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

Microbial Pathogenesis 45 (2008) 30–37

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Lipid body mobilization in the ExoU-induced release of inflammatory mediators by airway epithelial cells Maria-Cristina Plotkowski a, *, Bruno A. Branda˜o a, Maria-Cristina de Assis a, Luis-Filipe P. Feliciano a, Benoit Raymond c, Carla Freitas a, Alessandra M. Saliba a, Jean Marie Zahm d, Lhousseine Touqui c, Patrı´cia T. Bozza b a

Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Cieˆncias Me´dicas, Universidade do Estado do Rio de Janeiro, Avenue 28 de Setembro, 87 Fundos, 3 andar, 20 551-030, Rio de Janeiro, Brazil ˆmica, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil Departamento de Fisiologia e Farmacodina c Institut Pasteur, Unite´ de Defense Inne´e et Inflammation, INSERM U 874, Paris, France d INSERM U 903, Universite´ de Reims Champagne-Ardenne, Reims, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 November 2007 Received in revised form 22 January 2008 Accepted 25 January 2008 Available online 28 March 2008

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 PGE2 that was reduced by the bacterial treatment with MAFP, a cPLA2 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 PGE2. Also, in PA103-infected cells, PGE2 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 PGE2 and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Pseudomonas aeruginosa ExoU Lipid body Inflammatory response Arachidonic acid PGE2

1. Introduction Pseudomonas aeruginosa airway infections are usually associated with intense inflammatory response that plays a major role in the physiopathology of lung disease. P. aeruginosa pathogenicity involves virulence factors injected directly into eukaryotic cells via the type III secretory system. Prominent among them is ExoU, a toxin with phospholipase A2 (PLA2) activity that encompasses a broad range of substrates, including neutral lipids and phospholipids [1,2]. Following translocation into host cell cytosol, ExoU is activated by eukaryotic cell cofactors [3] and is targeted to cell plasma membranes [4,5]. Such targeting of the active enzyme to membranes rich in phospholipids is likely to be of importance in the cytotoxicity associated with ExoU, but can also favor the production of lipid second messengers that can subvert normal cellular processes [6].

* Corresponding author. Tel.: þ55 21 25876380; fax: þ55 21 2587 6476. E-mail address: [email protected] (M.-C. Plotkowski). 0882-4010/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2008.01.008

Enzymes with PLA2 activity catalyse the hydrolysis of ester bonds at the sn-2 position of cell glycerophospholipids, generating free fatty acids, including arachidonic acid (AA), and lysophospholipids. AA, a poly unsaturated fatty acid, is the precursor of eicosanoids, including the cyclooxygenase-derived prostaglandins and the lipoxygenase-derived leukotrienes [7]. AA metabolites, alone or in synergy with other mediators, control the exudate formation and cell influx in inflammatory reactions [8]. Although the biochemistry and pharmacology of the AA metabolites have been described in detail, questions remain about the early events leading to the formation of these molecules and, particularly, the subcellular sites within the cell from which AA can be mobilized. It is widely believed that cell membrane phospholipids are the major source of AA entering the cyclooxygenases or lipoxygenases pathway of oxidation, but alternative routes have been proposed. Indeed, studies suggested that lipid bodies represent major metabolic pools of eicosanoid precursors distinct from cellular membranes [9,10]. Lipid bodies are membraneless cytoplasmic inclusions found not only in fat-storage cells but also in many other cell types,

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bacteria, with its mutant PA103DexoU obtained by deletion of the exoU gene [17], with an ExoU-deficient mutant complemented with the exoU gene (PA103DUT/exoU), which secretes functionally active toxin, or with bacteria complemented with exoU coding for a toxin with a site-directed mutation in its serine catalytic site (PA103DUT/ S142A) [18]. At 1 h post-infection, cells infected with the ExoUproducing strains (PA103 and PA103DUT/exoU) released significantly more AA than control non-infected cells, than cells infected with the exoU-deficient strain or with bacteria complemented with the mutated gene (Fig. 1A). Previous treatment of bacteria with MAFP (methyl arachidonyl fluorophosphonate), a PLA2 inhibitor, reduced significantly their ability to induce the release of free AA (Fig. 1B), consistent with the phospholipase activity of ExoU. P. aeruginosa has been shown to induce the phosphorylation of cPLA2 from lung epithelial cells, resulting in increased release of free AA [19]. To ascertain the contribution of enhanced endogenous cPLA2 activity of PA103-infected cells to the over release of AA detected in our experiments, we analyzed, by immunoblot assays, the level of cPLA2 and phosphorylated cPLA2 proteins in control cells and in cells infected for 6 h and 24 h with the ExoU-producing PA103 and the exoU-deficient PA103DexoU strains. Similar to a previous study [20], neither cPLA2 nor phosphorylated cPLA2 were detected in BEAS-2B cell extracts (data not shown), further confirming the role of the ExoU in the AA release by PA103- and PA103DUT/exoU-infected airway epithelial cells. We next investigated whether AA released from cell phospholipids could be metabolized to eicosanoids. As shown in Fig. 1C, the ExoU-producing P. aeruginosa strains enhanced significantly the release of PGE2 by airway cells. Cells infected with the exoUdeficient mutant complemented with the mutated gene (PA103DUT/S142A) did not differ from those infected with the exoU-deficient mutant. Fig. 2 show that cells infected with the wild type bacteria released also increased concentrations of the cytokines IL-6 (A) and IL-8 (B).

composed of a core of neutral lipids covered by a monolayer of phospholipids, free cholesterol and several functionally diverse types of proteins [11,12]. Rather than being inert storage droplets, lipid bodies are dynamic organelles intimately linked to membrane transport pathways in the cell. Previous studies have shown that stimuli-elicited compartmentalization of lipids to form new lipid bodies is associated with enhanced cell capacity for eicosanoid generation (reviewed in Ref. [13]). Consistent with the hypothesis that the cellular responses leading to lipid body mobilization may be important in the eicosanoids synthesis during inflammation, lipid bodies have been shown to be enriched in lipoxygenase and cyclooxygenase eicosanoid-forming enzymes [9,14,15], cytosolic PLA2 (cPLA2) and their regulatory MAP kinases [16], as well as in AA esterified to both neutral lipids and phospholipids. We have recently shown that, due to its PLA2 activity, ExoU exhibits an eicosanoid-mediated proinflammatory activity by showing that endothelial cells infected with an ExoU-producing P. aeruginosa strain released high amounts of free AA and prostanoids [17]. We also showed that bronchoalveolar fluids from mice intratracheally infected with the ExoU-producing wild type bacteria exhibited more inflammatory cells and higher concentrations of PGE2 than fluids from control animals and from mice infected with an exoU-deficient mutant [17]. However, we did not investigate the contribution of the airway epithelial cells in the mice inflammatory response to ExoU. In the present study we investigated the capability of airway epithelial respiratory cells to contribute to an ExoU-induced inflammatory response and the role of lipid bodies as intracellular loci involved in eicosanoid synthesis by infected airway cells. 2. Results 2.1. Airway epithelial cells released increased concentrations of inflammatory mediators when challenged with ExoU-producing P. aeruginosa strains

2.2. Airway epithelial cells are enriched in lipid bodies To ascertain the capability of airway epithelial cells to contribute to the inflammatory response elicited in the airways of mice infected intratracheally with the ExoU-producing PA103 P. aeruginosa strain, we first assessed the presence of free AA in the supernatants of human cells from the BEAS-2B line previously labelled with [3H] AA, after infection with the wild type PA103

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Fig. 1. Release of [3H] AA (A) and PGE2 (C) by control BEAS-2B cells or by cells infected with different P. aeruginosa strains at 1 h and 24 h post-infection, respectively. In (B) it is shown the inhibitory effect of the bacterial treatment with the cPLA2 inhibitor MAFP on the release of [3H] AA by cells infected with the ExoU-producing strains. Data represent mean values of typical experiments out of two performed in triplicate. Bars represent standard errors of the means. ***p < 0.001 when data obtained with PA103 and PA103DUT/ exoU were compared with those from controls or from PA103DexoU- and PA103DUT/S142A-infected cells (A and C) or when the release of AA by cells infected with PA103 and PA103DUT/exoU previously treated with MAFP were compared with those from cells infected with untreated bacteria (B).

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cells, although some grains were also detected at the cell periphery. Moreover, infection with PA103 reduced significantly the content of silver grains over infected cells (Fig. 4C), consistent with the high amount of [3H] AA detected in the supernatants of PA103-infected cultures (Fig. 1A).

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Fig. 2. Release of IL-6 (A) and IL-8 (B) by control and P. aeruginosa-infected airway epithelial cells at 24 h post-infection. Data represent mean values of typical experiments out of two performed in triplicate. Bars represent standard errors of the means. *p < 0.05; ***p < 0.001.

membranes were labelled weakly or not at all [13,21]. We wondered, therefore, whether lipid bodies may have been involved in the ExoU-induced release of free [3H]-AA and PGE2 by infected cells. However, because to our knowledge these organelles have never been described in airway epithelial cells, before assessing the engagement of lipid bodies in ExoU-induced PGE2 production, we looked for their presence in BEAS-2B cells by using a standard protocol for lipid staining with OsO4 [14,22]. As shown in Fig. 3A, numberless small perinuclear lipid bodies were detected in noninfected BEAS-2B cells. Because transformed cells may differ substantially from cells in primary culture, we next investigated the presence of lipid bodies in mice tracheobronchial epithelial cells and in human cells newly isolated from nasal polyps, both in primary culture. As shown in Fig. 3B and C, both cell types exhibited lipid bodies in much higher numbers than described in nonstimulated human inflammatory cells [14,22]. 2.3. Lower amounts of cytosolic [3H] AA were detected in PA103-infected cells To address the question of the localization of exogenous [3H] AA in BEAS-2B cells, and of the ExoU capability to metabolize [3H] AA-containing cell lipids, radiolabelled cells were submitted to light microscopic autoradiography. As shown in Fig. 4A and B, silver grains were localized predominantly in the cytoplasm of BEAS-2B

The generation of lipid bodies can be rapidly elicited in vitro and in vivo by different stimuli, including bacterial LPS [14] and cis-unsaturated fatty acids, such as AA [15]. Since both stimuli were present during our assays, we anticipated at the start of our investigation that cells infected with the ExoU-producing bacteria would exhibit increased formation of these organelles. Surprisingly, PA103-infected cells exhibited fewer lipid bodies than control cells (Fig. 5A), as determined with an image-based quantitative analysis of OsO4-stained cells. Fig. 5B shows a representative microphotograph of BEAS-2B cells with countless lipid bodies, whereas Fig. 5C shows the image obtained after the micrograph treatment with the software. We next compared the content of lipid bodies in control and infected cells by FACS analysis of cell staining with the fluorescent probe BODIPY. Similar to the results obtained with OsO4stained cells, a significant reduction in the content of lipid bodies was detected in airway cells infected with the ExoU-producing PA103 strain (Fig. 6A and C). Complementation of the exoUdeficient mutant with the wild type gene (PA103DUT/exoU), but not with the gene with site-specific mutation in the toxin catalytic domain (PA103DUT/S142A), restored the bacterial ability to reduce the content of lipid bodies (Fig. 6A and C). Bacterial treatment with the PLA2 inhibitor MAFP abolished almost completely their ability to reduce the content of BODIPY-stained lipid bodies (Fig. 6B and D). Together, these results clearly show that the ability of ExoU to modulate the content of lipid bodies correlates with its PLA2 activity and is dependent on the enzyme hydrolase motif containing a catalytic serine residue. 2.5. The decrease in the content of lipid bodies correlated with the presence of intracellular PGE2 To explain the decrease in lipid bodies in cells infected with the ExoU-producing bacteria, we hypothesized that, in those cells, AA released from lipid bodies, phospholipids by the bacterial PLA2-like toxin was locally metabolized to eicosanoids reducing, somehow, the lipid body affinity for the fluorescent probe. To address this hypothesis, we first looked for immunocytochemical evidences of PGE2 synthesis in lipid bodies. Fig. 7A shows a phase contrast

Fig. 3. Light micrographs of BEAS-2B (A) and of mice (B) or human airway epithelial cells (C) in primary culture exhibiting numberless OsO4-stained lipid bodies.

M.-C. Plotkowski et al. / Microbial Pathogenesis 45 (2008) 30–37

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Fig. 4. Light microscopic autoradiograph of BEAS-2B cells incubated with [3H] AA and infected with the ExoU-producing PA103 (A) and ExoU-deficient PA103DexoU at 3 h postinfection (B). Note the distribution of silver grains mainly over the cell cytoplasm, although some grains were detected at the cell periphery. Infection with PA103 reduced the number of silver grains over the cells. (C) Exhibits the mean number of grains detected over 25 cells infected with each bacterial strain. Bars represent standard deviations. ***p < 0.001 when PA103-infected cells were compared with PA103DexoU-infected cells.

micrograph taken from a cell infected with PA103 containing many lipid bodies, whereas Fig. 7B shows the same cell labelled with antiPGE2 antibody. Merged image (Fig. 7C) shows colocalization of newly synthesized PGE2 with lipid bodies. We next looked for quantitative evidence of PGE2 synthesis in lipid bodies. Fig. 8A exhibits density plots representative of those obtained in different FACS assays, showing the distribution of control cells and of cells infected with PA103 and with PA103DexoU according to their simultaneous labelling with BODIPY and with the anti-PGE2 antibody. Whereas more than 95.0% of the cells from control cultures and from cultures infected with the exoU-deficient bacteria were labelled with BODIPY, in cultures infected with PA103 this percentage was lower than 30%. In contrast, in PA103-infected cultures, the percentage of cells containing intracellular PGE2 (12.7%) was higher than in control (4.0%) and in PA103DexoU-infected cultures (7.1%). Fig. 8B illustrates the distribution of PA103-infected cells according to their simultaneous reactivity with BODIPY and an antibody against PGE2 and defines the BODIPY-labelled cell population named R2. The mean number of cells in control cultures and in PA103DexoUinfected cultures in R2 (BODIPY labelled) was significantly higher than in cultures infected with PA103 (Fig. 8C). Nonetheless, the median of the PGE2 labelling intensity in PA103-infected cultures was significantly higher (Fig. 8D), clearly showing that although ExoU reduced somehow the number of lipid bodies in airway epithelial cells, the remaining organelles were intensely labelled with the anti-PGE2 antibody.

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3. Discussion and conclusions The response of airway epithelial cells to P. aeruginosa infection involves the release of proinflammatory cytokines and a marked recruitment of macrophages and neutrophils to the infected region. Different bacterial factors contribute to the early development of inflammatory response. Prominent among them is flagellin but other P. aeruginosa products, such as secreting quorum-sensing homoserine lactones, alginate, pyocyanin and/or other secreted factors like proteases and exotoxin A may also trigger proinflammatory signaling in airway epithelial cells (see Refs. [23] and [24] for review). In this study we report the ExoU-induced release of IL-6 and IL-8 as well as free AA and PGE2 by human airway epithelial cells and we speculate that overproduction of these proinflammatory mediators may contribute to the airway inflammation detected in the course of P. aeruginosa respiratory infections. Moreover, we suggest that lipid bodies may represent intracellular loci involved in ExoU-induced synthesis of prostanoids. To our knowledge, this is the first report suggesting the mobilization of these organelles by a bacterial toxin. Lipid bodies are known to be of importance in eicosanoid synthesis by human leukocytes and macrophages. They were shown to be increased in size and number in cells associated with human airway inflammatory diseases, including neutrophils recovered from the bronchoalveolar fluids of patients with sepsis [14] and adult respiratory distress syndrome [25] but, to our knowledge, they had never been described in airway epithelial cells.

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0 Fig. 5. Mobilization of lipid bodies in cells infected with ExoU-producing PA103 P. aeruginosa. (A) Semi-automatic quantification of cell area occupied by lipid bodies in cells from control cultures and from cultures infected with the ExoU-producing PA103 and with the ExoU-deficient PA103DexoU bacteria at 3 h post-infection. Data represent mean values of the results obtained with 25 cells from 12 different areas of each kind of culture. Bars represent standard errors of the means; *p < 0.05 and ***p < 0.001 when data from PA103DexoU- and PA103-infected cells were compared with those from control non-infected cells. (B) Light micrograph of lipid bodies in resting OsO4-stained cells; (C) binary images of the OsO4-stained lipid bodies obtained according to the so-called top-hat procedure.

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Fig. 6. (A) FACS detection of BODIPY-labelled cells in control non-infected cultures and in cultures infected with the different P. aeruginosa strains at 3 h post-infection. (B) Inhibitory effect of MAFP on the reduction of lipid body content in cells infected with the ExoU-producing PA103 and PA103DUT/exoU mutant. Data represent mean values of the results obtained in three different assays performed in triplicate. Bars represent standard errors of the means. In (A) ***p < 0.001 when data from PA103- and PA103DUT/exoU-infected cells were compared with those from control cells or from cells infected with the PA103DexoU mutant. In (B) *p < 0.05 and **p < 0.01 when data obtained from cells infected with MAFPtreated bacteria were compared with those obtained with untreated bacteria. (C) and (D) exhibit representative histograms obtained at 3 h post-infection of cells infected with MAFP-untreated and MAFP-treated bacteria, respectively. y-Axis corresponds to cell number whereas x-axis corresponds to log fluorescence intensity.

In normal host, airway epithelial cells provide barrier and surveillance functions and contribute to clear inhaled organisms by producing proinflammatory cytokines and chemokines to recruit and activate effector phagocytic cells [26,27]. Our finding that airway epithelial cells contain large numbers of lipid bodies that are readily mobilized in response to bacterial insult, resulting in the release of proinflammatory eicosanoids, raises another mechanisms by which airway epithelial cells can accomplish their surveillance function but also create a harmful inflammatory environment in the airway mucosa.

In this report, besides providing evidences of the marked expression of lipid bodies in airway epithelial cells, we showed the ability of ExoU to modulate their expression. However, in contrast with other reports that showed increased formation of lipid bodies in leukocytes exposed to Trypanosoma cruzi [28] or Mycobacterium bovis [22], the airway cells exposed to P. aeruginosa ExoU exhibited significantly decreased LB contents. Our findings are especially remarkable because this ExoU-induced consumption of lipid bodies was reduced by previous treatment of bacteria with a cPLA2 inhibitor.

Fig. 7. Light micrographs of PA103-infected BEAS-2B cell at 3 h post-infection showing lipid bodies detected by phase contrast microscopy (A) and newly synthesized PGE2 detected by cell labelling with a polyclonal anti-PGE2 antibody and with a secondary antibody-Texas Red complex (B). Merged image shows localization of PGE2 in lipid bodies (C).

M.-C. Plotkowski et al. / Microbial Pathogenesis 45 (2008) 30–37

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Fig. 8. (A) Representative density plot histograms obtained in 1 out of 5 FACS analysis of control and infected cells probed with BODIPY (x-axis) and anti-PGE2 antibody (y-axis) at 3 h post-infection. (B) Density plot histogram delineating the BODIPY-labelled cell population (R2 region) further analyzed. (C), Number of BODIPY-labelled cells in R2 population detected in control cultures and in cultures infected with the wild type bacteria and the ExoU-deficient mutant at 1 h and 3 h post-infection. (D) Median of the PGE2 labelling intensity in cells detected in the R2 population at 1 h and 3 h post-infection. Data in (C) and (D) represent mean values of the results obtained in five different FACS analysis performed in triplicate. Bars represent standard errors of the means. **p < 0.01 when data obtained with PA103-infected cells were compared with those from control and PA103DexoU-infected cultures.

Hydrolysis of triacylglycerol stored in lipid bodies, a major mechanism by which adipocytes modulate fatty acyl flux in response to host metabolic demands, depends on the lipases HSL (Hormone Sensitive Lipase) and ATGL (Adipose Triglyceride Lipase). ATGL function is also of importance for lipid body turnover in other mammalian cell type. As recently reported [29], ATGL overexpression in HeLa cells caused a decrease in the average size of lipid bodies, whereas the enzyme depletion by RNA interference led to a significant increase in the size of the organelles. In both adipocytes and non-adipocyte cells, ATGL was immunolocalized in the external surface of lipid bodies, known to be enriched in phospholipids [11,12]. Excitingly, studies [30,31] have shown significant sequence similarities in the catalytic domains of ATGL and of different serine esterases, including PLA2 enzymes and other members of the patatin superfamily exhibiting a serine–aspartate catalytic dyad. A number of observations suggest that serine–aspartate dyad accounts also for ExoU activity: (i) the alignment of ExoU with cPLA2 and iPLA2 identified serine 142 (S142) and aspartate 344 (D344) as putative catalytic amino acids [1,2]; (ii) inhibitors containing a serine-reactive group, such as MAFP, inhibit cPLA2 and ExoU activity; (iii) site-specific mutagenesis of either S142 or D344 to alanine suppresses the ExoU phospholipase activity and cytotoxicity [18]. In our study, the ability of ExoU to reduce the content of lipid bodies in infected cells was suppressed by MAFP treatment of bacteria. Moreover, exoU-depleted bacteria complemented with site-directed mutate exoU serine catalytic

motif [18] did not differ from exoU-depleted bacteria in their capability to reduce the content of lipid bodies. It would be, therefore, tempting to conclude that the decrease in the lipid body content of cells infected with PA103 and PA103DUT/exoU depended on the hydrolysis of triacylglycerol from the core of the organelles by ExoU. However, further studies will be required to clarify whether this toxin exert a direct effect on triacylglycerol turnover. A general problem faced by toxins injected into host cell cytoplasm is how to localize to their site of action without becoming degraded within the cell. In recent papers, it was shown that ExoU rapidly localize to plasma membranes of infected cells [4,5]. This subcellular location is likely to favor the hydrolysis of membrane phospholipids. Based on our results we propose that ExoU may be targeted to other phospholipid-enriched cell domains, i.e., lipid bodies, and that such targeting may have pathogenic relevance by contributing to the release of proinflammatory AA metabolites. In conclusion, we suggest that the ExoU-induced release of inflammatory cytokines and eicosanoids by airway epithelial cells may represent a novel mechanism by which P. aeruginosa maintains an intense inflammatory response in host lungs. Since ExoU is highly cytotoxic, epithelial damage is likely to favor both the leakage of inflammatory mediators into the general circulation and bacterial dissemination [32], which may explain the clinical significance of ExoU production, considered to be a marker for highly virulent bacteria isolated from patients with hospitalacquired pneumonia [33] and bacteremia [34].

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4. Materials and methods

determined. The concentrations of the inflammatory mediators were reported as pg per 105 cells.

4.1. Bacterial strains and culture conditions 4.5. Detection of cPLA2 activation The laboratory P. aeruginosa PA103 strain and its mutant PA103DexoU, constructed by deletion of the exoU gene [17], were used in this study. We also used two exoU-depleted mutants complemented with either wild type exoU gene (PA103DUT/exoU) or exoU with site-specific mutation from serine to alanine at aminoacid 142 of the toxin (PA103DUT/S142A) [18], a generous donation of Dr. Alan Hauser (Northwestern University, Chicago). Bacteria were grown in Luria–Bertani (LB) broth at 37  C for 14–16 h under mild agitation, harvested by centrifugation and resuspended in M-199 cell culture medium (Sigma–Aldrich) to an absorbance at 640 nm of 0.1, corresponding to about 108 colony forming units (CFU)/mL. 4.2. Cell culture and infection Human bronchial epithelial cells from the BEAS-2B line were cultured in cell culture medium containing 10% fetal calf serum (FCS), glutamine and antibiotics (complete culture medium). Confluent cultures were trypsinized, cells were suspended in complete culture medium, seeded in 24-well (2.0  105 cells per well) or 6-well (6.0  105 cells per well) tissue culture plates and cultured for 48 h. Cells were then infected at a multiplicity of infection of about 100 bacteria per cell. Since translocation of effector proteins from the type III secretory system depends on a close contact between bacteria and the host cells, bacteria were centrifuged (1000g for 10 min) onto the cell monolayers prior to incubation at 37  C for 1 h. Cells were then immediately processed or incubated with culture medium containing gentamicin at 300 mg/mL for different periods, to eliminate infecting bacteria. In some assays, bacteria were treated for 30 min with 100 mM of the cPLA2 inhibitor MAFP, before addition to the cell cultures. Primary cultures of cells from human nasal polyps were a generous donation of Dr. Jean Marie Tournier (INSERM U 514, Reims, France) and were obtained according to a modified protocol described by Chevillard et al. [35]. Primary cultures of mice traqueobronchial cells were obtained according to a modified protocol of Guillot et al. [36]. 4.3. AA release Cells cultured in 24-well plates for 24 h were incubated with H-AA (New England Nuclear, Boston, MA) at 0.2 mCi/mL for additional 24 h at 37  C. For the assays, cells were incubated with 500 mL of the bacterial suspensions or with culture medium. After 1 h at 37  C, the culture supernatants were removed and counted in 4 mL of scintillation counting liquid BCS (Amersham, Little Chalfont, UK) in a LKB scintillation counter. To confirm that the radioactivity detected in the supernatants corresponded to released free AA, and not to radiolabelled cell debris, lipid composition of the supernatants of infected and non-infected cultures was analyzed by thin layer chromatography, as described [17] (data not shown). 3

For immunoblotting analysis of cPLA2, cells were infected for 1 h and then treated with the gentamicin-containing culture medium for additional 19 h. Lysates from control cells and from cells infected with PA103 and the exoU-deficient mutant, containing 10 mg of cellular protein, were separated on 12% reducing polyacrylamide gels. Proteins were then transferred to Hybond-P membranes (Amersham–Biosciences). Nonspecific binding was blocked with 3% non-fat milk in 0.01 M PBS pH 7.4 containing 0.05% Tween 20 (PBST) at room temperature for 1 h. The membranes were then incubated with primary monoclonal antibody against cPLA2 (Santa Cruz Biotechnology; 1:500 dilution) or polyclonal antibody against phosphorylated cPLA2 (Cell Signaling; 1:1000 dilution) in PBS-T containing 3% non-fat milk. After overnight incubation at 4  C, the membranes were washed, incubated for 1 h with the corresponding horseradish peroxidase-conjugated secondary antibodies and exposed to the ECL Western blotting detection system (Amersham– Biosciences), according to the manufacturer’s instructions. 4.6. Lipid body detection Two different approaches were used to detect and quantify lipid bodies. In the first, cells cultured for 48 h on glass coverslips were infected or not for 1 h, rinsed and treated with the gentamicincontaining culture medium for additional 2 h. Cells were then fixed in 3.7% paraformaldehyde in PBS, rinsed, incubated with 1.5% OsO4 in 0.1 M cacodylate buffer pH 7.4 for 30 min, rinsed, incubated with 1.0% thiocarbohydrazide for 5 min, rinsed, restained in 1.5% OsO4 for 3 min, dried and observed in light microscope with oil immersion objective lens. Images from OsO4-stained cells were recorded with a CCD camera (CoolSnap HP, Roper Scientific) and a Axio-imager microscope (Zeiss), with a 63 objective. Binary images of the OsO4stained lipid bodies were obtained according to the so-called top-hat procedure, a semi-automatic procedure based on mathematical morphology tools [37]. Binary masks for the different cells were drawn by hand. Then, the relative surface occupied by the OsO4-stained lipid bodies was computed as the ratio of lipid body surfaces to cell surfaces. This was done for every cell fully present in the micrographs and the average of this relative surface was computed for any type of preparation. For each condition, 12 different areas of the cell cultures were analyzed. In the second approach used to quantify lipid bodies, cells infected or not as described above were detached from the microplate wells by trypsinization, fixed with 3.7% paraformaldehyde in PBS, rinsed, incubated with BODIPY [1-acyl-2-(7-octyl-BODIPY1-pentanoyl)-sn-glycerol; Molecular Probe] at 10 mg/mL in PBS for 30 min at room temperature, rinsed, resuspended in PBS-BSA 1% and submitted to flow cytometer analysis (FACS-calibur flow cytometer; Becton–Dickinson).

4.4. PGE2, IL-6 and IL-8 assays 4.7. Autoradiography Cells cultured in 24-well culture plates were incubated with 500 mL of the bacterial suspensions, or with culture medium, and submitted to centrifugation, as described above. After 1 h at 37  C, cells were rinsed and incubated with the gentamicin-containing culture medium. After 20 h, the concentrations of PGE2, IL-6 and IL-8 in the cell supernatants were determined by enzyme immunoassay (Cayman Chemical and R&D, respectively). Since cytotoxicity of the ExoU-producing strains led to the rapid killing of a high percentage of infected cells, after the 1 h infection period the cell concentrations in infected and in control culture wells were

Cells were labelled with 3H-AA (New England Nuclear) at 1.0 mCi/mL for 24 h, infected with PA103 or PA103DexoU suspensions, as described, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, post-fixed with 1.0% OsO4 in 0.1 M cacodylate buffer, dehydrated in a graded series of ethanol and embedded in Epon. Semithin sections (1.0 mm) were stained with ironhematoxylin, coated in a dark room with photographic emulsion, type LM1 (Amersham–Biosciences) and kept at 4  C in light-proof box, under dry atmosphere, for one week. Slides were then

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developed in D-170 (Kodak formula), fixed in 24% sodium thiosulphate, washed and mounted with Permount. The number of silver grains per cell was determined in microphotographs obtained with light microscopy using oil immersion objective. 4.8. Immunocytochemistry Immunolocalization of PGE2 in lipid bodies of cells cultured on glass coverslips infected for 1 h and treated with the antibioticcontaining culture medium for 2 h was performed as described [14]. Briefly, cells were fixed with 1% EDAC [N-(3-dimethylaminopropyl)N’-ethylcarbodiimide hydrochloride; Sigma–Aldrich] in PBS, used to cross-link eicosanoid carboxyl groups to amines of adjacent proteins. After 10 min at 37  C, cells were rinsed and incubated with mouse anti-PGE2 (Cayman) for 1 h at room temperature. Control coverslips were exposed to non-immune mouse serum for the same period. Coverslips were then rinsed, incubated with secondary antibody conjugated with Texas Red (Amersham–Biosceinces), and analyzed by both phase contrast and fluorescence microscopy. 4.9. FACS analysis of lipid body and PGE2 expression Cells infected for 1 h and treated with the gentamicin-containing culture medium for additional 2 h, as well as control cells, were trypsinized from the microplate wells, rinsed, fixed with 1% EDAC for 10 min at room temperature, rinsed, incubated with mouse anti-PGE2 antibody, rinsed and incubated with anti-mouse IgG conjugated with Texas Red. Cells were then rinsed, incubated with BODIPY solution for 30 min, rinsed and submitted to FACS analysis. 4.10. Statistical analysis Statistical analysis was performed using a one-way analysis of variance (ANOVA) with the Dunnett’s or Bonferroni’s test to determine significant statistical differences between groups, unless otherwise stated. p Values <0.05 were deemed to be significant. Acknowledgement The authors thank Dr. Antonio Haddad and Vani M. A. Correa (Departamento de Biologia Celular da Faculdade de Medicina de Ribeira˜o Preto/USP) for autoradiographic studies, Dr. Alan Hauser (Northwestern University, Chicago) for generously providing the P. aeruginosa mutants PA103DUT/exoU and PA103DUT/S142A and Dr. Jean Marie Tournier (INSERM U 514, Reims, France) for donation of human cells in primary cultures. Bruno Branda˜o was supported by fellowships from CAPES (Brazil). This work was supported by grants from CNPq and FAPERJ (Brazil). References [1] Sato H, Frank DW, Hillard CJ, Felix JB, Pankhaniya RR, Moriyama K, et al. The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin ExoU. EMBO J 2003;22:2959–69. [2] Phillips RM, Six DA, Dennis EA, Ghosh P. In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J Biol Chem 2003;278:41326–32. [3] Sato H, Feix JB, Frank DW. Identification of superoxide dismutase as a cofactor for the Pseudomonas type III toxin ExoU. Biochemistry 2006;45:10368–75. [4] Rabin SDP, Veesenmeyer JL, Bieging KT, Hauser AR. A C-terminal domain targets the Pseudomonas aeruginosa cytotoxin ExoU to the plasma membrane of host cells. Infect Immun 2006;74:2552–61. [5] Stirling FR, Cuzick A, Kelly SM, Oxley D, Evans TJ. Eukaryotic localization, activation and ubiquitinylation of bacteria type III secreted toxin. Cell Microbiol 2006;8:1294–309. [6] Sitkiewicz I, Stockbauer KE, Musser JM. Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis. Trends Microbiol 2007;15:63–9. [7] Williams KI, Higgs GA. Eicosanoids and inflammation. J Pathol 1988;156:101–10. [8] Diaz BL, Arm JP. Phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 2003;156:101–10.

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