THE MICROBIOTA AND MUCOSAL IMMUNE RESPONSES IN THE DEVELOPMENT OF ASTHMA

 描述(申请人提供)：哮喘是一种常见的过敏性疾病，以呼吸道炎症和阻塞为特征。虽然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。这个项目将检验这样一种假设，即呼吸轨迹和肠道微生物区系是增加对过敏性疾病的易感性或保护的关键因素， 包括哮喘。常规培养的哮喘小鼠模型将被用来检验这样一个假设，即免疫系统识别的细菌亚群负责调节哮喘的易感性。微生物区系先前已被证明在小鼠哮喘的发病机制中发挥作用，但对哪些微生物负有责任，甚至这些微生物驻留在哪里(哪些身体栖息地)知之甚少。在这笔赠款的第一个目标中，将对来自不同供应商、具有不同微生物区系的小鼠进行筛选，以确定其呼吸道易患过敏性炎症的微生物群落。卵清蛋白致敏后抗原激发可诱发哮喘。这些动物哮喘的严重程度将由它们肺部的呼吸道阻塞和组织炎症的程度来决定。然后，他们的呼吸道和肠道微生物区系将通过细菌16S rRNA测序和BugFACS进行表征，BugFACS是一种根据它们与免疫球蛋白A(IgA)的结合来分离和鉴定活细菌的技术，免疫球蛋白A是黏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，首先通过与两种类型的小鼠共居--那些含有与严重哮喘相关的微生物群的小鼠，以及那些具有与轻度疾病相关的微生物群的小鼠。微生物来源追踪算法应用于肠道和呼吸道微生物区系产生的16S rRNA数据集，将被用于将观察到的哮喘严重程度的变化与共生时细菌菌株在笼子之间的单或双侧入侵相关联。然后我会移植培养的侵入性 将这些菌株送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的严重程度有因果关系。在第二个目标中，一小部分表型良好的6-8岁儿童，有哮喘和没有哮喘，将被抽样，通过16S rRNA测序和BugFACS来表征他们的呼吸道和粪便微生物群。具有代表性的呼吸道和肠道微生物群落将从a)急性发作期间有代表性的哮喘捐赠者，b)基线时的同一哮喘患者，和c)有代表性的健康非哮喘患者，移植到无菌小鼠中。哮喘将在受体灵知生菌小鼠中诱发，然后将如上所述描述其特征。这些目的将有助于剖析呼吸道和肠道微生物区系在疾病发病机制中的相对贡献，并为鉴定可能具有诊断和治疗作用的细菌菌株提供一种方法。

 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.