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

描述（由申请人提供）：农业中的有机粉尘暴露，特别是来自大型动物养殖的有机粉尘，会导致严重的气道疾病，包括支气管炎，哮喘加重和阻塞性肺病。最初接触有机粉尘会引起强烈的气道炎症反应，随着时间的推移而减弱；然而，反复暴露在这些环境中的人表现出肺功能下降、持续炎症和进行性呼吸障碍的风险增加。我们已经在我们新开发的小鼠模型中证明，小鼠适应重复的有机粉尘暴露，但表现出慢性肺组织炎症和肺泡巨噬细胞功能受损的证据。我们还在体外建立了重复的有机粉尘暴露会严重损害抗原提呈细胞（APC）的表型和功能。由于APC在识别、响应和清除吸入的物质/毒素的同时介导适应性免疫，因此由于反复接触粉尘而导致APC功能受损可能会导致呼吸系统疾病的恶化。这些观察结果背后的机制尚不清楚，可能会导致新的治疗方法的发展，以预防和管理有机粉尘引起的农业工人气道疾病。灰尘的固有复杂性是定义有机粉尘诱导炎症反应机制的挑战。有机粉尘中一个已确定的炎症成分是内毒素；然而，流行病学和实验室研究未能将内毒素暴露与疾病表现联系起来。我们最近的分析显示，在大型动物养殖环境中，革兰氏阳性（而不是革兰氏阴性）细菌具有很强的优势，化学分析表明，细菌细胞壁中的一种肽聚糖（PGN）成分高浓度的菌胺酸。我们的研究还支持非内毒素成分，如PGN，正在驱动对大型动物养殖粉尘的先天免疫炎症反应。革兰氏阳性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）和细胞质核寡聚结构域分子（NOD2）。我们的初步数据表明，TLR2和NOD2是调节有机粉尘诱导炎症的重要信号转导分子。这些新的观察结果使我们假设负责识别革兰氏阳性肽聚糖的模式识别受体调节大型动物有机粉尘暴露的慢性炎症反应。为了验证这一假设，我们将进行三个特定目的的实验：1)确定暴露于有机粉尘的抗原提呈细胞表达NOD2的时间过程和可逆性，以及负责其表达的信号通路；2)明确NOD2和TLR2在体外和体内基因敲除小鼠对有机粉尘暴露后APC激活反应的调控作用；3)研究NOD2或TLR2缺失小鼠对有机粉尘暴露诱导的气道疾病的易感性是否发生改变。

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.