Dominique Leduc

Bacteriophages Can Treat and Prevent Pseudomonas aeruginosa Lung Infections

Antibiotic-resistant bacteria threaten life worldwide. Although new antibiotics are scarce, the use of bacteriophages, viruses that infect bacteria, is rarely proposed as a means of offsetting this shortage. Doubt also remains widespread about the efficacy of phage therapy despite recent encouraging results. Using a bioluminescent Pseudomonas aeruginosa strain, we monitored and quantified the efficacy of a bacteriophage treatment in mice during acute lung infection. Bacteriophage treatment not only was effective in saving animals from lethal infection, but also was able to prevent lung infection when given 24 h before bacterial infection, thereby extending the potential use of bacteriophages as therapeutic agents to combat bacterial lung infection.

Effect of antigen provocation of IL-5 transgenic mice on eosinophil mobilization and bronchial hyperresponsiveness

We investigated whether allergen-induced eosinophil recruitment into mouse airways modifies the in vivo bronchopulmonary responses to standard agonists, and adaptation of a technique described for larger animals. Swiss, CBA, and IL-5 transgenic mice were immunized with ovalbumin and challenged intranasally after 14 days. Immunization alone was followed by increased eosinophil counts in bone marrow and blood, whereas antigenic challenge induced eosinophil infiltration in lungs and bronchoalveolar lavage fluid, which was suppressed by dexamethasone. Despite the high eosinophil counts, no bronchopulmonary hyperreactivity to methacholine or serotonin was detected 3 to 96 hours after antigenic provocation. Our results demonstrate that immunization augments the production of eosinophils by mice, which is further increased by antigenic challenge, but that eosinophil overproduction and lung infiltration, per se, are not sufficient to induce bronchopulmonary hyperreactivity, even in constitutively hypereosinophilic IL-5 transgenic mice.

The Pseudomonas aeruginosa LasB metalloproteinase regulates the human urokinase-type plasminogen activator receptor through domain-specific endoproteolysis.

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala84-Val85 and Thr86-Tyr87 (D1-D2) and Gln279-Tyr280 (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa.

Anthrax Lethal Toxin Impairs IL-8 Expression in Epithelial Cells through Inhibition of Histone H3 Modification

Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis, the etiological agent of anthrax, whose pulmonary form is fatal in the absence of treatment. Inflammatory response is a key process of host defense against invading pathogens. We report here that intranasal instillation of a B. anthracis strain bearing inactive LT stimulates cytokine production and polymorphonuclear (PMN) neutrophils recruitment in lungs. These responses are repressed by a prior instillation of an LT preparation. In contrast, instillation of a B. anthracis strain expressing active LT represses lung inflammation. The inhibitory effects of LT on cytokine production are also observed in vitro using mouse and human pulmonary epithelial cells. These effects are associated with an alteration of ERK and p38-MAPK phosphorylation, but not JNK phosphorylation. We demonstrate that although NF-κB is essential for IL-8 expression, LT downregulates this expression without interfering with NF-κB activation in epithelial cells. Histone modifications are known to induce chromatin remodelling, thereby enhancing NF-κB binding on promoters of a subset of genes involved in immune response. We show that LT selectively prevents histone H3 phosphorylation at Ser 10 and recruitment of the p65 subunit of NF-κB at the IL-8 and KC promoters. Our results suggest that B. anthracis represses the immune response, in part by altering chromatin accessibility of IL-8 promoter to NF-κB in epithelial cells. This epigenetic reprogramming, in addition to previously reported effects of LT, may represent an efficient strategy used by B. anthracis for invading the host.

Edema Toxin Impairs Anthracidal Phospholipase A2 Expression by Alveolar Macrophages

Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A–dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deathly pathogen.

Plasmin cleaves the juxtamembrane domain and releases truncated species of the urokinase receptor (CD87) from human bronchial epithelial cells

The three‐domain (D1D2D3) urokinase receptor (CD87) is highly susceptible to cleavage within the D1–D2 linker sequence, but also within the juxtamembrane region by yet poorly characterized proteinases, allowing the release of D1 and D2D3 species in various (patho)physiological body fluids. Using immunoblot analysis and ELISA applied to a recombinant soluble CD87 and to CD87‐expressing epithelial cells, we establish that exogenous or in situ generated plasmin proteolyzes CD87 in the D1–D2 linker and D3 carboxyterminal sequences, producing a major soluble D2D3 species. Mass spectrometry analysis of the fragmentation of CD87‐related synthetic peptides, and aminoterminal sequencing of D2D3 reveal Arg83, Arg89, and Arg281 as residues targeted by plasmin within human CD87.

Characterization and pharmacological modulation of antigen‐induced peritonitis in actively sensitized mice

1 The intraperitoneal (i.p.) injection of 1 or 10 μg ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose‐dependent increase in the amounts of immunoreactive leukotriene (LT) C4 and LTB4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 μg ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non‐immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC4 and LTB4, but not histamine, release in mouse peritoneum 1 h after its injection. 2 Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels thereafter. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed. 3 Ovalbumin (1 μg)‐induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg−1, s.c.) or with the 5‐lipoxygenase inhibitor, BWA4C (20 mg kg−1, s.c.), whereas indomethacin (2 mg kg−1, s.c.) and the platelet‐activating factor (PAF)‐antagonist SR 27417 (10 mg kg−1, s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo‐oxygenase derivatives or PAF, mediate antigen‐induced eosinophil accumulation in the mouse peritoneum. 4 The histamine H1 receptor antagonist drug, cetirizine (15–30 mg kg−1, s.c.) markedly reduced ovalbumin‐induced eosinophil accumulation under conditions where terfenadine was ineffective, suggesting that the effect of cetirizine was not related to the inhibition of the H1 receptor effects of histamine. 5 The immunosuppressive agent, FK‐506 (1–2 mg kg−1, s.c.) and the protein synthesis inhibitor, cylcoheximide, when administered either in situ (0.06 ng/cavity) or systemically (5 mg kg−1, s.c.), prevented antigen‐induced eosinophil accumulation in the mouse peritoneum, contributing to the concept that substances (probably cytokines) originating from lymphocytes may be involved in the modulation of the eosinophilotactic response in this model. 6 The results of the present study indicate that the i.p. administration of ovalbumin to actively sensitized mice induced late eosinophil accumulation in the peritoneal cavity. This phenomenon, which may be in part mediated by the release of lipoxygenase metabolites and/or by newly generated factors, such as T‐lymphocytes‐derived eosinophilotactic cytokines, offers an interesting tool to investigate the mechanism of action of anti‐allergic and anti‐inflammatory drugs.

Effect of human recombinant interleukin‐5 on in vitro responsiveness to PAF of lung from actively sensitized guinea‐pigs

1 The intra‐tracheal (i.t.) administration of human recombinant interleukin‐5 (rhIL‐5; 100 or 300 ng) to isolated perfused lungs from guinea‐pigs actively sensitized to ovalbumin induced an increased bronchoconstriction and release of thromboxane A2 (TXA2) and histamine into the lung effluent following the subsequent (10 min) intra‐arterial injection of platelet‐activating factor (PAF). Lung responses to 5‐hydroxytryptamine were unaffected by rhIL‐5. 2 Hyperresponsiveness to PAF was observed when the lungs were obtained from guinea‐pigs used 2 or 7 days after a booster injection of the antigen and, to a lower extent, when they were from animals sensitized by a single antigen administration. By contrast, rhIL‐5 did not modify the responses to PAF of lungs from passively sensitized or from adjuvant‐treated guinea‐pigs, suggesting that immunological stimulation is required to allow the expression of synergism between rhIL‐5 and PAF. 3 Guinea‐pigs killed 2 and 7 days after the booster injection of the antigen exhibited a marked increase in the number of eosinophils in the bronchoalveolar lavage fluid (BAL), as compared to non‐sensitized animals. 4 Our results demonstrate that rhIL‐5 and PAF act synergistically to induce enhanced bronchoconstriction and mediator release exclusively when lungs are obtained from guinea‐pigs sensitized once to ovalbumin and then boosted. Since recruitment of eosinophils into the airways and the development of hyperresponsiveness to PAF are concomitant, it is suggested that eosinophils are the target cells for interaction between rhIL‐5 and PAF.

Critical role of cytosolic phospholipase A2α in bronchial mucus hypersecretion in CFTR-deficient mice

Cystic fibrosis (CF) is due to mutations in the CF transmembrane conductance regulator gene CFTR. CF is characterised by mucus dehydration, chronic bacterial infection and inflammation, and increased levels of cytosolic phospholipase A2&agr; (cPLA2&agr;) products in airways. We aimed to examine the role of cPLA2&agr; in the modulation of mucus production and inflammation in CFTR-deficient mice and epithelial cells. Mucus production was assessed using histological analyses, immuno-histochemistry and MUC5AC ELISA. cPLA2&agr; activation was measured using an enzymatic assay and lung inflammation determined by histological analyses and polymorphonuclear neutrophil counts in bronchoalveolar lavages. In lungs from Cftr-/- mice, lipopolysaccharide induced mucus overproduction and MUC5AC expression associated with an increased cPLA2&agr; activity. Mucus overproduction was mimicked by instillation of the cPLA2&agr; product arachidonic acid, and abolished by either a cPLA2&agr; null mutation or pharmacological inhibition. An increased cPLA2&agr; activity was observed in bronchial explants from CF patients. CFTR silencing induced cPLA2&agr; activation and MUC5AC expression in bronchial human epithelial cells. This expression was enhanced by arachidonic acid and reduced by cPLA2&agr; inhibition. However, inhibition of CFTR chloride transport function had no effect on MUC5AC expression. Reduction of CFTR expression increased cPLA2&agr; activity. This led to an enhanced mucus production in airway epithelia independent of CFTR chloride transport function. cPLA2&agr; represents a suitable new target for therapeutic intervention in CF.

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A2 expression through MAPK/NF-κB inactivation

Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.