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Effects of hyperoxia on lethality in mice with Pseudomonas aeruginosa pneumonia were defined, and protective roles of macrolides were examined both in vitro and in vivo. Sub-lethal hyperoxia accelerated lethality of mice with P. aeruginosa pneumonia. Bacterial number was not different in the lungs, but higher in the liver of mice in hyperoxic conditions. Filter-sterilized culture supernatants of bacteria induced loss of viability of alveolar epithelial cells, which was exaggerated in hyperoxia. Metalloprotease blocking by inhibitor or gene-disruption in bacteria resulted in partial reduction of cytotoxic activity in culture supernatants. Co-culture of bacteria with sub-inhibitory concentrations of macrolides, such as azithromycin, reduced cytotoxic activity in the culture supernatants. Azithromycin provided significant survival benefit in hyperoxia-pneumonia model, which was associated with suppression of bacterial dissemination to extra-pulmonary organs. These results suggest that hyperoxia serves as an important cofactor for bacterial dissemination and lethality of P. aeruginosa pneumonia. Our data identify the potential of macrolides to protect individuals with P. aeruginosa pneumonia in the setting of hyperoxia.

Legionella pneumophila is an intracellular organism and the major aetiological agent of Legionnaires' disease. Although recent progress has identified Toll-like receptors (TLRs) as receptors for recognition of pathogen-associated molecular patterns in a variety of micro-organisms, understanding the contribution of TLRs to the host response in L. pneumophila infection is still limited. This study examined the roles of TLR2 and TLR4 in murine L. pneumophila pneumonia and an in vitro infection model using bone-marrow-derived macrophages. TLR2-deficient mice, but not TLR4-deficient mice, demonstrated higher lethal sensitivity to pulmonary challenge with L. pneumophila than wild-type mice (P<0.05). Although no differences in pulmonary bacterial burden were observed among the mouse strains examined, lower values of macrophage inflammatory protein-2 (MIP-2), keratinocyte-derived cytokine and interleukin (IL)-6 and higher IL-12 levels were noted in lung homogenates of TLR2-deficient mice compared with the wild-type control and TLR4-deficient mice. Recruitment of inflammatory cells, particularly neutrophils, was severely disturbed in the lungs of TLR2-deficient mice. Reduced MIP-2 production was demonstrated in bone-marrow-derived macrophages from TLR2-deficient mice in response to live L. pneumophila and purified LPS of this strain, but not Escherichia coli LPS. These data highlight the involvement and importance of TLR2 in the pathogenesis of L. pneumophila pneumonia in mice. The results showed that TLR2-mediated recognition of Legionella LPS and subsequent chemokine-dependent cellular recruitment may be a crucial host innate response in L. pneumophila pneumonia.

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-L-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL-carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL-BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3 x 10(6) c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-alpha in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.