Mechanism of Mycoplasma-Induced Mast Cell Il-4 Synthesis

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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.