TLR Ligand and Pesticide Exposure in Farms: Role in Atopic Immune Responses

描述(由申请人提供)：NIEHS、NIOSH和NCI的研究人员最近在25,814名农场妇女中进行的农业健康研究报告称，在282名受试者中，农场中任何杀虫剂的使用与特应性哮喘之间存在高度显著的相关性。在农场长大的女性中，这种联系最为强烈。在农场长大和使用杀虫剂与特应性哮喘的总体风险共同相关，与农场环境的保护作用相反(正如基于卫生假说的预期)；同一项研究也证明了这一点，适用于在农场长大且从未使用杀虫剂的妇女。最近的另一项研究表明，在杀虫剂处理的小鼠中，内毒素诱导的几种细胞因子的产生受到抑制。农场环境中Toll样受体(TLR)配体的暴露，包括微生物和微生物产品中的微生物相关分子模式(MAMPs)，对过敏反应的抑制具有重要影响。这种抑制反应与最近修订的卫生假说对调节性T细胞(Treg)的诱导有关。因此，我们建议探索和确定在农场环境中，TLR配体和杀虫剂的联合呼吸道暴露如何影响与特应性哮喘相关的免疫反应。在第一个具体目标中，我们将表征环境TLR配体(内毒素、肽聚糖和细菌DNA)和两种与特应性哮喘相关的常规杀虫剂在俄亥俄州10个农场使用农药前后的暴露情况。将使用最先进的生物气溶胶采样方法。它包括一个可吸入取样器和一个由NIOSH开发的带有两级旋风分离器的大小选择采样系统(三种粒度的旋风分离器：&lt；1.05m、1-1.85m和&gt；1.85m)，用于收集空气中TLR配体和农药，以及用于从土壤表面收集可气雾化TLR配体和农药的新型微生物源强度测试仪。我们将使用LUDEP ICRP呼吸沉积模型来确定TLR颗粒配体和杀虫剂对男性、女性和儿童受试者的呼吸沉积潜力。因此，第一个具体目标的结果将使我们了解农场中同时接触可吸入TLR配体和杀虫剂的真实世界水平。在第二个特定目标中，我们将确定来自上述农场的未经净化但具有临床意义的空气和表面样本提取物如何影响宿主对卵蛋白诱导的超敏反应和Treg的诱导。这些影响的TLR4依赖性将通过卵蛋白致敏的野生型和TLR4-/-C57BL/6小鼠来确定。这些发现将提供重要的初步信息，说明农药暴露的不良反应与TLR配体暴露的保护作用在农业工人特应性哮喘发生中的关系。 公共卫生相关性：农药使用可能与农民中的特应性哮喘显著相关，因此耕作环境可能不会提供基于传统卫生学假说预测的保护效果。农业环境中存在着广泛的免疫刺激物质和农药，传统的用纯化材料和农药进行的调查可能不能准确地提供农药暴露的不良反应与TLR配体在农民中发生特应性哮喘的保护作用之间的关系的信息。在这个R21项目中，我们将探索在农场收集的未经净化但与临床相关的环境样本(施药前后)对卵蛋白过敏原致敏小鼠的免疫学活性。

DESCRIPTION (provided by applicant): The recent Agricultural Health Study among 25,814 farm women conducted by the researchers from NIEHS, NIOSH, and NCI reported a highly significant association between any use of pesticides in farms and atopic asthma in 282 subjects. This association was strongest among women who grew up on a farm. Growing up on farms and application of pesticides were jointly associated with higher overall risk for atopic asthma in contrary to the protective effect of farm environment (as expected based on the Hygiene Hypothesis); which was in the same study demonstrated for women who grew up on farms and never applied pesticides. Another recent study showed that the production of several cytokines induced by endotoxin was suppressed in pesticide treated mice. Exposures to toll like receptor (TLR) ligands in farm environment including MAMPs (microbe-associated molecular patterns) from microorganisms and microbial products have significant influence on the suppression of allergic response. This suppressive response was implicated to the induction of regulatory T cells (Treg) by recent revisions of the Hygiene Hypothesis. Therefore we propose to explore and determine how combined airway exposures to TLR ligands and pesticides influence immune responses associated with atopic asthma in farm environments. In the first specific aim we will characterize exposures of environmental TLR ligands (endotoxin, peptidoglycan, and bacterial DNA) and two conventional pesticides relevant to atopic asthma in 10 farms of Ohio before and after pesticide application. A state-of-the-art bioaerosol sampling methodology will be used. It includes an inhalable sampler and a NIOSH-developed size- selective sampling system with two-stage cyclone (three particle size fractions: <1.0 5m, 1 - 1.8 5m, and >1.8 5m), for the collection of airborne TLR ligands and pesticides and a novel microbial source strength tester for the collection of aerosolizable TLR ligands and pesticides from soil surface. We will determine the respiratory deposition potential of particulate TLR ligands and pesticides for male, female, and children subjects using the LUDEP ICRP respiratory deposition model. Results from the first specific aim will thus allow us to understand the real world simultaneous exposure levels to inhalable TLR ligands and pesticides in farms. In second specific aim we will determine how unpurified but clinically relevant air and surface sample extracts from above-mentioned farms analogous to real world exposures influence host susceptibility towards ovalbumin induced hypersensitivity and induction of Tregs. The TLR4 dependence of these influences will be determined by using ovalbumin-sensitized wild type and TLR4 -/- C57BL/6 mice. The findings will provide vital preliminary information on the relationship between adverse effects of pesticide exposure versus protective effects of TLR ligand exposure in the development of atopic asthma among agricultural workers. PUBLIC HEALTH RELEVANCE: Pesticide usage can be significantly associated with atopic asthma among the farmers and therefore farming environment may not provide the protective effect predicted based on the traditional Hygiene Hypothesis. Wide range of immunostimulatory materials and pesticides are present in farming environments and traditional investigations with purified materials and pesticides may not provide precise information on relationship between adverse effect of pesticide exposures versus protective effect of TLR ligands in developing atopic asthma among farmers. In this R21 project we will explore immunological activities of unpurified but clinically relevant environmental samples collected in farms (before and after pesticide application) in ovalbumin allergen sensitized mice.