Mycoplasma pneumoniae activation of airway epithelium

描述(申请人提供)：肺炎支原体是人类肺部疾病的主要原因，最初与呼吸道上皮细胞的相互作用可能有助于随后的疾病发展。显然，随着免疫介导性炎症反应在呼吸道粘膜下层的募集和发展，针对支原体的免疫反应是疾病的主要组成部分。因此，呼吸道上皮细胞产生的因子可能影响这些免疫介导的损伤的发病机制；然而，由呼吸道上皮细胞产生的细胞因子/趋化因子的全部补体尚不清楚。此外，参与这些细胞激活的机制和支原体成分尚不清楚。如果了解，这些信息可能有助于开发干预支原体和其他肺部疾病的新方法。该项目的长期目标是确定支原体呼吸道疾病免疫反应产生的核心分子机制。该项目建议测试肺炎支原体刺激人类呼吸道支气管上皮细胞产生趋化因子或其他细胞因子的假设，这些细胞因子可能导致炎症损害。此外，我们认为支原体黏附蛋白促进支原体脂蛋白与呼吸道上皮细胞Toll样受体(TLR)之间的相互作用，进而刺激上皮细胞产生这些因子。具体地说，我们提出了以下问题：1)肺炎支原体膜组分是否激活或损伤人支气管上皮细胞，这是否依赖于黏附分子？2)肺炎支原体是否通过Toll样受体(TLR)刺激人支气管上皮细胞？实验设计如下：1)有活力的支原体、支原体膜和脂蛋白将刺激人支气管上皮细胞系。将测定细胞因子/趋化因子的mRNA和蛋白水平。我们将比较粘附性双亲支原体和非粘附性支原体的活性。2)监测支原体刺激前后支气管上皮细胞TLR的表达情况。将表达每个人TLR的质粒导入缺乏TLR的细胞系，以确定它们在支原体(细胞、膜和脂蛋白)刺激细胞中的作用。TLR在人支气管上皮细胞上的作用将通过阻断抗体或用非活性形式的TLR进行转基因来确定。这些研究将为进一步阐明参与这些反应的分子事件，以及研究上皮源性趋化因子/细胞因子在疾病发病机制中的作用奠定基础。此外，这些事件很可能会加剧哮喘和其他呼吸道疾病。因此，调节这些事件中的每一个都可能导致针对支原体和其他慢性呼吸道疾病的新疗法。这种名为肺炎支原体的细菌导致了“行走肺炎”，它至少占美国肺炎病例的30%，并与严重的哮喘有关。这项拟议的研究将研究这种细菌导致肺部疾病和更严重的哮喘的机制。最终，我们预计这些研究将通过促进患者肺部的有利反应而不是有害反应来更有效地治疗由这种细菌引起的疾病。

DESCRIPTION (provided by applicant): Mycoplasma pneumoniae is a major cause of lung disease in humans, and the initial interaction with airway epithelial cells is likely to contribute to subsequent disease development. It is clear that immune responses against mycoplasma are a major component of disease with the recruitment and development of immune-mediated inflammatory responses in the submucosa of the respiratory tract. Thus, the factors produced by airway epithelial cells are likely to influence the pathogenesis of these immune-mediated lesions; however, the full complement of cytokines/chemokines produced by airway epithelial cells is unknown. Furthermore, the mechanisms and mycoplasma components involved in the activation of these cells is uncharacterized. If understood, this information could facilitate the development of new approaches to intervene in mycoplasma and other pulmonary diseases. The long-term objective of this project is to determine the molecular mechanisms central to the generation of immune responses in mycoplasma respiratory disease. This project proposes to test the hypothesis that M. pneumoniae stimulate human respiratory bronchoepithelial cells to produce chemokines or other cytokines that are likely to contribute to the inflammatory lesions. Furthermore, we propose that mycoplasma adhesion protein promote the interaction between mycoplasma lipoproteins and toll-like receptors (TLR) of respiratory epithelial cells, which in turn stimulate the production these factors by epithelial cells. Specifically, we proposed to address the following questions: 1) Do M. pneumoniae membrane components activate or damage human bronchoepithelial cells and is this dependent on adhesion molecules? 2) Does M. pneumoniae stimulate human bronchoepithelial cells via toll-like receptors (TLR)? The experimental designs are: 1) Viable mycoplasma, mycoplasma membranes and lipoproteins will stimulate a human bronchoepithelial cell line. Cytokine/chemokine mRNA and protein levels will be determined. We will compare the activity of an adherent parental mycoplasma strain with a nonadherent strain. 2) The expression of TLR by bronchoepithelial cells will be monitored before and after mycoplasma stimulation. Cell lines lacking TLR will be transfected with plasmids expressing each of the human TLR to determine their roles in mycoplasma (cell, membranes and lipoproteins) stimulation of cells. The role of the TLR on human bronchoepithelial cells will be determined using blocking antibodies or transfection with inactive forms of TLR. These studies will provide the foundation for future studies to further elucidate the molecular events involved in these responses, and examine the role of epithelium-derived chemokines/cytokines in disease pathogenesis. Furthermore, it is likely that these events contribute to the exacerbation of asthma and other respiratory diseases. Thus, modulation of each of these events may lead to novel therapies against mycoplasma and other chronic respiratory diseases. The bacterium, Mycoplasma pneumoniae, is responsible for "walking pneumonia" which comprises at least 30% of the pneumonia cases in the United States and associated with severe asthma. The proposed research will study the mechanism through which this bacterium causes lung disease and more severe asthma. Ultimately, we expect these studies will lead to more effective treatments of diseases due to this bacterium by means of promoting favorable, rather than harmful, responses within the lungs of patients.