Maria-Cristina Plotkowski

Lipid body mobilization in the ExoU-induced release of inflammatory mediators by airway epithelial cells.

This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE(2) that was reduced by the bacterial treatment with MAFP, a cPLA(2) inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE(2). Also, in PA103-infected cells, PGE(2) was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE(2) and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.

ExoU-induced vascular hyperpermeability and platelet activation in the course of experimental Pseudomonas aeruginosa pneumosepsis.

To address the question whether ExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, can induce hemostatic abnormalities during the course of pneumosepsis, mice were instilled i.t. with the ExoU-producing PA103 P. aeruginosa or with a mutant obtained by deletion of the exoU gene. Control animals were instilled with sterile vehicle. To assess the role of ExoU in animal survival, mice were evaluated for 72 h. In all the other experiments, animals were studied at 24 h after infection. PA103-infected mice showed significantly higher mortality rate, lower blood leukocyte concentration, and higher platelet concentration and hematocrit than animals infected with the bacterial mutant, as well as evidences of increased vascular permeability and plasma leakage, which were confirmed by our finding of higher protein concentration in bronchoalveolar lavage fluids and by the Evans blue dye assay. Platelets from PA103-infected mice demonstrated features of activation, assessed by the flow cytometric detection of higher percentage of P-selectin expression and of platelet-derived microparticles as well as by the enzyme immunoassay detection of increased thromboxane A2 concentration in animal plasma. Histopathology of lung and kidney sections from PA103-infected mice exhibited evidences of thrombus formation that were not detected in sections of animals from the other groups. Our results demonstrate the ability of ExoU to induce vascular hyperpermeability, platelet activation, and thrombus formation during P. aeruginosa pneumosepsis, and we speculate that this ability may contribute to the reported poor outcome of patients with severe infection by ExoU-producing P. aeruginosa.

Stenotrophomonas maltophilia Interaction with Human Epithelial Respiratory Cells In Vitro

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co‐incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co‐infection was carried out with heat‐killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.

TLR 5, but neither TLR2 nor TLR4, is involved in lung epithelial cell response to Burkholderia cenocepacia

Burkholderia cenocepacia is known to induce a harmful inflammatory response in the airways of cystic fibrosis patients. Toll-like receptors (TLRs) play key roles in sensing microbial-associated molecular patterns and initiating host innate immunity, but their role in the inflammatory response elicited by B. cenocepacia has not been precisely examined. In this study, we evaluated the contribution of TLR2, TLR4 and TLR5 to the signaling pathways triggered by B. cenocepacia in human bronchial epithelial cells. By quantitative reverse transcriptase (RT)-PCR, we demonstrated that the expression of both TLR2 and TLR4 was significantly upregulated by B. cenocepacia infection, whereas TLR5 expression remained unchanged. Using a dominant-negative approach and airway epithelial cells isolated from MyD88(-/-) mice, we found that B. cenocepacia activated a signaling complex that required the adapter molecule MyD88. Moreover, using epithelial cells from TLR2(-/-), TLR4(-/-) or TLR2/4(-/-) mice or cells overexpressing a functional form of TLR5, we established that TLR5, but neither TLR2 nor TLR4, critically regulated B. cenocepacia-induced lung epithelial inflammatory response.

Lack of MyD88 Protects the Immunodeficient Host Against Fatal Lung Inflammation Triggered by the Opportunistic Bacteria Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen of major concern for cystic fibrosis patients as well as immunocompromised cancer patients and transplant recipients. The mechanisms by which B. cenocepacia triggers a rapid health deterioration of the susceptible host have yet to be characterized. TLR and their key signaling intermediate MyD88 play a central role in the detection of microbial molecular patterns and in the initiation of an effective immune response. We performed a study to better understand the role of TLR-MyD88 signaling in B. cenocepacia-induced pathogenesis in the immunocompromised host, using an experimental murine model. The time-course of several dynamic parameters, including animal survival, bacterial load, and secretion of critical inflammatory mediators, was compared in infected and immunosuppressed wild-type and MyD88−/− mice. Notably, when compared with wild-type mice, infected MyD88−/− animals displayed significantly reduced levels of inflammatory mediators (including KC, TNF-α, IL-6, MIP-2, and G-CSF) in blood and lung airspaces. Moreover, despite a higher transient bacterial load in the lungs, immunosuppressed mice deficient in MyD88 had an unexpected survival advantage. Finally, we showed that this B. cenocepacia-induced life-threatening infection of wild-type mice involved the proinflammatory cytokine TNF-α and could be prevented by corticosteroids. Altogether, our findings demonstrate that a MyD88-dependent pathway can critically contribute to a detrimental host inflammatory response that leads to fatal pneumonia.

Pseudomonas aeruginosa -induced production of free radicals by IFNγ plus TNFα-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?

We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNγ plus TNFα to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed. In this study we investigated the fate of IFNγ plus TNFα‐activated HUVEC that have harboured IC bacteria for up to 24 h. At 10 h, the endothelial cell killing of P. aeruginosa isolates exceeded 90%. IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC. P. aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH). Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability. Treatment of cells with the antioxidant α‐lipoic acid significantly increased the survival of infected cells. Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells. Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.

ExoU activates NF-κB and increases IL-8/KC secretion during Pseudomonas aeruginosa infection.

ExoU, a Pseudomonas aeruginosa cytotoxin injected into host cytosol by type III secretion system, exhibits a potent proinflammatory activity that leads to a marked recruitment of neutrophils to infected tissues. To evaluate the mechanisms that account for neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We demonstrate that ExoU increases IL-8 mRNA and protein levels in P. aeruginosa-infected epithelial and endothelial cell lines. Also, ExoU induces the nuclear translocation of p65/p50 NF-κB transactivator heterodimer as well as NF-κB-dependent transcriptional activity. ChIP assays clearly revealed that ExoU promotes p65 binding to NF-κB site in IL-8 promoter and the treatment of cultures with the NF-κB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. These results were corroborated in a murine model of pneumonia that revealed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage when mice were treated with Bay 11-7082 before infection with an ExoU-producing strain. In conclusion, our data demonstrate that ExoU activates NF-κB, stimulating IL-8 expression and secretion during P. aeruginosa infection, and unveils a new mechanism triggered by this important virulence factor to interfere in host signaling pathways.

Pseudomonas aeruginosa binds to soluble cellular fibronectin

We have investigatedPseudomonas aeruginosa binding to plasma and cellular fibronectin (FN), in both their soluble and insoluble forms. Bacterial binding to insoluble FN was studied by exposing coverslips coated with FN to radiolabeled microorganisms.P. aeruginosa binding to soluble FN was investigated (1) by comparing radiolabeled bacteria treated with FN with PBS-treated bacteria in their adhesion to a collagen matrix; (2) by analyzing the reactivity ofP. aeruginosa with plasma or cellular FN adsorbed to gold particles with transmission electron microscopy (TEM).P. aeruginosa did not bind significantly to insoluble plasma or cellular FN, or to soluble plasma FN. In contrast, bacterial treatment with soluble cellular FN significantly increased the adhesion to the collagen matrix. With TEM, we confirmed the reactivity ofP. aeruginosa with soluble cellular FN. Because there is a marked secretion of cellular FN during wound repair, we speculate that this reactivity may account for the propensity ofP. aeruginosa to infect repairing tissues.

Pseudomonas aeruginosa toxin ExoU induces a PAF-dependent impairment of alveolar fibrin turnover secondary to enhanced activation of coagulation and increased expression of plasminogen activator inhibitor-1 in the course of mice pneumosepsis

BackgroundExoU, a Pseudomonas aeruginosa cytotoxin with phospholipase A2 activity, was shown to induce vascular hyperpermeability and thrombus formation in a murine model of pneumosepsis. In this study, we investigated the toxin ability to induce alterations in pulmonary fibrinolysis and the contribution of the platelet activating factor (PAF) in the ExoU-induced overexpression of plasminogen activator inhibitor-1 (PAI-1).MethodsMice were intratracheally instilled with the ExoU producing PA103 P. aeruginosa or its mutant with deletion of the exoU gene. After 24 h, animal bronchoalveolar lavage fluids (BALF) were analyzed and lung sections were submitted to fibrin and PAI-1 immunohistochemical localization. Supernatants from A549 airway epithelial cells and THP-1 macrophage cultures infected with both bacterial strains were also analyzed at 24 h post-infection.ResultsIn PA103-infected mice, but not in control animals or in mice infected with the bacterial mutant, extensive fibrin deposition was detected in lung parenchyma and microvasculature whereas mice BALF exhibited elevated tissue factor-dependent procoagulant activity and PAI-1 concentration. ExoU-triggered PAI-1 overexpression was confirmed by immunohistochemistry. In in vitro assays, PA103-infected A549 cells exhibited overexpression of PAI-1 mRNA. Increased concentration of PAI-1 protein was detected in both A549 and THP-1 culture supernatants. Mice treatment with a PAF antagonist prior to PA103 infection reduced significantly PAI-1 concentrations in mice BALF. Similarly, A549 cell treatment with an antibody against PAF receptor significantly reduced PAI-1 mRNA expression and PAI-1 concentrations in cell supernatants, respectively.ConclusionExoU was shown to induce disturbed fibrin turnover, secondary to enhanced procoagulant and antifibrinolytic activity during P. aeruginosa pneumosepsis, by a PAF-dependent mechanism. Besides its possible pathophysiological relevance, in vitro detection of exoU gene in bacterial clinical isolates warrants investigation as a predictor of outcome of patients with P. aeruginosa pneumonia/sepsis and as a marker to guide treatment strategies.

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection

BackgroundBurkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays.MethodsB. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase.ResultsET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs.ConclusionB. cenocepacia were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for B. cenocepacia-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care B. cenocepacia-infected patients.