Mycoplasma pneumoniae activation of airway epithelium

描述（由申请人提供）：肺炎支原体是人类肺部疾病的主要原因，与气道上皮细胞的初始相互作用可能导致随后的疾病发展。很明显，针对支原体的免疫反应是呼吸道粘膜下层免疫介导炎症反应的募集和发展的疾病的主要组成部分。因此，气道上皮细胞产生的因子可能影响这些免疫介导病变的发病机制；然而，气道上皮细胞产生的细胞因子/趋化因子的完整补体尚不清楚。此外，参与这些细胞活化的机制和支原体成分尚未确定。如果了解这些信息，将有助于开发新的方法来干预支原体和其他肺部疾病。该项目的长期目标是确定在支原体呼吸道疾病中产生免疫反应的分子机制。本项目拟验证肺炎支原体刺激人呼吸道支气管上皮细胞产生趋化因子或其他可能导致炎性病变的细胞因子的假设。此外，我们提出支原体粘附蛋白促进支原体脂蛋白与呼吸道上皮细胞toll样受体（TLR）的相互作用，进而刺激上皮细胞产生这些因子。具体来说，我们提出解决以下问题：1)肺炎支原体膜成分是否激活或损害人支气管上皮细胞，这是否依赖于粘附分子？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)活的支原体、支原体膜和脂蛋白将刺激人支气管上皮细胞系。测定细胞因子/趋化因子mRNA和蛋白水平。我们将比较贴壁亲本支原体菌株与非贴壁菌株的活性。2)监测支原体刺激前后支气管上皮细胞TLR的表达。用表达人类TLR的质粒转染缺乏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对人支气管上皮细胞的作用将通过阻断抗体或转染无活性TLR来确定。这些研究将为进一步阐明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提供基础。此外，这些事件可能会加剧哮喘和其他呼吸系统疾病。因此，调节这些事件可能导致针对支原体和其他慢性呼吸系统疾病的新疗法。这种名为肺炎支原体的细菌是“行走的肺炎”的罪魁祸首，在美国，至少30%的肺炎病例是由“行走的肺炎”引起的，并与严重哮喘有关。拟议的研究将研究这种细菌导致肺部疾病和更严重哮喘的机制。最终，我们期望这些研究将通过促进患者肺部有益而不是有害的反应，从而导致更有效的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.