Subversion of a Lysosomal Pathway Regulating Neutrophil Apoptosis by a Major Bacterial Toxin, Pyocyanin

Abstract

Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramatically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. Here, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunction, shown by altered lysosomal pH, within 15 mins of exposure. Lysosomal disruption was followed by mitochondrial membrane permeabilization, caspase activation and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin D (CTSD), abrogated pyocyanin-induced apoptosis and translocation of CTSD to the cytosol followed pyocyanin treatment and lysosomal disruption. A stable analogue of cyclic AMP (dbcAMP) impeded the translocation of CTSD and prevented the destabilization of Mcl-1 by pyocyanin. Thus pyocyanin activated a co-ordinated series of events dependent upon lysosomal dysfunction and protease release, the first description of a bacterial toxin utilising a lysosomal cell death pathway. This may be a pathological pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses.

Introduction

Neutrophils are the predominant inflammatory cells recruited during the innate immune response to bacterial infection and are critical for bacterial clearance. Subsequent resolution of inflammation requires removal of these potentially toxic leukocytes to prevent a dysregulated immune response. Induction of apoptosis is a crucial mechanism of homeostasis, down-regulating proinflammatory functions(1) and resulting in macrophage-mediated clearance of apoptotic cells(2). Some bacteria, however, induce inappropriate or premature apoptosis of phagocytes, particularly macrophages, depleting cell numbers and function, with associated impairment of host defense(3),(​4).

Pseudomonas aeruginosa is an important human pathogen. Chronic infection with P. aeruginosa is a major cause of pulmonary damage and mortality in cystic fibrosis, and acute infection is observed both in the immunocompromised host and in patients with ventilator-associated pneumonia(5). In patients with cystic fibrosis, persistent P. aeruginosa colonisation of the lung demonstrates inadequate mechanisms of bacterial clearance despite profound neutrophilic inflammation(6). Whilst immune defenses in cystic fibrosis may be impaired at multiple levels, an excess of apoptotic neutrophils in this setting implies a neutrophil defect may contribute significantly to unresolved infection(7). The prominence of P. aeruginosa sepsis in neutropenic patients(8) also highlights both the role of the neutrophil in defence against this organism and the clinical importance of understanding how this pathogen subverts the innate immune response. P. aeruginosa generates highly diffusible toxic secondary metabolites known as phenazines, which are critical for P. aeruginosa virulence and cytotoxicity in C. elegans and mouse infection models(9), and it is the only organism to produce a specific phenazine, named pyocyanin(10). We have shown pyocyanin, at concentrations detected in sputum of cystic fibrosis patients (11), induces a rapid, profound, and selective acceleration of neutrophil apoptosis in vitro(12). In a murine model of pulmonary P. aeruginosa infection, mice infected with a pyocyanin-producing strain, as compared with a pyocyanin-deficient but otherwise genetically identical strain, also showed accelerated neutrophil apoptosis and impaired bacterial clearance(13).

Neutrophils are short-lived cells. Two major pathways to apoptosis are recognised: one proceeds through death receptor signalling, via membrane-associated signalling complexes and caspase-8 activation, and a second “stress” pathway, known to be regulated by oxidant stress, is mediated by mitochondria and regulated by bcl-2 family members(14). The mechanisms of pyocyanin-induced acceleration of neutrophil apoptosis are largely unknown but may involve ROI generation and altered redox status(12). It is also unclear why neutrophils are exquisitely sensitive to pyocyanin. We therefore investigated the mechanisms of pyocyanin-induced apoptosis in neutrophils, and describe a novel pathway of pathogen-mediated neutrophil apoptosis, characterised by lysosomal acidification and activation of cathepsin D (CTSD).

Materials and Methods

Neutrophil isolation and culture

Human neutrophils were isolated by dextran sedimentation and plasma-Percoll (Sigma, Poole, UK) gradient centrifugation from whole blood of normal volunteers(15). The studies were approved by the South Sheffield Research Ethics Committee and subjects gave written, informed consent. Purity of neutrophil populations (>95%) was assessed by counting >500 cells on duplicate cytospins. Neutrophils were suspended at 2.5 × 10⁶/ml in RPMI with 1% penicillin/streptomycin and 10% FCS (all Invitrogen, Paisley, UK) and cultured in 96 well “Flexiwell” plates (BD Pharmingen, Oxford, UK).

Preparation and analysis of pyocyanin

Pyocyanin was prepared by photolysis of phenazine methosulphate (Sigma) and purified and characterised as previously described(16).

Assessment of viability and apoptosis

Nuclear morphology was assessed on Diff-Quik-stained cytospins, with blinded observers counting >300 cells per slide on duplicate cy tospins. Necrosis was assessed by trypan blue exclusion and was <2% unless indicated. Alternatively, neutrophils were washed in PBS and stained with PE-labelled Annexin V (BD Biosciences, San Jose, CA) and TOPRO®-3 iodide (Molecular Probes, Paisley, UK) to identify apoptotic (Annexin V+) and necrotic (TOPRO-3+) cells(17). Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Twenty thousand events were recorded and data was analyzed by CellQuest software (BD Biosciences).

Caspase-3 activity assay

Caspase 3 activity was determined by measuring enzymatically cleaved fluorescent substrate (DEVD-AMC, Bachem, Weil am Rhein, Germany) as previously described(18). Neutrophil lysates were prepared by re-suspension of treated cells in lysis buffer (100 mM HEPES, pH 7.5, 10% w/v sucrose, 0.1% CHAPS, 5 mM DTT) at a concentration of 1×10⁸/ml. Lysates were frozen at −80°C until required. Using the FLUSYS software package for the Perkin-Elmer LS-50B fluorimeter, lysate equivalents of 5 million neutrophils were co-incubated with 20 μM Ac-DEVD-AMC in DMSO. Kinetic data was collected for at least 20 minutes to ensure stability of activity. A known amount of free AMC was used to calibrate the system and allowed calculation of caspase-3 activity. In separate experiments, executioner caspase (caspases 3 and 7) activity was measured using a Caspase-Glo 3/7 Assay (Promega, Madison, WI). Neutrophils were cultured at 5×10⁶/ml and treated with media (control), pyocyanin [50 μM] and pyocyanin with dbcAMP (100 μM) for 3 h. Cells were directly transferred to a white 96-well flat-bottomed plate (Dynex Technologies) at a density of 62,500 cells per well in a 25 μl volume.) An equivalent volume of caspase-Glo 3/7 buffer mixed with substrate reagent was added to each well. The plate was read using a Lumistar Galaxy Luminometer (BMG Labtechnologies, Offenburg, Germany) at 25°C for 200 cycles.

ATP and glucose measurements

ATP was measured using a commercially-available bioluminescent kit (Sigma) using a Lumistar Galaxy Luminometer. Glucose was assayed by detecting change in glucose concentration in lysates and culture supernatants using a commercial kit (Sigma), as previously described(19). Neutrophils were cultured in RPMI alone, with a glucose concentration of 2 mg/ml. Both assays were standardised using known concentrations of ATP and glucose respectively (data not shown).

Modulation of pyocyanin-induced apoptosis

Neutrophils were incubated in the presence and absence of pyocyanin following pre-incubation with candidate modulators of pyocyanin-induced apoptosis. Except where indicated, a concentration of 50 μM pyocyanin was used since it significantly accelerates neutrophil apoptosis approximately 5 fold at 5 h(12). The pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD.fmk)(​18) was obtained from Enzyme Systems Products (Livermore, CA) and Boc-D.fmk [50 μM] was obtained from Calbiochem (San Diego, CA). MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone (MeOSuc, Bachem) [10 μM] & elastatinal (Sigma) [10 μM] were used as neutrophil elastase inhibitors, with optimal inhibitory concentrations determined by assessment of inhibition of fluorogenic substrate digestion by purified neutrophil elastase (data not shown). BB94 [1 μM] (gift from Dr. D. Buttle, University of Sheffield, UK) was used as a pan metalloproteinase inhibitor(20). Specific cathepsin inhibitors were CA-074Me [25 μM] for cathepsin B (Calbiochem), pepstatin A [10 μM] (Sigma) and DAME (Diazoacetyl-DL-2-aminohexanoic acid-methyl ester, Bachem) for CTSD and CK-08 [1 μM] (Enzyme Systems Products) for cathepsin G(21). Bafilomycin A1 [100nM] (Sigma) inhibits the membrane vacuolar ATPase(22).

ROI production

ROI production was assessed by incubating 5 × 10⁵ neutrophils in 200 μl RPMI with 5 μM DHR (dihydrorhodamine, Sigma) for 30 minutes at 37°C, and measuring fluorescence in the FL-1 channel by flow cytometry.

Hypoxia Experiments

Peripheral blood neutrophil culture in hypoxia was performed as previously described(23). Neutrophils were resuspended at 5 ×10⁶/ml in RPMI + 10% FCS and incubated in normoxic (19 kPa) or hypoxic (3 kPa) environments in the presence or absence of pyocyanin [50 μM] for 6 h. Normoxia was controlled using a humidified 5% CO₂/air incubator, and hypoxia, by pre-gassing medium for 30 min in a sealed hypoxic work station with 5% CO₂/balance N₂ gas mix and subsequent culture in a humidified hypoxic (CO₂/N₂) incubator. Cytospins were prepared and apoptosis scored by light microscopy.

Assessment of mitochondrial and lysosomal membrane permeability

To detect loss of ΔΨ_m neutrophils were incubated with 10 μg/ml JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide, Molecular Probes) at 37°C. Loss of ΔΨ_m was assayed by observing a shift in fluorescence emission from red (∼590 nm) to green (∼525 nm) using flow cytometry(24). Neutrophils were treated with valinomycin (100 μM, Sigma) as a positive control. Lysosomal pH was measured by incubating neutrophils with 1 mg/ml FITC-dextran 70S (Sigma), a pH-sensitive fluorescent probe, for 30 mins at 37°C. Increasing pH (i.e. loss of acidification) within the lysosomal compartment is associated with increased green fluorescence detected in the FL-1 channel(25). Loss of lysosomal acidification was determined by incubating neutrophils with 5 μM acridine orange (Sigma) for 30 minutes at 37°C, and loss of FL-3 fluorescence was measured by flow cytometry(21). Cytospins were also prepared and viewed by fluorescence microscopy. Neutrophils treated with 100 nM bafilomycin A1 a known inhibitor of vacuolar ATPase, a key regulator of lysosomal pH Ran, 2003 #965}, were used as a positive control in these experiments.

CTSD translocation

Boron dipyrromethane difluoride (BODIPY FL)-pepstatin A (Molecular Probes, Eugene, OR) is a fluorescent pH-sensitive probe used to measure the subcellular distribution of CTSD(26). Neutrophils were treated with media (control), bafilomyin A1 [100 nM] or pyocyanin [50 μM] for 30 mins. The cells were washed and incubated with 1 μM BODIPY FL-pepstatin A (in media) at 37°C for 30 mins after which they were washed, resupended in media and incubated at 37°C for a further 60 mins to allow endosomal trafficking. Cytospins were prepared and viewed under a fluorescent microscope (Leica AF6000, x63 objective). CTSD-labelled BODIPY FL-Pepstatin A was visible as punctate fluorescent inclusions.

SDS-PAGE and Western Immunoblotting

Whole-cell extracts were used for Mcl-1 and actin immunoblots and prepared as described(27). Cytosolic and membrane fractions used in CTSD and CTSG immunoblots were prepared by sonication (three 10 second bursts in hanks balanced salts solution supplemented with protease inhibitor cocktail III (Calbiochem) followed by 25,000 RPM microcentrifugation for 45 mins. Proteins were separated by 15% v/v SDS-PAGE, blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Hemel Hempstead, UK) and protein transfer confirmed by Ponceau S (BDH, UK) staining. Blots were incubated overnight at 4°C for Mcl-1 (S-19, Santa Cruz Biotechnology, CA, USA) and at RT for 2 h for actin (Sigma), CTSG (Abcam, Cambridge, UK) and CTSD (Calbiochem), protein detection was with HRP-conjugated IgG (Dako, Ely, UK) and ECL (Amersham Biosciences, Little Chalfont, UK).

Statistics

For multiple comparisons, means and standard error of the mean (SEM) were analysed by ANOVA with post-test as indicated (GraphPad Prism, CA, USA). For comparison of two sample means paired Student’s t tests were used.

Results

Pyocyanin-induced neutrophil apoptosis is caspase dependent

Pyocyanin-induced cell death in neutrophils displays both morphological features (nuclear condensation, cell shrinkage) and cell surface changes (Annexin V binding to exposed phosphatidylserine) of apoptosis(12). We therefore investigated whether pyocyanin-induced neutrophil death was also associated with caspase activation, using both morphology and Annexin V binding to quantify neutrophil apoptosis. A pan-caspase inhibitor, zVAD.fmk, inhibited pyocyanin-induced apoptosis in a concentration-dependent manner, with significant reduction in neutrophil death at concentrations from 5 μM (Fig 1A). Fifty μM zVAD.fmk delayed pyocyanin-induced death up to 10 h (Fig 1B), thereafter secondary necrosis in pyocyanin-treated cells made estimations of apoptosis unreliable. Apoptosis of control neutrophils at 5 h was 5.7±1.2% and was not significantly inhibited by zVAD.fmk at any concentration used (data not shown). A second pan-caspase inhibitor, Boc-D.fmk, also inhibited pyocyanin-induced apoptosis at 5 h (pyocyanin-induced apoptosis in the absence (34.8±3.8%) and presence (21.9±5.5%) of 50μM Boc-D.fmk). Since zVAD-fmk has some caspase-independent effects in neutrophils(28), we confirmed caspase activation by demonstrating cleavage of a caspase-3 specific fluorescent substrate, DEVD-AMC. Pyocyanin treatment (4 h) caused a significant increase in neutrophil intracellular caspase-3 activity compared with untreated controls (Fig 1C). Thus, pyocyanin-induced neutrophil apoptosis is delayed by caspase inhibition and associated with caspase-3 activation.

Figure 1. Pyocyanin-induced neutrophils apoptosis is caspase dependent.

A. Neutrophils were treated with pyocyanin (50 μM) in the presence or absence of a concentration range of zVAD.fmk for 4 h. Apoptosis was assessed by morphologic criteria and chart shows mean ±SEM % apoptosis from 3 independent experiments. Baseline control apoptosis in these experiments was 5.7±1.2% and was not significantly inhibited by any concentration of zVAD.fmk (data not shown). Statistically significant inhibition of pyocyanin-induced apoptosis was observed at concentrations of zVAD.fmk of 5 μM and greater (*p<0.05 and **p<0.01 ANOVA with Dunnett’s post-test). B. Neutrophils were treated with 50 μM pyocyanin in the absence (empty bars) or presence (black bars) of zVAD.fmk [50 μM] for 0, 2, 5 and 10 h. Apoptosis was assessed by flow cytometry, and the chart shows mean ±SEM % apoptosis for 3 independent experiments. Significant inhibition of pyocyanin-induced apoptosis was observed at 5 h (*p<0.05) and 10 h (***p<0.001, ANOVA, Bonferroni’s post-test). C. Lysates were prepared from neutrophils treated with media (control, open bars) or 50 μM pyocyanin (black bars) for 1 and 4 h. Caspase 3 activity was detected using a specific fluorogenic substrate and measured kinetically (Flusys software). Pyocyanin significantly increased caspase-3 activity at 4 h (*p=0.0355, Student’s t test).

Pyocyanin induced oxidative stress mediates neutrophil apoptosis

The cytotoxic effects of pyocyanin on bacteria and eukaryotic cells are linked to its ability to undergo non-enzymatic redox cycling within cells, with resultant ROI formation, and further loss of reducing capacity by direct oxidation of NADH/NADPH and GSH(29). We found that pyocyanin induces prolonged generation of ROI in neutrophils as measured by oxidation of DHR (Fig 2A), in keeping with our previous observations (12). To further corroborate the role of oxidative stress in pyocyanin-induced apoptosis, neutrophils were incubated in a hypoxic environment in the presence of pyocyanin for 5 h. We hypothesised that reduced availability of oxygen would hinder the ability of pyocyanin to induce apoptosis. Figure 2B shows pyocyanin is unable to induce neutrophil apoptosis in hypoxia.

Figure 2. Oxidative stress is essential for pyocyanin -induced neutrophil apoptosis.

A. DHR-loaded neutrophils were treated with media (control, empty bars) or 50 μM pyocyanin (black bars) for a range of timepoints; 30, 60, 120 and 180 mins. Increases in fluorescence were measured by flow cytometry and indicated enhanced ROI production. The chart shows mean ±SEM of mean channel fluorescence (MFI) of 3 independent experiments. Statistically significant differences were seen between control and pyocyanin-treated cells at all timepoints (***p<0.001, ANOVA, Bonferroni’s post-test). B. Neutrophils incubated in normoxic (empty bars) or hypoxic (black bars) conditions were cultured with media (control) or pyocyanin [50 μM] for 6 h. Apoptosis was assessed by light microscopy. Pyocyanin treatment of normoxic neutrophils induced apoptosis (**p<0.01, ANOVA, Bonferroni’s post-test). Hypoxic neutrophils survived in the presence of pyocyanin, to levels comparable with the control.

Metabolic activity is maintained in pyocyanin-treated neutrophils

Pyocyanin-induced ROI production is linked to depletion of intracellular ATP in epithelial cells(30),(​29). To determine whether neutrophil death was associated with cellular ATP depletion in response to the profound ROI induction, we measured intracellular ATP in the neutrophil by a bioluminescence technique. We did not detect a reduction in ATP levels at time points up to 3 h following pyocyanin treatment (Fig 3A). Metabolic pathways in pyocyanin-treated neutrophils remained viable as shown by this maintenance of intracellular ATP, and by our subsequent experiments, which showed increased glucose uptake (Fig 3B) and maintenance of intracellular glucose concentrations in these cells (Fig 3C).

Figure 3. Pyocyanin leads to metabolic cell stress in the neutrophil.

A. Neutrophils were treated with media (control, empty bars) or 50 μM pyocyanin (black bars) for 1 and 3 h following which lysates were prepared and intracellular ATP measured. No significant differences were observed between the groups. B-C. Neutrophils were treated with media (control, empty bars) or 50 μM pyocyanin (black bars) for 2 h and glucose was measured in extracellular (B) and intracellular (C) compartments. The charts show mean ±SEM from 4 independent experiments. A statistically significant decrease in extracellular glucose was detected in pyocyanin-treated cells (*p<0.05 Student’s t test).

ROI are a feature of the “stress” pathway of apoptosis and ROI production leads to loss of mitochondrial membrane potential (ΔΨ_m)(​14). Since pyocyanin can interrupt mitochondrial respiration in epithelial cells as a result of ROI generation (29), we determined whether pyocyanin-mediated ROI production was inducing neutrophil apoptosis via loss of mitochondrial inner transmembrane potential (ΔΨ_m). We measured ΔΨ_m in neutrophils using the mitochondrial dye, JC-1. Figure 4A shows flow cytometry dot plots illustrating the distribution of high-FL-1 fluorescent neutrophils (associated with a loss of ΔΨ_m (24),(​31)) in control, pyocyanin- and valinomycin-treated populations at 4 h. Pyocyanin induced only modest changes in the proportions of cells showing loss of mitochondrial inner transmembrane potential, and these non-significant changes were only observed at later time points (Fig 4B). These observations were confirmed using a second mitochondrial dye, DiOC₆ (data not shown). Thus, although loss of ΔΨ_m appears to occur in pyocyanin-induced neutrophil apoptosis, changes are not apparent until later time points, when there is already a significant increase in apoptosis. Loss of ΔΨ_m was therefore unlikely to be an initiating factor in the engagement of apoptosis.

Figure 4. Pyocyanin-induced neutrophil apoptosis does not depend on early mitochondrial dysfunction.

Neutrophils were treated with media (control, empty bars), 50 μM pyocyanin (shaded bars) or 1 μM valinomycin (black bars) for 1, 2 and 4 h. The fluorescent dye, JC-1 [10 μg/ml] was used to determine ΔΨ_m and changes in FL-1 fluorescence were measured by flow cytometry. A. Representative dot plots showing distribution of cells staining high and low for JC-1 at 4 h. B. The charts show mean ±SEM % cells with loss of ΔΨ_m (high JC-1) from 3 independent experiments. Valinomycin caused significant loss of ΔΨ_m at every timepoint (**p<0.01, ***p<0.001, ANOVA, Bonferroni’s post-test).

Pyocyanin-induced apoptosis is preceded by changes in lysosomal pH but these are not sufficient to induce death

A pathway of cell death is now recognised, activated primarily in pathological rather than homeostatic circumstances, in which lysosmal dysfunction may precede loss of ΔΨ_m(32). Lysosomal pH was measured using a pH-sensitive marker (FITC-conjugated dextran) that is taken up by acidic structures and increases in FL-1 channel fluorescence as pH rises due to loss of protonation(33),(​25). Exposure of neutrophils to pyocyanin or bafilomycin A1 (an inhibitor of the vacuolar (H+)-ATPase that maintains normal lysosomal pH gradients(34)) increased lysosomal pH compared to untreated controls (Fig 5A and 5B).

Figure 5. Pyocyanin induces loss of lysosomal acidification.

A. Illustrative flow cytometry histogram showing right-shifts in FL1-H fluorescence of FITC-dextran labelled neutrophils treated with either media (control), pyocyanin [50 μM] or bafilomycin A1 [100 nM] for 2 h. Increasing fluorescence in FL-1 reflects elevated intralysosomal pH secondary to reduced protonation. B. FITC-dextran stained neutrophils were incubated with media (control, empty bars) or 50 μM pyocyanin (black bars) for the indicated times. The chart shows fold change of MFI (mean ±SEM, n=3) vs. control at 30 mins. Pyocyanin treatment significantly elevated lysosomal pH at 120 (***p<0.001) and 240 mins (**​<​p0.01, ANOVA, Bonferroni’s post-test).

We confirmed loss of lysosomal acidification in neutrophils by staining with acridine orange, which is lysosomotropic and accumulates in acidic organelles(35). On fluorescence microscopy (Fig 6A), a punctate staining pattern was seen in the cytosol of control neutrophils, consistent with lysosomal accumulation of the stain. In neutrophils treated with pyocyanin or bafilomycin A1, the punctate staining pattern was lost, in keeping with loss of lysosomal acidification(21, 32). Statistically significant losses of fluorescence were detected at 15 min following pyocyanin treatment (Fig 6B). Once again, bafilomycin A1 was used as a positive control in these experiments and, at a concentration (100 nM) that inhibits V-ATPase function in neutrophils(36), caused loss of lysosomal acidification to an even greater degree than pyocyanin. However, this concentration of bafilomycin A1 was without effect on constitutive neutrophil apoptosis (Fig. 6C), in keeping with previous studies(37), although higher concentrations of bafilomycin are pro-apoptotic to neutrophils (data not shown). Lysosomal alkalinization alone is not, therefore, sufficient to induce neutrophil apoptosis.

Figure 6. Pyocyanin induces early loss of lysosomal acidification.

A. Representative fluorescent microscopy images illustrating typical punctate staining with acridine orange which is lost after treatment with pyocyanin or baflinomycin A1. B. Neutrophils were treated with media (control, empty bars), 50 μM pyocyanin (shaded bars) and baflinomycin A1 (100 nM, black bars) for 15, 30 and 60 mins. Loss of lysosomal acidification was assessed with the fluorescent lysosomal dye, acridine orange and fluorescence measured by flow cytometry. Decreases in FL-3 reflect loss of acridine orange due to lysosomal disruption. The chart shows mean ±SEM MFI from 3 independent experiments. Pyocyanin caused significant loss of lysosomal acidification at all timepoints (**p<0.01, ANOVA, Bonferroni’s post-test). C. Neutrophils were incubated with media (control, empty bars) or 100 nM bafilomycin A1 for varying times. Apoptosis was assessed by flow cytometry, chart shows mean ±SEM % apoptosis (as defined by annexin V positivity) for 3 independent experiments.

CTSD translocation and activation is associated with pyocyanin-induced apoptosis

Alterations in lysosomal pH alone do not induce apoptosis and release of lysosomal proteases are critical for completion of the apoptotic programme in a range of cell types(21, 35). No attenuation of pyocyanin-induced neutrophil apoptosis was seen by treatment with elastase inhibitors or a pan-metalloproteinase inhibitor (Fig 7A,B). In contrast, significant reductions in pyocyanin-induced apoptosis were observed with pepstatin A, an inhibitor of aspartyl proteases(21), both alone and in combination with inhibitors of the cysteinyl cathepsins B, G and L (Fig 7C). The latter inhibitors were without effect, either alone or in combinations that excluded pepstatin A, on constitutive neutrophil apoptosis (data not shown) or on pyocyanin-induced apoptosis (Fig 7C). Although pepstatin A is a specific inhibitor of CTSD, it has been reported to cause neutrophil activation similar to changes induced by FMLP treatment(38), and we found it also had an anti-apoptotic effect upon neutrophils in the absence of pyocyanin (data not shown). We therefore investigated the potential of a second and unrelated CTSD inhibitor, DAME (39), to modulate pyocyanin-induced neutrophil cell death. DAME treatment significantly abrogated pyocyanin-induced neutrophil apoptosis (Fig 7D) at concentrations that were without effect upon constitutive neutrophil death. CTSD is bound within the matrix of neutrophil azurophilic granules but on activation is released to the cytosol(40).

Figure 7. Pyocyanin-induced neutrophil apoptosis is mediated by activation of CTSD.

A-B. Media (control) and pyocyanin [50 μM]-treated neutrophils were incubated in the absence (empty bars) or presence of inhibitors of elastase (A; elastatinal, [10 μM] shaded bars and Me-O-Suc [10 μM] black bars) or metalloproteinases (B; BB94 [1 μM], shaded bars) for 5 h and apoptosis assessed morphologically. None of the inhibitors significantly inhibited apoptosis (ANOVA, Bonferroni’s post-test). C. Pyocyanin [50 μM]-treated neutrophils were incubated in the presence or absence of a range of cathepsin (CAT) inhibitors for 5 h and apoptosis assessed by flow cytometry. The inhibitor of CTSD and combinations including the CTSD inhibitor significantly inhibited pyocyanin-induced apoptosis (*p<0.05, **p<0.01, ANOVA, Dunnett’s post-test). D. Neutrophils were incubated for 5 h with media (open bars) or pyocyanin [50 μM] (black bars) both alone and in the presence of a concentration range of the CTSD inhibitor, DAME. Apoptosis was assessed by morphology and is expressed as fold change from either control or pyocyanin alone. Five micromolar DAME significantly inhibited pyocyanin-induced apoptosis and was without effect on constitutive apoptosis (*p<0.05, ANOVA, Dunnett’s post-test).

BODIPY FL-pepstatin A is a pH dependent fluorescent probe which binds to CTSD within the acidified lysosomal compartment(26). We demonstrated the expected pattern of CTSD localization with punctate cytosolic inclusions in control neutrophils (Fig 8A). Neutrophils treated for 30 mins with pyocyanin or bafilomycin A1 lost this typical granular staining pattern. To determine whether the decrease in lysosomal BODIPY FL-pepstatin A staining reflected alteration in lysosomal pH alone or LMP we assessed translocation of CTSD by Western immunoblotting. Figure 8B shows CTSD distribution in neutrophil lysates separated into membrane (upper panel) and cytosolic (lower panel) fractions. The predicted 44 kDa and 31 kDa forms(41) are present within the membrane fraction of both control and bafilomycin A1 treated cells, whereas pyocyanin-treated cells show translocation of the 31 kDa form into the cytosol. We also studied translocation of another cathepsin, CTSG, and demonstrated that treatment with pyocyanin but not bafilomycin caused translocation to the cytosolic fraction (Fig 8C), although as shown in Figure 7C, CTSG inhibition did not abrogate pyocyanin-mediated apoptosis.

Figure 8. CTSD is translocated to the cytosol following pyocyanin treatment.

A. Neutrophils were treated with media (control), pyocyanin [50 μM] or bafilomycin A1 [100 nM] for 30 mins before staining with the CTSD probe, BODIPY FL-pepstatin A [1 μM]. Cytospins were prepared and the cells viewed by fluorescence microscopy. Cells without BODIPY FL-pepstatin A were used to measure background fluorescence (not shown). Cells treated with pyocyanin or bafilomycin A1 lost the punctate fluorescent inclusions that are typical of granular localisation, indicating translocation of CTSD and/or loss of lysosomal acidifcation. B. Membrane (upper panel) and cytosolic (lower panel) fractions were prepared from 4 × 10⁶ neutrophils treated with media or pyocyanin [50 μM] for 30 mins and subjected to SDS-PAGE immunoblotting. Blots were probed for CTSD and showed the appearance of the 31 kD fragment of CTSD in the pyocyanin-treated cytosolic fractions that was not present in the cytosol of control or bafilomycin A1-treated cells. C. Cytosolic fractions were prepared from neutrophils treated with media (control), pyocyanin [50 μM] or bafilomycin A1 [100 nM] for 30 mins. SDS-PAGE immunoblots were probed for CATG and actin (loading control) and showed significantly more CATG in the pyocyanin-treated sample compared with control or bafilomycin A1.

A stable cAMP analogue retards pyocyanin-mediated neutrophil apoptosis by regulating CTSD translocation, caspase activity and Mcl-1 expression

We previously reported that the synthetic cyclic AMP (cAMP) analogue dibutyryl cAMP (dbcAMP) is able to significantly abrogate pyocyanin-induced apoptosis of neutrophils(12). Having identified pathways of pyocyanin-induced apoptosis, we asked how dbcAMP might inhibit pyocyanin-induced death. Dibutyryl cAMP was unable to inhibit pyocyanin-induced ROI generation or loss of lysosomal acidification (Fig 9A,B). However, translocation of CTSD to the cytosol in pyocyanin-treated cells was reduced in cells also treated with dbcAMP (Fig. 9C). Pyocyanin-induced caspase activity was also prevented by dbcAMP (Fig 9D). Another potentially important effect of dbcAMP was also studied. The anti-apoptotic bcl-2 family member, Mcl-1, plays a critical role in the regulation of neutrophil apoptosis(42) and cAMP analogues have been shown to stabilize Mcl-1 protein levels in neutrophils(43). We showed pyocyanin treatment of neutrophils reduced Mcl-1 protein levels, as did cycloheximide and sodium salicylate treatment as previously described(44) but this effect of pyocyanin was reversed by co-incubation with dbcAMP (Fig 9E). These data suggest dbcAMP modulates pyocyanin-induced neutrophil apoptosis via multiple downstream mechanisms that include reduced CTSD translocation, caspase activation and stabilization of Mcl-1.

Figure 9. A stable cAMP analogue retards pyocyanin-mediated apoptosis.

A. Neutrophils were preloaded with DHR and incubated with pyocyanin in the presence (black bars) or absence (empty bars) of dbcAMP [100 μM] for 3 h. Increases in fluorescence were measured by flow cytometry (FL-1) and indicated enhanced ROI production. Chart shows mean±SEM MFI from 4 individual experiments. Pyocyanin-induced ROI production was unaffected by dbcAMP (ANOVA, Bonferroni’s post-test). B Neutrophils were treated with media (control), bafilomycin A1 [100 nM] or pyocyanin [50 μM] alone or in combination with dbcAMP [100 μM] for 30 mins. Loss of lysosomal acidification was assessed with acridine orange and fluorescence measured by flow cytometry. Decreases in FL-3 reflect loss of acridine orange due to lysosomal disruption. The chart shows mean ±SEM MFI from 3 independent experiments (**p<0.01, ***p<0.001, ANOVA, Bonferroni’s post-test). Pyocyanin-induced loss of lysosomal acidification was not prevented by dbcAMP. C. Membrane (upper panel) and cytosolic (lower panel) fractions were prepared from 4 × 10⁶ neutrophils treated for 30 mins with media (control), bafilomycin A1 [100 nM] or pyocyanin [50 μM] alone or in combination with dbcAMP [100 μM]. SDS-PAGE immunoblots were probed for CTSD and show the translocation of the 31 kDa form of CTSD to the cytosol in pyocyanin-treated lysates, which was reduced in the presence of dbcAMP. D. Neutrophils were incubated for 3 h with media (control) or pyocyanin [50 μM] alone or in combination with dbcAMP [100 μM]. Executioner caspase (3 and 7) activity was measured using a Caspase-Glo 3/7 Assay. Chart shows fold change from control. Caspase activity induced by pyocyanin is significantly greater than control and is inhibited by dbcAMP (***p<0.001, ANOVA, Bonferroni’s post-test). E Whole cell protein lysates were prepared from neutrophils treated for 2h with pyocyanin [50 μM], cycloheximide (CHX, 10 μg/ml) + sodium salicylate (sal, 10 μM) or pyocyanin + dbcAMP [100 μM] and subjected to SDS-PAGE immunoblotting. Blots were probed for Mcl-1 (upper panel) and actin (loading control, lower panel). Dibutyryl cAMP prevented pyocyanin-induced degradation of Mcl-1.

Discussion

In these studies we describe a novel mechanism of pathogen-induced subversion of neutrophil apoptosis that is critically dependent upon disruption of intracellular organelles and protease activity. This pathway is highly analogous to the recently-described lysosomal death pathway, used in the regulation of cell survival in a range of pathological processes (reviewed by Guicciardi et al(32)), providing additional insights into the pathways capable of regulating neutrophil survival.

Pyocyanin is a low molecular weight, bluish-green pigment secreted by P.aeruginosa that determines the characteristic colour of infected pus and sputum. It is a major factor responsible for oxidant-dependent killing of C. elegans by P. aeruginosa through its ability to undergo redox cycling and to cause superoxide generation(45, 46), and production of pyocyanin is an important determinant of severity in murine models of sepsis (9). The important potential for pyocyanin to be an agent of immune subversion by prevention of neutrophil-mediated bacterial clearance has only recently been realised. To this end, we have shown pyocyanin both acclerates neutrophil apoptosis in vitro(12) and in vivo(13) and impairs clearance of P. aeruginosa from the lung(13).

In this work, we show that pyocyanin-induced neutrophil death is caspase-dependent and associated with increased caspase-3 activity, in keeping with evidence that caspase-3 is the major “executioner” caspase in human neutrophils(47, 48). These data provided biochemical confirmation that the cell death induced was apoptotic and represented a subversion of important normal regulatory pathways controlling neutrophil lifespan. We then sought the apoptotic pathways upstream of caspase activation to determine the mechanism of pyocyanin-induced neutrophil killing. The dependence upon ROI for pyocyanin-induced death was implied by effects of anti-oxidants in our previous studies (12) and here we further show the pro-apoptotic effects of pyocyanin were prevented by culture in hypoxia. Recent studies in epithelial cells found pyocyanin, in addition to causing intracellular ROI generation, can directly oxidise both NADH and NAPDH(49). This loss of cellular reducing capacity is associated with impaired glycolysis(50) and reduced levels of cyclic nucleotides, particularly ATP(29, 51). Although detailed experiments studying the energetic status of the cell were not performed, intracellular levels of ATP and glucose were maintained following pyocyanin treatment, data that is in keeping with the requirement of neutrophils to maintain function in very demanding environments of low pH, low glucose, low oxygen tension, and high oxidative stress such as in purulent secretions(52). Indeed, preservation of ATP is necessary for co-ordinated execution of apoptotic programmes and ATP depletion favours necrotic cell death rather than “classical” apoptosis(53), providing further support for our data showing no significant loss of intracellular ATP. Neutrophils, again because of the environments in which they must be active, are unique in that the majority of ATP generation in these cells occurs via oxygen-independent glycolysis(54) and we found intracellular glucose levels were maintained in pyocyanin-treated neutrophils. Glucose uptake from the extracellular medium was measured and, for untreated neutrophils, was comparable to previous studies using the same methodology(19), with an increased uptake following pyocyanin treatment that was comparable to that of LPS or PMA activated neutrophils(55, 56).

We then sought evidence for mitochondrial inner transmembrane permeabilization, which is characteristic of the stress pathway of apoptosis characterised by ROI generation(14). Although mitochondria have a minimal role in ATP generation in neutrophils they do have a critical role in apoptosis induction(24, 57). Studies in Jurkat T cells have highlighted the ability of ATP derived by glycolysis to maintain ΔΨ_m for some hours following a metabolic insult, a process that is critically dependent upon enhanced uptake of extracellular glucose(58). We detected increased ΔΨ_m following pyocyanin treatment, but this occurred in concert with, rather than preceding, onset of apoptosis. Thus, whilst ΔΨ_m may be part of the amplification mechanism leading to executioner caspase activation, it is unlikely to be part of the programme initiating apoptosis.

A number of studies have shown oxidative stress-induced cell death is associated with lysosomal destabilization(32) and that ROI can induce lysosomal permeabilization(35, 59). Within 15 mins of pyocyanin treatment of neutrophils there was evidence of alkalinization of the lysosomal compartment that preceded any detectable changes in ΔΨ_m or caspase-3 activity. A lysosomal pathway of apoptosis that precedes mitochondrial changes is recognised in other cell types, with critical proteases translocating from lysosomes and other secretory vesicles into the cytoplasm(21, 60). This pathway is activated primarily in pathological rather than homeostatic circumstances(32) and can be activated by death receptors or lipid mediators(61) and following accumulation of lysosomotropic agents(21). The azurophilic or primary granules are generally regarded as the lysosomal structures within neutrophils, since they are the major cellular reservoir of acid-dependent hydrolases, contain lysosomal membrane proteins and are abnormal in Chediak-Higashi Syndrome, a disorder of lysosomes and related structures(62, 63). However, since they lack classical lysosomal membrane markers such as LAMP-1 and LAMP-2(62, 64) they are sometimes described as lysosme-related organelles(63).

Recent studies by Ran et al provided important insights into the actions of pyocyanin(22). Yeast mutants with reduced sensitivity to pyocyanin frequently had mutations in the V-ATPase, an enzyme complex involved in mitochondrial electron transport and ATP synthesis but also a major regulator of lysosomal pH(65). Ran et al found pyocyanin both induced lysosomal membrane permeabilization and inhibited V-ATPase function in epithelial cells, with high concentrations (2 mM) of a V-ATPase inhibitor, bafilomycin A1, having similar effects to pyocyanin. In neutrophils, bafilomycin A1, at a concentration (100 nM) that inhibits neutrophil V-ATPase function(36), reduced lysosomal acidification to an even greater degree than pyocyanin. This concentration of bafilomycin A1 was, however, without effect on neutrophil apoptosis, in keeping with previous studies(37). Two other global regulators of intracellular pH, amiloride (an inhibitor of Na⁺/H⁺ exchangers) and zinc chloride (an inhibitor of NADPH-oxidase associated proton channels) were also without effect on pyocyanin-induced apoptosis (data not shown). Our findings support those of Ran et al(22) in demonstrating pyocyanin-induced loss of lysosomal acidification in neutrophils that is likely mediated via the lysosmal V-ATPase, but also show lysosomal alkalinization alone is not sufficient to induce apoptosis. It is not clear whether the effect of pyocyanin upon V-ATPase function is indirect, perhaps resulting from ROI generation, or whether pyocyanin is lysosomotropic and binds and directly inhibits V-ATPase function, as has been described for other agents, e.g. quinolones(21) and concanamycin(66).

Lysosomes contain multiple potent proteases that contribute to bacterial killing(67), several of which have been associated with onset of apoptosis(32). Using a series of broad and narrow-spectrum protease inhibitors, we identified a role for CTSD in pyocyanin-induced apoptosis. We found that a specific CTSD inhibitor, pepstatin A, delayed pyocyanin-induced death, but also caused activation of neutrophils ((38) and our data not shown). We therefore used a second specific CTSD inhibitor (DAME) that also delayed pyocyanin-induced neutrophil apoptosis. We identified CTSD staining in neutrophils, with the expected distribution in subcellular organelles and we detected translocation of a 31kDa fragment of CTSD from the membrane to the cytosolic fraction of neutrophils following pyocyanin treatment. These data are in keeping with data showing a role for CTSD in apoptosis of fibroblasts(59, 68) and endothelial cells(69) following oxidant stress. Importantly, neutrophil primary granules contain significant amounts of CTSD(40, 70), accounting for 38% of acidic protease activity of neutrophils(71). Although CTSG was also released into the cytosol by pyocyanin treatment, CTSG inhibition did not abrogate apoptosis, suggesting a particular role for CTSD in this system. Our work showing that pyocyanin destablises neutrophil granules and releases CTSD into the cytosol may therefore explain the exquisite susceptibility of neutrophils to pyocyanin-induced apoptosis(12), since cells with lower or absent numbers of CTSD-containing granules (such as epithelial cells) are not stimulated to die when exposed to pyocyanin. The mechanisms by which CTSD induces apoptosis are uncertain. CTSD release is upstream of caspase activation(72) although a recent over-expression study suggests that the catalytic activity of CTSD is not essential for its pro-apoptotic role(73). The recent studies of Blomgran et al show a role for cysteinyl cathepsins, including CTSG, in mediating E. coli-induced neutrophil apoptosis, with evidence of cathepsin-mediated Bid cleavage and down-regulation of Mcl-1(74). Our studies also demonstrate reductions in Mcl-1 protein and that restoration of Mcl-1 protein levels by dbcAMP is associated with delay of pyocyanin-induced apoptosis.

A number of pathogens such as E. coli(75), Staphylococcus aureus(76) and Streptococcus pyogenes(77) are associated with neutrophil apoptosis following phagocytosis; this is likely to be a host-mediated process to prevent intracellular persistence of bacteria(78). However, our studies and those of Blomgran et al(74) identify a relationship between lysosome function and apoptosis in bacterial infection has not previously been recognised, despite the prominent role of lysosomal proteases in anti-bacterial responses of neutrophils. In these studies, pathogens effectively subvert these processes, with release of granule proteases triggered by bacterial-driven ROI generation.

In summary, we have demonstrated pyocyanin induces apoptosis by engagement of lysosomal pathways of cell death and we provide evidence it may be a pathological mechanism of cell death to which neutrophils are particularly susceptible. Furthermore, this is the first description of a bacterial toxin utilising this pathway of mammalian cell apoptosis to subvert host defense. Our findings are of clinical relevance, since understanding mechanisms to inhibit pyocyanin-induced neutrophil death in P. aeruginosa infections may lead to development of therapies that favour an effective immune response to this major human pathogen.