Role of pattern recognition receptors in organic dust-induced airway inflammation

描述(申请人提供)：农业中的有机粉尘暴露，特别是大型动物养殖场的有机粉尘暴露，会导致严重的呼吸道疾病，包括支气管炎、哮喘加重和阻塞性肺部疾病。最初接触有机粉尘会引起强烈的呼吸道炎症反应，这种反应会随着时间的推移而减弱；然而，反复暴露在这些环境中的人会增加肺功能下降、持续性炎症和进行性呼吸功能障碍的风险。在我们新开发的小鼠模型中，我们证明了小鼠适应了重复的有机粉尘暴露，但显示出慢性肺组织炎症和肺泡巨噬细胞功能受损的证据。我们还在体外证实，反复接触有机粉尘会严重损害抗原提呈细胞(APC)的表型和功能。由于APC在调节适应性免疫的同时具有识别、反应和清除吸入剂/毒素的功能，因此由于反复接触粉尘而导致的APC功能受损可能会导致呼吸道疾病恶化。这些观察结果背后的机制尚不清楚，可能导致开发新的治疗方法来预防和管理农业工人的有机粉尘引起的呼吸道疾病。粉尘固有的复杂性是定义有机粉尘引起的炎症反应机制的一个挑战。有机粉尘中已证实的炎性成分之一是内毒素；然而，流行病学和基于实验室的研究未能将接触内毒素与疾病表现联系起来。我们最近的分析显示，革兰氏阳性(而不是革兰氏阴性)细菌占主导地位，化学分析表明，在大型动物养殖环境中，细菌细胞壁中的肽聚糖(PGN)组分-胞壁酸浓度很高。我们的研究还支持，非内毒素成分，如PGN，正在推动对大型动物养殖粉尘的先天免疫炎性反应。革兰氏阳性的PGN及其降解产物可通过多种模式识别受体发挥作用，包括细胞表面Toll样受体2(TLR2)和胞浆核寡聚化结构域分子(NOD2)。我们的初步数据表明，TLR2和NOD2是调节有机粉尘诱导的炎症反应的重要信号转导分子。这些新颖的观察结果使我们假设，负责识别革兰氏阳性多肽的模式识别受体可以调节大型动物有机粉尘暴露的慢性炎症反应。为了验证这一假说，我们将进行三个特定目标的实验：1)确定有机粉尘暴露下抗原提呈细胞NOD2表达的时程和可逆性，以及导致其表达的信号通路；2)确定NOD2和TLR2在调节体外和体内有机粉尘暴露后APC激活反应中的功能作用；以及3)研究NOD2或TLR2缺陷小鼠是否对有机粉尘暴露引起的呼吸道疾病表现出改变的易感性。 与公共健康相关：这项建议的意义在于确定有机粉尘中存在的革兰氏阳性微生物成分介导炎症的机制，这代表了与目前专注于内毒素驱动机制的教条的范式转变。该项目将帮助确定新的炎症目标，这些目标可能导致对反复接触有机粉尘的个人采取新的预防和治疗策略。

DESCRIPTION (provided by applicant): Organic dust exposure in the agricultural industry, particularly from large animal farming, results in significant airway disease including bronchitis, exacerbation of asthma, and obstructive lung disease. Initial exposure to organic dust induces an intense airway inflammatory response that wanes over time; however, persons repetitively exposed to these environments exhibit an increased risk of lung function decline, persistent inflammation and progressive respiratory impairment. We have demonstrated in our newly developed murine model that mice adapt to repetitive organic dust exposure, yet manifest evidence of chronic lung tissue inflammation and impaired alveolar macrophage function. We have also established in vitro that repetitive organic dust exposure profoundly impairs antigen presenting cell (APC) phenotype and function. As APCs function to recognize, respond, and clear inhaled agents/toxins while mediating adaptive immunity, a functionally impaired APC due to repetitive dust exposure would likely contribute to worsening respiratory disease. The mechanisms underlying these observations are not clear and could lead to the development of novel treatments to prevent and manage organic dust-induced airway disease in agriculture workers. The inherent complexity of the dust is a challenge in defining mechanisms of organic dust-induced inflammatory responses. One established inflammatory component in organic dusts is endotoxin; however, epidemiologic and laboratory-based studies have failed to link endotoxin exposure to disease manifestations. Our recent analysis has revealed a strong predominance of Gram-positive (rather than Gram-negative) bacteria and chemical analysis demonstrated a high concentration of muramic acid, a component of peptidoglycan (PGN) from the bacterial cell wall, in large animal farming environments. Our studies also support that non-endotoxin components, such as PGN, are driving the innate immune inflammatory responses to large animal farming dusts. Gram-positive PGN and its degradation products can act through several pattern-recognition receptors including cell surface Toll-like receptor 2 (TLR2) and the cytosolic nuclear oligomerization domain molecule (NOD2). Our preliminary data suggest that TLR2 and NOD2 are important signal transduction molecules key to the regulation of organic dust-induced inflammation. These novel observations led us to hypothesize that pattern recognition receptors responsible for recognizing Gram-positive peptidoglycan regulate the chronic inflammatory response to large animal organic dust exposure. To test this hypothesis we will perform experiments outlined in three specific aims: 1) determine the time course and reversibility of NOD2 expression by antigen presenting cells with organic dust exposure and the signaling pathways responsible for its expression; 2) define the functional role of NOD2 and TLR2 in modulating APC response to activation following organic dust exposure in knock-out mice in vitro and in vivo; and 3) investigate whether mice deficient in either NOD2 or TLR2 exhibit altered susceptibility to organic dust exposure induced airway disease. PUBLIC HEALTH RELEVANCE: The significance of this proposal lies in identifying mechanisms by which the Gram-positive microbial components present in organic dust mediate inflammation, which represents a paradigm shift from the current dogma focused on endotoxin-driven mechanisms. This project will help identify novel inflammatory targets that could lead to new prevention and treatment strategies of individuals subjected to repeated organic dust exposure.