Critical Role of TLR-MyD88 Signaling in the Macrophage Response to Mycoplasma pneumoniae

RATIONALE: An association between airway Mycoplasma pneumoniae (Mp) and persistent asthma is well established. To define mechanisms for normal clearance of Mp from the airways, we developed an experimental model of Mp clearance in mice.METHODS: Mice were inoculated with 4x106 Mp intranasally. Whole lungs were harvested for extraction of total RNA and preparation of cell suspensions. Numbers of Mp were determined by Real Time PCR. Recruited leukocytes were characterized by flow cytometry. Bone marrow-derived macrophages (BMM) were infected in vitro with Mp at 1:100 MOI and 1-h later were fixed and stained with anti-NFkB for assessment of nuclear translocation, or lysed for analysis of NFkB phosphorylation by Western blotting. Mp carrying a transposon encoding yellow fluorescent protein (YFP) permitted visualization of interactions between macrophages and the microbe.RESULTS: 24-h after inoculation with Mp, subpopulations of CD11b+ macrophages/monocytes had been recruited into the lungs. Clearance of Mp was impaired in macrophage-deficient Csf1op/op mice. Furthermore, mice deficient in MyD88, a major adaptor for TLR signaling, showed dramatically delayed clearance of Mp, associated with reduced activation and recruitment of macrophages into the lungs. Incubation of macrophages in vitro with Mp induced nuclear translocation and phosphorylation of NFκB, and clearance of YFP-labeled Mp from the cultures. These responses were impaired in MyD88-deficient BMM.CONCLUSIONS: These data establish that macrophages play a central role in the clearance of Mp from the lungs and that MyD88 signaling is essential for activation of the NFkB pathway in the macrophage response to this pathogen. Supported by NIH grant P01HL073907. RATIONALE: An association between airway Mycoplasma pneumoniae (Mp) and persistent asthma is well established. To define mechanisms for normal clearance of Mp from the airways, we developed an experimental model of Mp clearance in mice. METHODS: Mice were inoculated with 4x106 Mp intranasally. Whole lungs were harvested for extraction of total RNA and preparation of cell suspensions. Numbers of Mp were determined by Real Time PCR. Recruited leukocytes were characterized by flow cytometry. Bone marrow-derived macrophages (BMM) were infected in vitro with Mp at 1:100 MOI and 1-h later were fixed and stained with anti-NFkB for assessment of nuclear translocation, or lysed for analysis of NFkB phosphorylation by Western blotting. Mp carrying a transposon encoding yellow fluorescent protein (YFP) permitted visualization of interactions between macrophages and the microbe. RESULTS: 24-h after inoculation with Mp, subpopulations of CD11b+ macrophages/monocytes had been recruited into the lungs. Clearance of Mp was impaired in macrophage-deficient Csf1op/op mice. Furthermore, mice deficient in MyD88, a major adaptor for TLR signaling, showed dramatically delayed clearance of Mp, associated with reduced activation and recruitment of macrophages into the lungs. Incubation of macrophages in vitro with Mp induced nuclear translocation and phosphorylation of NFκB, and clearance of YFP-labeled Mp from the cultures. These responses were impaired in MyD88-deficient BMM. CONCLUSIONS: These data establish that macrophages play a central role in the clearance of Mp from the lungs and that MyD88 signaling is essential for activation of the NFkB pathway in the macrophage response to this pathogen. Supported by NIH grant P01HL073907.