Cathepsin L protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis.

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.

Mycoplasma pulmonis and lymphoma in bioassays in rats.

Lymphomas were reported to be induced in rats in bioassays of aspartame, methyl-tertiary-butyl ether (MTBE), and other chemicals conducted by a nonprofit cancer research organization. European regulatory authorities concluded that lymphomas in the aspartame study were caused by Mycoplasma pulmonis and suggested that this also was the case for the MTBE bioassay. To assess the role of M. pulmonis in these bioassays, we reviewed the tumor data for the aspartame and MTBE bioassays and, additionally, the organization's bioassay of methanol. For all 3 studies, the most frequently reported hematopoietic neoplasm was lympho-immunoblastic lymphoma, the most frequently affected organ was the lung, and, in almost half of the rats with this diagnosis, the lung was the only affected organ. Lesions diagnosed as lymphoma in published illustrations had pleomorphic cellular morphology and appeared to contain neutrophils. Information from these reports and other sources indicated that lesions typical of M. pulmonis disease were prevalent among the aspartame and MTBE study rats and that the rats were not specific-pathogen-free. Because the lymphoma type, cellular morphology, and organ distribution reported in these studies are atypical of lymphoma in rats, because lymphocyte and plasma cell accumulation in the lung is characteristic of M. pulmonis disease, and because M. pulmonis disease can be exacerbated by experimental manipulations, including chemical treatment, we suggest that a plausible alternative explanation for the reported results of these bioassays is that the studies were confounded by M. pulmonis disease and that lesions of the disease were interpreted as lymphoma.

Effect of experimental genital mycoplasmosis on production of matrix metalloproteinases in membranes and amniotic fluid of Sprague-Dawley rats.

PROBLEM: Preterm, premature rupture of membranes (PPROM) is a dire pregnancy outcome that is frequently associated with infection by the genital mycoplasmas, Mycoplasma hominis, Ureaplasma parvum, and U. urealyticum. One potential mechanism by which these microorganisms may cause PPROM is by increasing the concentration of matrix metalloproteinases (MMPs) in the membranes and amniotic fluid. We tested this hypothesis in a well-defined model system of genital infection with M. pulmonis, a natural reproductive pathogen of rats. METHOD OF STUDY: Timed-pregnant, specific pathogen-free, Sprague-Dawley rats were infected with 10(7) CFU M. pulmonis at gestation day (gd) 14. Controls received an equivalent volume (100 microL) of sterile medium. At gd 18, rats were euthanized, and membranes and amniotic fluids were harvested and stored at -70 degrees C until analysis. Proteinase activity of amniotic fluid and membranes was resolved on discontinuous 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography gels. Band intensity was determined using a digital gel documentation system and the manufacturer's software (Kodak). RESULTS: Gelatinolytic activity associated with a band similar in molecular weight to ProMMP-9 (92 kDa, the inactive precursor of MMP-9) was significantly increased in amniotic fluids and membranes harvested from M. pulmonis-treated pups at gd 18 when compared with tissues harvested from control pups. Both ProMMP-9 and ProMMP-2 (72 kDa, the inactive precursor of MMP-2) were increased in infected animals at gd 21. CONCLUSION: Our study suggests that the genital mycoplasmas can increase MMP-9 production in vivo.

Neutrophil histamine contributes to inflammation in mycoplasma pneumonia.

Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell-deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.

T-bet deficiency facilitates airway colonization by Mycoplasma pulmonis in a murine model of asthma.

Epidemiological and clinical evidence suggest a correlation between asthma and infection with atypical bacterial respiratory pathogens. However, the cellular and molecular underpinnings of this correlation remain unclear. Using the T-bet-deficient (T-bet(-/-)) murine model of asthma and the natural murine pathogen Mycoplasma pulmonis, we provide a mechanistic explanation for this correlation. In this study, we demonstrate the capacity of asthmatic airways to facilitate colonization by M. pulmonis and the capacity of M. pulmonis to exacerbate symptoms associated with acute and chronic asthma. This mutual synergism results from an inability of T-bet(-/-) mice to mount an effective immune defense against respiratory infection through release of IFN-gamma and the ability of M. pulmonis to trigger the production of Th2-type cytokines (e.g., IL-4 and IL-5), and Abs (e.g., IgG1, IgE, and IgA), eosinophilia, airway remodeling, and hyperresponsiveness; all pathophysiological hallmarks of asthma. The capacity of respiratory pathogens such as Mycoplasma spp. to dramatically augment the pathological changes associated with asthma likely explains their association with acute asthmatic episodes in juvenile patients and with adult chronic asthmatics, >50% of whom are found to be PCR positive for M. pneumoniae. In conclusion, our study demonstrates that in mice genetically predisposed to asthma, M. pulmonis infection elicits an inflammatory milieu in the lungs that skews the immune response toward the Th2-type, thus exacerbating the pathophysiological changes associated with asthma. For its part, airways exhibiting an asthmatic phenotype provide a fertile environment that promotes colonization by Mycoplasma spp. and one which is ill-equipped to kill and clear respiratory pathogens.