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Published ahead of print on October 4, 2007, doi:10.1164/rccm.200612-1804OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 35-43, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200612-1804OC

Original Article

Impairment of Apoptotic Cell Engulfment by Pyocyanin, a Toxic Metabolite of Pseudomonas aeruginosa

Stephen M. Bianchi1,*, Lynne R. Prince1,*, Kathleen McPhillips2, Lucy Allen3, Helen M. Marriott1, Graham W. Taylor4, Paul G. Hellewell3, Ian Sabroe1, David H. Dockrell5, Peter W. Henson2 and Moira K. B. Whyte1

1 Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; 2 Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado; 3 Academic Unit of Cardiovascular Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; 4 Department of Medicine, Hampstead Campus, Royal Free and University College School of Medicine, London, United Kingdom; and 5 Academic Unit of Infectious Diseases, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom

Correspondence and requests for reprints should be addressed to Prof. Moira Whyte, F.R.C.P., Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, M Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. E-mail: m.k.whyte{at}sheffield.ac.uk


   ABSTRACT
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AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Cystic fibrosis lung disease is characterized by accumulation of apoptotic neutrophils, indicating impaired clearance of dying cells. Pseudomonas aeruginosa, the principal microbial pathogen in cystic fibrosis, manipulates apoptosis induction via production of toxic metabolites. Whether these metabolites, particularly pyocyanin, can also modulate apoptotic cell engulfment is unknown.

Objectives: To assess the effects of pyocyanin on apoptotic cell engulfment by macrophages in vitro and in vivo and to investigate potential mechanisms of the observed effects.

Methods: Human monocyte–derived macrophages were treated with pyocyanin before challenge with apoptotic neutrophils, apoptotic Jurkat cells, or latex beads, and phagocytosis was assessed by light microscopy and flow cytometry. Effects of pyocyanin production on apoptotic cell clearance in vivo were assessed in a murine model, comparing infection by wild-type or pyocyanin-deficient P. aeruginosa. Oxidant production was investigated using fluorescent probes and pharmacologic inhibition and Rho GTPase signaling by immunoblotting and inhibitor studies.

Measurements and Main Results: Pyocyanin treatment impaired macrophage engulfment of apoptotic cells in vitro, without inducing significant macrophage apoptosis, whereas latex bead uptake was preserved. Macrophage ingestion of apoptotic cells was reduced and late apoptotic/necrotic cells were increased in mice infected with pyocyanin-producing P. aeruginosa compared with the pyocyanin-deficient strain. Inhibition of apoptotic cell uptake involved intracellular generation of reactive oxygen species (ROS) and effects on Rho GTPase signaling. Antioxidants or blockade of Rho signaling substantially restored apoptotic cell engulfment.

Conclusions: These studies demonstrate that P. aeruginosa can manipulate the inflammatory microenvironment through inhibition of apoptotic cell engulfment, and suggest potential strategies to limit pulmonary inflammation in cystic fibrosis.

Key Words: macrophages • phagocytosis • apoptosis • inflammation • cystic fibrosis



   AT A GLANCE COMMENTARY
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METHODS
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 DISCUSSION
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Scientific Knowledge on the Subject
Engulfment of apoptotic cells plays a crucial role in the resolution of inflammation and infection.

What This Study Adds to the Field
This study reports that a bacterial toxin modulates the ingestion of apoptotic cells, and identifies an additional mechanism by which pathogens subvert the host response to favor their own survival.

 
Resolution of inflammation involves apoptosis of recruited inflammatory cells and their recognition and clearance by professional phagocytes (1). These mechanisms can clear substantial inflammatory infiltrates without significant inflammatory cell necrosis or bystander tissue injury (2), but dysregulation of these efficient clearance systems is now widely described in inflammatory disease (3). The pathology of cystic fibrosis (CF) lung disease is characterized by a massive chronic neutrophilic inflammation of the airways, with the infiltrate containing excessive numbers of both apoptotic and necrotic neutrophils on a scale not seen in other inflammatory lung diseases (4). These findings could reflect increased neutrophil apoptosis, or impairment of apoptotic cell clearance, or a combination of these processes. This accumulation of effete neutrophils has important functional consequences, particularly the liberation of granule proteases (4, 5), and there is evidence in CF that neutrophil elastase can impair apoptotic cell clearance (4).

Pseudomonas aeruginosa is an opportunistic pathogen that causes a range of infections in the immunocompromised host and is the principal cause of mortality in CF lung disease (6). P. aeruginosa produces a range of factors that modify host immune responses and contribute to its pathogenicity (7). We have begun to dissect the impact of P. aeruginosa on both induction of apoptosis and clearance of apoptotic cells. In host–pathogen interactions, pathogen-driven neutrophil apoptosis is a well-recognized mechanism of immune evasion used by a number of bacteria (8), and we and others have shown P. aeruginosa can induce neutrophil apoptosis (9, 10). There are, however, no reports of a microbial factor modulating the engulfment of apoptotic cells by professional phagocytes such as macrophages. P. aeruginosa produces highly diffusible, pigmented toxic secondary metabolites, known as phenazines, that play a major role in killing infected organisms such as Caenorhabditis elegans and mice (11) and, in pneumonia models, cause extensive tissue damage (12). We have shown that pyocyanin, the principal phenazine generated by P. aeruginosa, induces rapid apoptosis of neutrophils (9, 13). Excess apoptotic neutrophils are detected in mice infected with a pyocyanin-producing, wild-type P. aeruginosa as compared with pyocyanin-deficient strains (13), confirming acceleration of apoptosis but also raising the possibility that pyocyanin might alter clearance of apoptotic neutrophils.

We therefore examined whether pyocyanin modulated uptake of apoptotic neutrophils by macrophages. We show significant reductions in apoptotic cell engulfment in vitro that are confirmed in a murine pneumonia model in vivo and are not due to reduced macrophage viability. We further show that impairment of apoptotic cell engulfment is dependent on reactive oxygen species (ROS) generation by macrophages and modulation of GTPase activity. To our knowledge, this is the first description of a microbial factor modulating apoptotic cell engulfment and identifies a potentially important mechanism of host tissue damage in infection.


   METHODS
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Materials
Pyocyanin was prepared by photolysis of phenazine methosulphate (Sigma, Poole, UK) and purified and characterized as described (14). Dihydroethidium (DHE), dihydrorhodamine (DHR), carboxyl-modified green fluorescent latex beads were from Sigma. Hoechst 33342, MnTBAP, and the Rho-kinase inhibitor (Y-27632) were from Calbiochem (San Diego, CA), C3 transferase (C3T) protein from Cytoskeleton (Denver, CO), and Rho/Rac activity assay from Upstate (Charlottesville, VA). All media, antibiotics and sera were from Life Technologies (Glasgow, UK). The terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) ApopTag Plus Peroxidase In Situ Apoptosis Detection kit used for in vivo studies was from the Intergen Co. (Oxford, UK), the TUNEL ApopTag Fluorescein Direct In Situ Apoptosis Detection kit used for assessment of human monocyte–derived macrophage (HMDM) apoptosis from Chemicon (Hampshire, UK), the Caspase-Glo 3/7 assay was from Promega (Southampton, UK), annexin V was from BD PharMingen (Oxford, UK), and To-Pro3 was from Invitrogen (Paisley, UK).

Cell Isolation and Culture
Human peripheral blood cells, neutrophils and PBMCs, were isolated from whole blood of healthy volunteers by dextran sedimentation and plasma–Percoll gradient centrifugation (9). Ethical approval was obtained from the South Sheffield Research Ethics Committee (Sheffield, UK), and all subjects gave informed consent. Resulting neutrophil populations were more than 97% pure, with the majority of contaminating cells being eosinophils. PBMCs were plated and matured to human monocyte-derived macrophages (HMDM) as previously described (1) and studied at Days 6–8 in culture. Jurkat cells were obtained from American Type Culture Collection (www.atcc.org) and grown in RPMI with 10% fetal calf serum.

Induction and Assessment of Apoptosis
Polymorphonuclear leukocytes (PMNs) (>95% purity) were cultured at 2.5 x 106/ml in RPMI with 1% penicillin/streptomycin and 10% fetal calf serum in 96-well Flexiwell plates (BD PharMingen) for 24 hours. At the 24-hour time point, cells were washed and shown to be typically 60 to 70% apoptotic by assessment of nuclear condensation on Giemsa-stained cytospins and necrosis was less than 2% by trypan blue exclusion. Jurkat cells were exposed to ultraviolet irradiation at 254 nm for 10 minutes, with similar levels of apoptosis. HMDM numbers and apoptosis were assessed by identification of characteristic nuclear morphology of apoptosis in Hoechst 33342–stained cells on fluorescence microscopy (15). In further experiments, HMDMs grown on coverslips were assessed for apoptosis by TUNEL, using an ApopTag Fluorescein Direct In Situ Apoptosis Detection kit following the manufacturer's recommended protocol. Coverslips were mounted on slides using VectaShield mounting media (Vector Labs, Peterborough, UK) containing 4'-6-diamidino-2-phenylindole (DAPI) (nuclear counterstain). Caspase 3/7 activity of HMDMs was measured using a Caspase-Glo 3/7 assay, following the manufacturer's recommended protocol. Briefly, 24 hours post-treatment, HMDMs were incubated with Caspase-Glo reagent for 1 hour in the dark, and the luminescence was measured with a Lumistar Galaxy luminometer (BMG Lab Technologies Ltd, Offenburg, Germany).

Phagocytosis Assays
HMDMs were cocultured for 1 hour with "aged" neutrophils (range, 60–70% apoptotic) suspended in 500 µl of Iscove's medium (without serum) in 24-well plates. Uningested cells were removed by washing with Hanks balanced salt solution (HBSS), then plates were fixed and stained for myeloperoxidase (1). Phagocytosis was determined by visual counting of 500 HMDMs in duplicate wells and was scored both as percentage of HMDM ingesting and as phagocytic index, generated by multiplying the percentage of macrophages that had phagocytosed cells by the average number of apoptotic cells ingested per macrophage (16). Uptake of fluorescent Cell Tracker (Invitrogen)–loaded apoptotic neutrophils or Jurkat cells was measured by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson), as previously described (17). Uptake of mixed serum-opsonized carboxyl-modified green fluorescent latex beads was scored by fluorescence microscopy, and we confirmed there were no differences in ingested rather than adherent cells by confocal microscopy (data not shown). In experiments using pharmacologic inhibitors, HMDMs were treated with the inhibitor before the addition of pyocyanin for the following times: MnTBAP (1–100 µM), 30 minutes; Y-27632 (10 µM), 30 minutes; C3T (1 µg/ml), 24 hours. The inhibitors also remained present throughout the pyocyanin pretreatment period.

Murine Model of P. aeruginosa Infection
This model of acute P. aeruginosa infection has been previously described (13). Briefly, C57BL6 mice (8–12 wk) were instilled with 1 x 107 cfu of bacteria via the trachea, either a pyocyanin-producing wild-type strain, PA14, or a pyocyanin-deficient but otherwise genetically identical strain, {Delta}phnAB (11). At the time points indicated, mice were killed by overdose and bronchoalveolar lavage (BAL) performed to obtain total and differential cell counts, including the proportion of neutrophils that were apoptotic (18). Macrophage ingestion of apoptotic cells was identified by morphologic criteria (16) and by TUNEL staining of BAL cytospins. Apoptotic and necrotic macrophages and neutrophils in BAL were identified by flow cytometry using annexin V and ToPro-3 staining, respectively (19).

Assessment of ROS Production
Production of ROS by HMDMs was assessed using cell-permeable DHR staining (5 µM for 30 min) and cells were analyzed by flow cytometry (20), with each sample run in triplicate. HMDMs were also stained with the oxidant-sensitive fluorescent probe (DHE), and ROS production assessed by nuclear fluorescence via microscopy (21).

Rho/Rac Activity Assays
Rho/Rac activity assays were performed according to the manufacturer's instructions. Briefly, 2.5 x 107 HMDMs were stimulated for time points as indicated. Active Rho and Rac were isolated by incubating lysates with sepharose beads bound to Rhotekin or p-21 activated kinase (PAK), respectively, which were run using a sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Whole cell lysates were run on separate gels and total Rho/Rac levels were evaluated using mouse anti-Rho (Cytoskeleton) or mouse anti-Rac (Upstate). The densitometry was measured using Image J software (National Institutes of Health, Bethesda, MD) and expressed as a percentage of the untreated control.

Statistical Analysis
Results are expressed as mean ± SEM. Analysis of variance (ANOVA) was applied for multiple comparisons, and posttests were applied where appropriate. Student's t tests were used for comparison of two sample means. Where important data comparisons did not reveal significant differences, this is indicated by NSD (no significant difference). All data were analyzed using GraphPad Prism version 4.0b (San Diego, CA).


   RESULTS
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DISCUSSION
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Pyocyanin Impairs Engulfment of Apoptotic Cells
To determine whether pyocyanin could inhibit apoptotic cell engulfment, we assayed phagocytosis of apoptotic PMNs (APMNs) by HMDMs in vitro. Pyocyanin caused a concentration- and time-dependent reduction in phagocytosis of APMNs. Of note, pyocyanin reduced both the proportion of HMDMs that ingested APMNs and the average number of APMNs ingested (Figure 1). In three further experiments, we confirmed that the defect was of ingestion rather than adherence of apoptotic cells to HMDMs using a flow-based assay to detect engulfment of Cell Tracker–labeled APMNs (see Table E1 in online supplement). Because pyocyanin causes acceleration of neutrophil apoptosis (9) and loss of macrophage viability would lead to loss of phagocytic capacity, we confirmed that pyocyanin was not causing macrophage death using three independent methods. First, pyocyanin-treated HMDMs were stained with a fluorescent nuclear dye, Hoechst 33342, and no significant loss of cell numbers was detected in pyocyanin-treated HMDMs compared with untreated control cells (Figures 2A and 2B). Second, TUNEL staining showed there was minimal, if any, increase in nuclear changes of apoptosis (Figure 2C), and third a caspase 3/7 activity assay detected no increase in caspase activation after pyocyanin treatment, in contrast to a positive control, staurosporine (Figure 2D).


Figure 1
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  		Figure 1. Pyocyanin impairs macrophage uptake of apoptotic neutrophils. Human monocyte–derived macrophages (HMDMs) were exposed to pyocyanin for up to 24 hours before incubation with apoptotic polymorphonuclear leukocytes (APMNs) for 1 hour and phagocytosis of APMNs was quantified by staining for myeloperoxidase (MPO). (A) Pyocyanin inhibition of apoptotic neutrophil engulfment is concentration dependent. Phagocytosis is expressed as mean ± SEM phagocytic index (% HMDMs phagocytosing x mean number APMNs per phagocytosing HMDM) after exposure to varying concentrations of pyocyanin for 24 hours. Data shown are from three independent experiments and significant differences from untreated cells are indicated for each concentration of pyocyanin (significance calculated by analysis of variance [ANOVA] with Dunnet's posttest). (B) The percentage of HMDMs ingesting APMNs after varying lengths of preincubation with pyocyanin (50 µM) in five independent experiments and significant differences from cells harvested at time 0 are indicated (significance calculated by ANOVA with Dunnet's posttest). (C, D) Ingestion of apoptotic cells is quantified by counting MPO-stained inclusions in HMDMs by light microscopy. Arrowheads highlight the presence of MPO-stained apoptotic neutrophils within untreated HMDMs (control; C), whereas apoptotic neutrophils are largely extracellular in HMDMs pretreated with pyocyanin for 24 hours (D). PYO = pyocyanin. Statistical significance is illustrated by *P < 0.05, **P < 0.01.

 

Figure 2
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  		Figure 2. Pyocyanin-treated monocyte-derived macrophages remain viable. Human monocyte–derived macrophage (HMDM) viability was assessed using Hoechst 33342 staining and fluorescence microscopy. Cell counts were performed for cell number and cells were also assessed for morphologic changes of apoptosis. (A) There was no loss of cell number in HMDMs pretreated with pyocyanin (50 µM) for 24 hours (solid bar) relative to controls (open bar) in three independent experiments. NSD signifies no significant difference. Chart shows data normalized to 100% in controls. (B) Photomicrographs illustrating typical morphology of viable cells in both control and pyocyanin-treated populations. (C) HMDM apoptosis was assessed by TUNEL staining in cells pretreated with pyocyanin (50 µM; solid bar) or staurosporine (10 µg/ml; shaded bar) for 24 hours relative to controls (open bar) in four independent experiments. The percentage of apoptotic cells was not significantly increased in pyocyanin-treated cells. (D) Caspase 3/7 activity was measured in HMDMs pretreated with pyocyanin (50 µM; solid bar) or staurosporine (10 µg/ml; shaded bar) for 24 hours compared with controls (open bar) (n = 4).

 
We addressed whether the phagocytic defect was specific for apoptotic cell uptake or more generalized. Engulfment of fluorescent-labeled apoptotic Jurkat cells was assessed by flow cytometry. Ingestion of these cells by control macrophages was significantly greater than ingestion of apoptotic neutrophils, 80% compared with 35%, but was significantly reduced after pyocyanin pretreatment (Figure 3A). In contrast, uptake of serum-opsonized carboxyl-modified green fluorescent latex beads, an assay of Fc-receptor–mediated phagocytosis, did not differ between pyocyanin-treated and control HMDMs (Figure 3B).


Figure 3
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  		Figure 3. The pyocyanin-mediated phagocytic defect is specific for apoptotic cells. (A) Apoptotic Jurkat cells were labeled with Cell Tracker (Invitrogen) and coincubated with human monocyte–derived macrophages (HMDMs) for 1 hour after pretreatment of HMDMs with 50 µM pyocyanin (solid bar) or media control (open bar) for 24 hours. Engulfment of apoptotic cells was measured by flow cytometry in three independent experiments and was significantly reduced by pyocyanin treatment. (B) HMDMs were pretreated with media (open bar) or 50 µM pyocyanin (solid bar) for 24 hours before being coincubated with carboxyl-modified green fluorescent latex beads at a ratio of 1:5 (HMDM:bead) for 1 hour. Phagocytosis was scored by fluorescence microscopy based on the percentage of macrophages containing at least one latex bead and did not differ significantly between the groups.

 
Mice Infected with Pyocyanin-producing P. aeruginosa Have Reduced Clearance of Apoptotic Cells
To determine whether pyocyanin impaired clearance of apoptotic cells in an acute P. aeruginosa pneumonia model (13), mice infected with a wild-type, phenazine-producing strain were compared with mice infected with a phenazine-deficient strain that has only 10% of wild-type pyocyanin production (11). The model is associated with rapid neutrophil influx and, in mice infected with phenazine-producing bacteria, with accelerated neutrophil apoptosis by 18 hours after bacterial instillation (13). To determine macrophage engulfment of apoptotic cells, we assessed apoptotic cell uptake by alveolar macrophages using TUNEL staining (Figure 4A) at 18 and 30 hours after instillation of bacteria. At 18 hours, total neutrophil counts were similar in mice infected with both strains (PA14, 1.92 ± 0.23 x 106; {Delta}phnAB, 1.80 ± 0.12 x 106) but were increased in the pyocyanin-deficient infection at 30 hours (PA14, 2.02 ± 0.14 x 106; {Delta}phnAB 3.90 ± 0.54 x 106; P < 0.05). There were increased numbers of apoptotic PMNs in BAL from mice infected with the wild-type strain at 18 hours (PA14, 8.20 ± 0.91 x 105; {Delta}phnAB, 1.96 ± 0.08 x 105; P < 0.01) and at 30 hours (PA14, 3.96 ± 0.80 x 105; {Delta}phnAB, 1.57 ± 0.37 x 105; P < 0.05), in keeping with our previous findings (13). Despite the excess of apoptotic cells in the infected PA14 mice, the proportion of macrophages containing apoptotic cells was lower in these mice (Figures 4B and 4C) and ingesting macrophages also contained reduced numbers of apoptotic bodies (Figures 4D and 4E) in wild-type infection compared with mice infected with the phenazine-deficient strain. Importantly, increased numbers of late apoptotic/necrotic (To-Pro3 positive) cells were detected at 30 hours in mice infected with the pyocyanin-producing strain (Figures 4F and 4G), suggesting the wave of early apoptotic cells detected at 18 hours had not been efficiently cleared. Moreover, these data support the view that the reduced numbers of apoptotic cells detected within macrophages in wild-type P. aeruginosa infection reflect impaired engulfment rather than increased efficiency of apoptotic body degradation by macrophages. In keeping with our previous data (13), there was no difference in BAL macrophage numbers in the two groups of mice, and we detected no differences in macrophage death, assessed by To-Pro3 staining, between mice infected with the different strains (see Figure E1). There was a trend toward increased bacterial numbers in mice infected with wild-type P. aeruginosa (log10 cfu: PA14, 5.82 ± 0.47; {Delta}phnAB, 4.09 ± 0.55 at 18 h; and PA14, 4.98 ± 0.16; {Delta}phnAB, 4.02 ± 0.54 at 30 h). These colony numbers were not significantly different, perhaps reflecting the sample size, although significant differences were found only at 48 hours and beyond in our previous study (13).


Figure 4
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  		Figure 4. Pyocyanin production is associated with impaired apoptotic cell clearance in vivo. C57BL6 mice were instilled with 107 cfu live wild-type strain (PA14; open bars) or phenazine-deficient strain ({Delta}phnAB; solid bars) of P. aeruginosa, and bronchoalveolar lavage (BAL) was performed after 18 (B, D, F) or 30 (C, E, G) hours. Total and differential cell counts were obtained by hemocytometer and cytospin counts. Data shown were obtained from three independent experiments. (A) Cytospins were TUNEL stained and macrophage phagocytosis of apoptotic bodies (TUNEL positive inclusions, indicated by arrowheads) visualized by microscopy. (B, C) Phagocytosis of APMNs by macrophages was assessed and represented as percentage of macrophages engulfing APMNs (D, E). Phagocytic index was similarly calculated from TUNEL-stained cytospins. (F, G) The presence of necrotic cells in BAL was assessed by ToPro-3 uptake on flow cytometry. Statistical significance was calculated by Student's t test and is illustrated by {dagger}P < 0.05, {dagger}{dagger}P < 0.01.

 
ROS Production Mediates Reduced Apoptotic Cell Uptake by Pyocyanin-treated Macrophages
The cytotoxic effects of pyocyanin on bacteria and eukaryotic cells are linked to its ability to undergo nonenzymatic redox cycling within cells, with resulting ROS generation (22). P. aeruginosa–induced killing of C. elegans and in a murine model of sepsis is dependent on both pyocyanin production and ROS generation (11), and pyocyanin-mediated neutrophil apoptosis is associated with massive and sustained generation of ROS (9). We measured ROS production in HMDMs using a fluorescent probe, DHR. We observed a significant increase in ROS 30 minutes after pyocyanin treatment (Figures 5A and 5B) that remained elevated to 24 hours. The magnitude of ROS generation by macrophages appeared less than that of neutrophils (data not shown), possibly reflecting their greater antioxidant levels (23), which may also explain their resistance to pyocyanin-induced apoptosis. An antioxidant and superoxide dismutase-mimetic, MnTBAP, significantly reduced ROS levels at 6 hours (Figure 5C) at concentrations of 1 µM and above. We confirmed these findings using a second fluorescent probe, DHE, demonstrating nuclear fluorescence after pyocyanin treatment that was inhibited by MnTBAP (Figure 5C). A previous study showed that some fluorescent probes, including DHR, can be directly oxidized by redox-active compounds, including pyocyanin, but the increased fluorescence signal that results is not prevented by antioxidants (24). Our data show that the pyocyanin-induced increase in macrophage fluorescence is significantly inhibited by antioxidant treatment (Figure 5C). Crucially, MnTBAP, at a concentration that inhibited ROS production, also reversed pyocyanin-mediated impairment of apoptotic cell uptake, demonstrating dependence on ROS generation (Figure 5D).


Figure 5
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  		Figure 5. Pyocyanin impairment of macrophage apoptotic cell uptake is mediated via reactive oxygen species (ROS) generation. (A) ROS production by human monocyte–derived macrophages (HMDMs) was measured by flow cytometry. Dihydrorhodamine-loaded HMDMs were treated with 50 µM pyocyanin over a range of time points (30, 60, 240, and 360 min). Representative histograms show increases in fluorescence (oxidant production) as right shifts on the x axis (FL-1). (B) Mean data for ROS production, expressed as geometric mean fluorescence intensity (MFI) over time, in HMDMs treated with 50 µM pyocyanin in the presence (solid bars) or absence (open bars) of an antioxidant, MnTBAP (1 µM). Data were from three independent experiments and significant differences are indicated for pyocyanin-treated cells (open bars) for each time point compared with cells at time 0. Results for cells incubated with MnTBAP and pyocyanin compared with pyocyanin alone were significantly different only at 360 minutes as indicated by the bar (significance calculated by analysis of variance with Bonferroni's posttest). (C) HMDMs were treated for 6 or 24 hours with media (control), pyocyanin (50 µM), MnTBAP (100 µM), or pyocyanin and MnTBAP in combination. Nuclear fluorescence was visualized by dihydroethidium staining and fluorescence microscopy. Representative images are shown from a single experiment of a set of three. (D) Media- and pyocyanin-treated HMDMs were cultured in the presence (solid bars) or absence (open bars) of MnTBAP (1 µM). HMDMs were subsequently incubated with APMNs for 1 hour and ingestion scored by myeloperoxidase staining and microscopy. Data from three independent experiments are shown as phagocytic index. Statistical significance was calculated by ANOVA and is illustrated by *P < 0.05, **P < 0.01, ***P < 0.001.

 
Pyocyanin Inhibits Apoptotic Cell Uptake by Effects on Small GTPases
Pathways originally defined in C. elegans involve the Rho family of low-molecular-weight GTPases in signaling pathways mediating apoptotic cell engulfment (25). The Rho GTPase Rac facilitates engulfment of apoptotic cells, whereas Rho inhibits uptake (25). Rho-kinase is a downstream mediator of RhoA inhibition of uptake of apoptotic cells, and blockade of Rho-kinase enhances apoptotic cell uptake (26). We therefore investigated whether pyocyanin-induced ROS might mediate its actions by alterations in Rho signaling. Rho activity was measured using Rhotekin to pull down active Rho, and we analyzed both total and active Rho by Western blotting. We found that Rho activity was significantly increased after pyocyanin treatment (Figures 6A and 6B), whereas Rac-1 activity, measured using PAK to pull down active Rac, was significantly reduced by pyocyanin treatment (Figures 6C and 6D). Thus, pyocyanin treatment of HMDMs inhibited apoptotic cell uptake, inhibited Rac, and activated Rho.


Figure 6
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  		Figure 6. Pyocyanin inhibits apoptotic cell uptake via effects on Rho/Rac GTPases. Human monocyte–derived macrophages (HMDMs) were treated with pyocyanin for the indicated times and analyzed for active and total Rho (A, B) and Rac (C, D). Representative gels are shown from a single experiment of a set of four and the mean ± SEM amounts of active Rho/Rac were quantified using Image J densitometry software (n = 4; National Institutes of Health). *Amounts of Rho/Rac significantly different from levels in untreated cells. (E) HMDMs were treated with media (control) or pyocyanin (50 µM) (open bars) for 24 hours in the presence or absence of Y-27632 (10 µM) (solid bars) or C3 transferase (1 µg/ml) (shaded bars). HMDMs were subsequently incubated with APMNs and ingestion scored by microscopy (times indicated above represent incubation periods before addition of APMNs) and expressed as phagocytic index. Data were obtained from seven independent experiments (significance calculated by analysis of variance with Bonferroni posttest). PYO = pyocyanin. Statistical significance was calculated by ANOVA and is illustrated by *P < 0.05, **P < 0.01, ***P < 0.001

 
We next examined the ability of C3 transferase (from Clostridium botulinum), which inactivates RhoA, as well as Y-26732, a specific inhibitor of Rho-kinase, to reverse pyocyanin-impaired macrophage engulfment of apoptotic neutrophils. We found that both compounds, at concentrations previously shown to inhibit their respective targets (26), substantially restored engulfment of APMNs by pyocyanin-treated macrophages (Figure 6E). These data place regulation of GTPase activity downstream of induction of ROS in macrophages and are consistent with other recent data from one of our labs that identified a role for ROS production and inactivation of small GTPases in impairment of apoptotic cell uptake after tumor necrosis factor-{alpha} treatment of macrophages (21).


   DISCUSSION
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that pyocyanin, a major secondary metabolite of P. aeruginosa, impairs macrophage engulfment of apoptotic cells as a result of intracellular ROS generation and modulation of small GTPase signaling. This finding identifies a novel and potentially important mechanism by which pathogens could disrupt efficient clearance of inflammatory cells, increasing host tissue injury.

Chronic infection with P. aeruginosa is a major cause of pulmonary damage and mortality in patients with CF (27), and a number of different P. aeruginosa products have been shown to modify host immune responses (7). A central feature of CF lung disease is abnormal neutrophil recruitment and persistence in the airway, often beginning early in childhood (28). There is also evidence for aberrant neutrophil death in the CF lung, leading to DNA release and sputum hyperviscosity (29), and leakage of major proteases such as neutrophil elastase that exacerbate lung injury (30). The proportion of neutrophils in CF sputum that are apoptotic is in the region of 30 to 40% (4), vastly in excess of the levels of less than 1% found in patients with community-acquired pneumonia (31). This in turn suggests delayed clearance of apoptotic cells in CF airways, which could reflect either macrophage capacity for phagocytosis being overwhelmed by vast numbers of effete neutrophils or a more specific defect of macrophage engulfment. Against the former possibility is the observation of effective clearance of apoptotic cells and subsequent resolution of acute lobar pneumonia where the burden of neutrophils is also very substantial (32). Vandivier and colleagues showed that the presence of neutrophil elastase in CF sputum specifically impairs engulfment of apoptotic neutrophils, demonstrating a "vicious circle" in which the presence of large numbers of effete neutrophils will further impair apoptotic cell clearance (4).

We investigated the possibility that P. aeruginosa might directly impair apoptotic cell engulfment. There is some evidence that dysregulation of clearance mechanisms may be pathogen dependent. Watt and colleagues (34) analyzed sputum samples from patients with CF and identified a large excess of late apoptotic or secondarily necrotic neutrophils in patients infected with P. aeruginosa or Burkholderia cenocepacia compared with those infected with other gram-negative pathogens. The findings suggest these pathogens are causing accelerated neutrophil apoptosis, as previously described for both P. aeruginosa (9) and B. cenocepacia (33), as well as other bacterial pathogens (8), but could also imply that phagocytosis of effete neutrophils by macrophages was impaired in these infections (34). The patients with CF studied by Vandivier and coworkers, who showed evidence both of profoundly impaired apoptotic cell clearance and of neutrophil necrosis, were also all infected with P. aeruginosa (4). We studied the major toxic metabolite of P. aeruginosa, pyocyanin, because it has been shown to be a key agent of P. aeruginosa pathogenicity in multiple experimental models and to cause massive oxidant stress by intracellular redox generation (11, 35). We found that pyocyanin impaired macrophage engulfment of apoptotic cells in vitro at concentrations that have been reported in sputum from patients chronically colonized with P. aeruginosa (36). Although there are reports of P. aeruginosa inducing apoptosis of a macrophage-like cell line in vitro (37), pyocyanin treatment did not cause significant macrophage apoptosis. Using a murine model of acute resolving P. aeruginosa, we showed that infection with a pyocyanin-producing strain of P. aeruginosa was associated with reduced numbers of apoptotic cells within airway macrophages and with increased numbers of necrotic neutrophils that had not been engulfed. There were no differences in macrophage numbers or evidence of increased macrophage apoptosis in mice infected with the pyocyanin-producing strain of P. aeruginosa, compared with a phenazine-deficient strain in which pyocyanin production was shown to be 10% of that in wild-type strains (11). There is a previous report of P. aeruginosa inducing apoptosis of a macrophage-like cell line via type III secretion-dependent mechanisms (37). We cannot exclude effects of other P. aeruginosa virulence factors on macrophage numbers, but there were no differences in macrophage numbers between the two strains, so the impact of any such factors was similar between wild-type and phenazine-deficient bacteria. The reduction in apoptotic cell engulfment in mice infected with the wild-type strain was observed at both time points studied, but was more marked at 30 hours. Because we were unable to measure pyocyanin production in vivo in the murine model, we cannot determine whether this reflects a delayed pyocyanin effect or that an increase in "available" apoptotic cells "unmasks" the defect at 30 hours. However, it is notable that pyocyanin produces long-lasting effects in vitro: oxidant production persisted 6 hours after pyocyanin treatment (the latest time point at which it was measured), down-regulation of Rac activity was significant only at 24 hours after pyocyanin treatment, and maximal inhibition of apoptotic cell engulfment was also detected after 24 hours. These data identify a pathologic process that could exacerbate host tissue injury, both directly by release of proteases and other toxic products from secondarily necrotic neutrophils (4) and indirectly by failure of apoptotic cell uptake to generate macrophage release of antiinflammatory cytokines (38). A more chronic model of P. aeruginosa infection, as opposed to the acute, resolving model used in these experiments, could further address effects on host tissue injury.

As predicted from other cell types, including neutrophils (9, 35) and pulmonary epithelial cells (22), pyocyanin treatment caused generation of ROS in macrophages, using pyocyanin concentrations detected in clinical samples. Pyocyanin-induced ROS generation induces apoptosis in neutrophils (9) and in pulmonary epithelial cells, although over a longer time course (9, 22, 39). We did not detect significant macrophage apoptosis after pyocyanin treatment, and it is possible that the much greater sensitivity of neutrophils to pyocyanin-induced apoptosis reflects their low basal activity of antioxidant enzymes, in contrast to macrophages which have much higher levels, particularly of glutathione (GSH) and glutathione peroxidase (23). The macrophage thus exhibits a more subtle phenotype of impaired apoptotic cell engulfment, without loss of viability. Engulfment was partially restored by treatment with an antioxidant, MnTBAP, confirming that the effects of pyocyanin were ROS dependent. A previous study had shown that monocyte-derived macrophage engulfment of apoptotic cells was impaired by hydrogen peroxide treatment, supporting a specific role for ROS in impairing apoptotic cell engulfment (40). The phagocytic defect appears to be specific to apoptotic cell uptake, as evidenced by the signaling pathway implicated, and also by preserved Fc-mediated uptake of fluorescent beads. The latter is in keeping with studies by Muller and colleagues (41), who showed macrophage uptake of bacteria was preserved in the presence of pyocyanin.

A large number of receptors for surface changes on apoptotic cells have been implicated in the tethering, engulfment, and signaling uptake of apoptotic cells in mammalian systems, with evidence for considerable redundancy (42). Studies of deficiencies of engulfment in C. elegans, in contrast, have identified a number of genes in signaling pathways for cell clearance, including evidence the Rho-family GTPases are involved in engulfment of apoptotic cells (43) and in regulation of subsequent maturation of the phagosome (44). We found that pyocyanin treatment of HMDMs increased Rho activity and reduced Rac-1 activity, a pattern previously associated with impaired apoptotic cell engulfment (25, 26). There is evidence the Rho/Rac balance within cells might determine the rate of phagosome maturation and that, in monocyte-derived macrophages, inhibition of Rho signaling pathways delays intracellular disposal of ingested apoptotic cells (44). Theoretically, therefore, activation of these pathways by pyocyanin might accelerate degradation of ingested apoptotic cells and thus contribute to the finding of smaller numbers of apoptotic cells detected within macrophages after pyocyanin treatment or in the P. aeruginosa infection model. However, the increased numbers of uningested late apoptotic/necrotic neutrophils observed in the mice infected with pyocyanin-producing P. aeruginosa support the view that there is impaired engulfment of apoptotic cells. Moreover, inhibition of Rho signaling pathways does not alter the numbers of ingested apoptotic neutrophils detected within monocyte-derived macrophages in vitro, as shown in Figure 6E, showing accelerated degradation of apoptotic cells is not an explanation for our findings.

We showed that the effects of pyocyanin on apoptotic cell engulfment could be reversed by treatment with an antioxidant, MnTBAP, or by Rho-kinase inhibition, and thus are amenable to therapeutic targeting. The specific defect of apoptotic cell engulfment could be abrogated by specific Rho-kinase inhibitors, novel compounds which have been used as vasodilators in clinical trials in patients with stable angina (45) and ischemic stroke (46). Recent evidence also suggests that statins, which inhibit prenylation of Rho GTPases, can also enhance apoptotic cell engulfment by both monocyte-derived macrophages and, importantly, human alveolar macrophages (47). More generally, there is evidence of increased oxidative stress in the airways in CF, which is a major factor in lung damage and is believed to be caused by excessive neutrophil activation and oxidant release (48). Oxidative stress is associated with airway inflammation (49) and is increased in infection with P. aeruginosa or B. cenocepacia (50). Treatment with antioxidants may be of clinical benefit (48) and has specifically been shown to reduce neutrophilic inflammation and neutrophil elastase activity in the airways (48). Our data suggest that one important effect of antioxidant treatment might be restoration of apoptotic cell engulfment that has been impaired by P. aeruginosa infection, in addition to other beneficial effects of these compounds.

In conclusion, we show that pyocyanin, a major toxin produced by P. aeruginosa, impairs macrophage engulfment of apoptotic cells both in vivo and in vitro and that these effects are substantially reversed by antioxidants or by blockade of Rho signaling pathways. Because more than 95% of those with significant CF lung disease are infected with P. aeruginosa and pyocyanin also accelerates neutrophil apoptosis, therapies directed at the toxic effects of pyocyanin could abrogate both acceleration of neutrophil apoptosis and impairment of apoptotic cell clearance, with the potential to reduce chronic tissue injury.


   Acknowledgments
 
The authors thank Dr. Frederick Ausubel (Harvard Medical School) for the gift of PA14 and {Delta}phnAB strains of P. aeruginosa and Professor John Savill and Dr. Simon Brown (University of Edinburgh) for very helpful discussions.


   FOOTNOTES
 
Supported by a Wellcome Trust Clinical Training Fellowship to S.M.B. (ref. 064997) and the Sheffield Hospitals Special Trustees. D.H.D. is a Wellcome Senior Clinical Fellow and I.S. holds a Medical Research Council Senior Clinical Fellowship.

* These authors contributed equally to this article and are joint first authors. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200612-1804OC on October 4, 2007

Conflict of Interest Statement: S.M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.R.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.W.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.G.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.S. has received support from GlaxoSmithKline and AstraZeneca for conference attendance, and has received lecture fees from GlaxoSmithKline, Boehringer Ingelheim, and AstraZeneca. He has also received an unrestricted research fellowship from GlaxoSmithKline. D.H.D. has received support from GlaxoSmithKline, Gilead, Boehringer Ingelheim, Abbott, and Roche for conference attendance and has received lecture fees from GlaxoSmithKline to facilitate conference attendance. P.W.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.K.B.W. has received a research grant from GlaxoSmithKline, relating to a multicenter asthma genetics study. She has received support from Boehringer Ingelheim for conference attendance and lecture fees from AstraZeneca.

Received in original form December 13, 2006; accepted in final form October 3, 2007


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TOP
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 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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