Mechanism of Mycoplasma-Induced Mast Cell Il-4 Synthesis

该项目的总体目标是确定肺炎支原体在慢性呼吸道高反应性和炎症发展中的先天和获得性免疫机制，重点是肥大细胞的作用。 具体目的1：克隆肺炎支原体P1粘附素，并在大肠杆菌中表达，获得一株缺失该分子的突变株。参与肥大细胞IL-4反应的P1粘附素或部分片段将被克隆并在大肠杆菌中表达。利用转座子诱变，将产生一株P1缺陷的肺炎支原体，用于肥大细胞刺激实验和小鼠感染模型。 具体目标2：鉴定和鉴定肥大细胞表面在P1糖蛋白诱导产生IL-4过程中所必需的表面分子，检测肺微环境中其他分子的调节作用，验证在人肥大细胞上观察到的效应，并表征在肥大细胞系中参与肺炎支原体细胞因子反应的特定分子的表达下调的影响。将鉴定和表征肺炎支原体诱导肥大细胞因子产生的靶分子，检测突变缺失的影响，分析介质在肺微环境中的调节作用，并研究肺炎支原体对一种新的人类肥大细胞株的影响。 具体目标3：确定肺炎支原体呼吸道感染是否诱导TH2 CD4淋巴细胞重新聚集到呼吸道，并确定这是否通过依赖于肥大细胞来源的IL-4或其他肥大细胞产物的机制发生。肺炎支原体在肥大细胞诱导的TH2细胞向肺和呼吸道的募集中合作的能力将通过过继转移抗原特异性T细胞来研究。 具体目的4：在肺炎支原体与肥大细胞之间的相互作用已被取消或肥大细胞产生IL-4被阻止的条件下，表征感染对小鼠模型的影响。肺炎支原体P1缺陷株(S)将在小鼠身上测试对慢性感染模型中呼吸道高反应性的影响。将研究肥大细胞上靶分子在肺炎支原体诱导的细胞因子产生中的作用。 在突变的小鼠品系中。

The overall goals of this project are to define innate and adaptive immune mechanisms by which Mycoplasma pneumoniae contributes to the development of chronic airway hyperreactivity and inflammation, focusing on the role of mast cells. Specific Aim 1: To clone and express the P1 adhesin of M. pneumoniae in E. coli and to produce a mutant M. pneumoniae strain deficient in that molecule. The P1 adhesin or select fragments involved in the mast cell IL-4 response will be cloned and expressed in E. coli. Using transposon mutagenesis, a P1-deficient strain of M. pneumoniae will be produced for use in mast cell stimulation experiments and the mouse model of infection. Specific Aim 2: To identify and characterize surface molecules on mast cells that are essential for induction of IL-4 production by P1 glycoprotein, examine modulating effects of other molecules in the pulmonary microenvironment, validate the effects observed using human mast cells, and characterize the effects of knockdown of expression of specific molecules involved in the M. pneumoniae cytokine response in mast cell lines. Target molecules for M. pneumoniae-induced mast cell cytokine production will be identified and characterized and the effects of mutational deletion examined, modulating effects of mediators in the pulmonary microenvironment will be analyzed, and effects of M. pneumoniae in a new human mast cell line will be studied. Specific Aim 3: To determine if respiratory tract infection by M. pneumoniae induces the recruitment to the airways of TH2 CD4 + lymphocytes, and to determine if this occurs using a mechanism that is dependent on mast cell-derived IL-4 or other mast cell products. The ability of M. pneumoniae to cooperate in the mast cell-induced recruitment of TH2 cells to the lungs and airways will be studied using adoptive transfer of antigen specific T cells. Specific Aim 4: To characterize the effects of infection in a mouse model under conditions in which interactions between M. pneumoniae and mast cells have been abrogated or in which mast cell IL-4 production is prevented. P1 deficient strain(s) of M. pneumoniae will be tested in mice for effects on airway hyperreactivity in a chronic infection model. The role of target molecules on the mast cells for M. pneumoniae-induced cytokine production will be studied in mutant strains of mice.