Abstract
Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.
Highlights
Cystic fibrosis (CF) is the most common genetic disorder affecting the Caucasian population
The live bacteria within the lung CF macrophages did not differ significantly over time, with a median survival of 98% and 92% at 2 h and 4 h, respectively, after infection. Statistical evaluation of these data demonstrated that bacteria survival in CF macrophages was significantly higher than in non-CF macrophages at 4 h after infection, and no significant difference was observed at 2 h, there was a trend to a greater fraction of surviving bacteria in the CF macrophages as compared to the non-CF macrophages
As well as the contribution of epithelial cells to CF lung disease, other immune cells that are either resident or recruited appear to contribute to lung defence. They lack the characteristic CF lung disease, murine models, in particular, provide evidence of reduced elimination of intracellular P. aeruginosa by macrophages [14,15] and alterations in macrophage signalling that contribute to elevated inflammatory responses [16]. In line with these observations, we have recently demonstrated that human MDMs from CF patients infected by P. aeruginosa show a significant increase in intracellular bacteria survival compared to non-CF cells [21]
Summary
Cystic fibrosis (CF) is the most common genetic disorder affecting the Caucasian population. With the aim to identify the bactericidal mechanism affected by the CFTR mutations, we focused our study on the role of NADPH-oxidase-dependent reactive oxygen species (ROS) in P. aeruginosa elimination by human macrophages. We assessed the contribution of ROS to human macrophage activity against P. aeruginosa, by evaluating the generation of the oxidative burst and the effects of NADPH oxidase inhibition on the intracellular bacterial survival in non-CF and CF macrophages. Our data demonstrate that NADPH-dependent ROS are involved in the elimination of intracellular P. aeruginosa within the first few hours after infection This activity was detected in both non-CF and CF macrophages, which suggests that this pathway is not affected by CFTR dysfunction. We provide evidence that as well as ROS, other non-oxidative mechanisms are involved in the elimination of P. aeruginosa by macrophages, as demonstrated by the efficient elimination of these bacteria later in the infection with NADPH-inhibitor treatment of the macrophages
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