Benoit Raymond

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium‐independent (iPLA2) and cytosolic phospholipase A2 (cPLA2), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC‐1 line infected with the ExoU‐producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA2 inhibitor, as well as significant amounts of the cyclooxygenase (COX)‐derived prostaglandins PGE2 and PGI2. Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non‐infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 104 cfu of PA103 exhibited a marked influx of inflammatory cells and PGE2 release that could be significantly reduced by indomethacin, a non‐selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid–mediated inflammatory response of host organisms.

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.

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.

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host.

Type-IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA(2)-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA(2)-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA(2)-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA(2)-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA(2)-IIA. Transgenic mice over-expressing human sPLA(2)-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA(2)-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA(2)-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA(2)-IIA, indicating that instilled sPLA(2)-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA(2)-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.

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.