种类型的小鼠-那些携带与严重哮喘相关的微生物群和那些携带与轻度疾病相关的微生物群。应用于从肠道和呼吸道微生物群生成的16S rRNA数据集的微生物来源跟踪算法将用于将共饲养后观察到的哮喘严重程度变化与笼舍之间细菌菌株的单侧或双侧侵袭相关联。然后我会将培养的侵入性 菌株进入无菌小鼠的呼吸道和/或肠道，以确定这些菌株是否与哮喘严重程度有因果关系。在第二个目标中，将对一小组具有良好表型的6 - 8岁儿童（有和没有哮喘）进行采样，以通过16S rRNA测序和BugFACS表征他们的呼吸和粪便微生物群。将代表性呼吸道和肠道微生物群落从a）急性加重期间的代表性哮喘供体、B）基线时的相同哮喘患者和c）代表性健康非哮喘患者移植到无菌小鼠中。将在受体无菌小鼠中诱导哮喘，然后如上所述表征。这些目标将有助于剖析呼吸道和肠道微生物群对疾病发病机制的相对贡献，并提供一种鉴定可能具有诊断和治疗效用的细菌菌株的方法。

 DESCRIPTION (provided by applicant): Asthma is a common allergic disorder characterized airway inflammation and obstruction. Though the prevalence of allergic diseases such as asthma is increasing, the reasons are incompletely understood. Environmental, rather than genetic, factors are thought to be primarily responsible, in part because of the association of allergy with Western lifestyles. This project will examine the hypothesis that the respiratory trac and gut microbiota is a key factor in conferring susceptibility or protection to allergic diseases, including asthma. Conventionally-raised and gnotobiotic mouse models of asthma will be used to test the hypothesis that a subset of bacteria, recognized by the immune system, is responsible for modulating susceptibility to asthma. The microbiota has previously been demonstrated to play a role in asthma pathogenesis in mice, but little is known about which microbes are responsible or even where (which body habitat) these organisms reside. In the first aim of this grant, mice from various vendors with different microbiota will be screened for microbial communities that predispose to allergic inflammation of their airways. Asthma will be induced by ovalbumin sensitization followed by antigen challenge. Severity of asthma in these animals will be determined by the degree of airway obstruction and tissue inflammation in their lungs. Their respiratory and intestinal microbiota will then be characterized by bacterial 16S rRNA sequencing, and BugFACS, a technique that allows isolation and identification of viable bacteria based on their binding to immunoglobulin A (IgA), the major antibody produced at mucosal surfaces. I will identify candidate bacterial strains that impact asthma phenotypes, initially by co-housing two types of mice - those harboring a microbiota associated with severe asthma and those with a microbiota associated with mild disease. Microbial source tracking algorithms, applied to 16S rRNA datasets generated from gut and respiratory tract microbiota, will be used to correlate observed changes in asthma severity upon co-housing with uni- or bilateral invasion of bacterial strains between cagemates. I will then transplant cultured invasive strains into the respiratory tract and/or gut of germ-free mice to establish whether these strains are causally related to asthma severity. In the second aim, a small group of well phenotyped 6-8 year old children, with and without asthma, will be sampled to characterize their respiratory and fecal microbiota by 16S rRNA sequencing and BugFACS. Representative respiratory tract and gut microbial communities will be transplanted from a) a representative asthmatic donor during an acute exacerbation, b) the same asthmatic at baseline, and c) a representative healthy non-asthmatic patient, into germ-free mice. Asthma will be induced in the recipient gnotobiotic mice that will then be characterized as described above. These aims will help dissect the relative contributions of respiratory tract and gut microbiota to disease pathogenesis and provide an approach for identifying bacterial strains that may have diagnostic and therapeutic utility.