Toll-like receptor 5 (TLR5), IL-1 secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing

Unité de Défense Innée et Inflammation, Institut Pasteur, 75724 Paris, France.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2012; 109(5):1619-24. DOI: 10.1073/pnas.1108464109
Source: PubMed


A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation.

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Available from: Michel Chignard, Mar 18, 2014
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    • "Bacterial phagocytosis was performed as previously described in our laboratory [9]. A total of 5×105 AMs were infected with bacteria (MOI = 10) for 1 h. "
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    ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. While a number of studies have demonstrated the roles of TLR2, TLR4 and TLR5 in host defense againt P. aeruginosa infection, the implication of TLR9 in this process has been overlooked. Here, we show that P. aeruginosa DNA stimulates the inflammatory response through TLR9 pathway in both a cell line and primary alveolar macrophages (AMs). This activation requires asparagine endopeptidase- and endosomal acidification. Interestingly, TLR9-/- mice resisted to lethal lung infection by P. aeruginosa, compared to WT C57BL/6 mice. The resistance of TLR9-/- mice to P. aeruginosa infection was associated with: (i) a higher ability of TLR9-/- AMs to kill P. aeruginosa; (ii) a rapid increase in the pro-inflammatory cytokines such as TNFα, IL-1β and IL-6 production; and (iii) an increase in nitric oxide (NO) production and inductible NO synthase expression in AMs. In addition, inhibition of both IL-1β and NO production resulted in a significant decrease of P. aeruginosa clearance by AMs. Altogether these results indicate that TLR9 plays a detrimental role in pulmonary host defense toward P. aeruginosa by reducing the AMs clearance activity and production of IL-1β and NO necessary for bacteria killing.
    PLoS ONE 03/2014; 9(3):e90466. DOI:10.1371/journal.pone.0090466 · 3.23 Impact Factor
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    • "Furthermore, in healthy subjects TLR-4 is mainly expressed by monocytic cells, whereas in patients with CF the infiltrating neutrophils are the major source of TLR-4 (38). Regarding TLR-5, its involvement has been shown in the recognition of flagelled bacteria like P. aeruginosa and it is thus considered as a critical factor for alveolar macrophage phagocytosis [33]. In our study, CF macrophages did not express TLR‑5 on their membrane compared to non-CF macrophages, suggesting that this toll-like receptor is one of the limiting factors for P. aeruginosa recognition and phagocytosis in CF. "
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    ABSTRACT: Early in life, cystic fibrosis (CF) patients are infected with microorganisms. The role of macrophages has largely been underestimated in literature, whereas the focus being mostly on neutrophils and epithelial cells. Macrophages may however play a significant role in the initiating stages of this disease, via an inability to act as a suppressor cell. Yet macrophage dysfunction may be the first step in cascade of events leading to chronic inflammation/infection in CF. Moreover, reports have suggested that CFTR contribute to altered inflammatory response in CF by modification of normal macrophage functions. In order to highlight possible intrinsic macrophage defects due to impaired CFTR, we have studied inflammatory cytokines secretions, recognition of pathogens and phagocytosis in peripheral blood monocyte-derived macrophages from stable adult CF patients and healthy subjects (non-CF). In CF macrophage supernatants, concentrations of sCD14, IL-1β, IL-6, TNF-α and IL-10 were strongly raised. Furthermore expression of CD11b and TLR-5 were sorely decreased on CF macrophages. Beside, no difference was observed for mCD14, CD16, CD64, TLR-4 and TLR1/TLR-2 expressions. Moreover, a strong inhibition of phagocytosis was observed for CF macrophages. Elsewhere CFTR inhibition in non-CF macrophages also led to alterations of phagocytosis function as well as CD11b expression. Altogether, these findings demonstrate excessive inflammation in CF macrophages, characterized by overproduction of sCD14 and inflammatory cytokines, with decreased expression of CD11b and TLR-5, and impaired phagocytosis. This leads to altered clearance of pathogens and non-resolution of infection by CF macrophages, thereby inducing an exaggerated pro-inflammatory response.
    PLoS ONE 09/2013; 8(9):e75667. DOI:10.1371/journal.pone.0075667 · 3.23 Impact Factor
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    • "Then, 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. Indeed, among the molecular mechanisms and effector molecules relevant to P. aeruginosa elimination by macrophages, which include autophagy, asparagine endopeptidase, NO and ROS, this last has been shown to be negatively affected by CFTR mutations in mice [22]–[24]. Thus, 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. "
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    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.
    PLoS ONE 08/2013; 8(8):e71717. DOI:10.1371/journal.pone.0071717 · 3.23 Impact Factor
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