Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47phox and p40phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia. These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

Cystic fibrosis (CF) is a multiorgan disease caused by defects in chloride transport caused by defective CF transmembrane regulator (CFTR) conductance (1). Clinically, patients characteristically suffer from recurrent polymicrobial lung infections with eventual respiratory demise (2). Members of the Burkholderia cepacia complex cause fatal septicemia and rapid outbreaks in patients with CF (35), and their multidrug-resistant phenotype has rendered many antibiotic regimens ineffective. B. cenocepacia is thought to be the most virulent species of the complex and is associated with poor posttransplant survival in chronically infected patients leading to exclusion from life-saving lung transplant eligibility (610). In addition to playing an important role in CF, Burkholderia species (including B. cenocepacia and B. multivorans) are increasingly recognized as the causative agents of serious hospital-acquired infections in non-CF patients (1113). The inability of patients with and without CF to clear Burkholderia is a major factor in the disease course and is predicated on the inability of macrophages to kill ingested B. cenocepacia or B. multivorans (14). The vital role of macrophages in CF host–pathogen interactions has been highlighted recently by several groups (1419). Although macrophage-mediated clearance of B. cenocepacia is defective in CF (20, 21), little is known regarding differences when compared with clearance of the less clinically virulent B. multivorans (8, 22, 23).

Intracellular killing of other pathogens besides Burkholderia is also defective in CF, despite abundant neutrophil-mediated production of reactive oxygen species (ROS) in lung tissue (24, 25). CF airways are characterized by constitutive ROS production that is dependent on both failed bacterial clearance and defective CFTR, and can be alleviated with autophagy stimulation (26). However, it remains unclear why intracellular pathogen killing in CF macrophages is defective, despite the abundant production of ROS in lung tissue (24, 25). Macrophages are more resistant to damage from ROS-induced oxidative stress compared with neutrophils and monocytes (27); therefore, intracellular organisms may persist within macrophages even in environments of continuous host inflammatory production such as the CF lung, providing a potential replicative niche for Burkholderia.

In addition to CF macrophage deficits in host immune responses, Burkholderia can scavenge ROS (28) and affect key oxidase components (29, 30) in non-CF cells. Generation of ROS by assembly of the NADPH in macrophages in response to pathogens is a fundamental host defense strategy. Despite this, little is known about the impact of CFTR on NADPH assembly and activation in human macrophages, and no studies have demonstrated an inherent defect in ROS production in human CF macrophages. In addition, how generation of an oxidative burst occurs in CF macrophages after initial contact with Burkholderia is not clear, including assembly of the NADPH oxidase complex and subsequent production and function of ROS, despite the fact that these macrophages do indeed ingest and harbor bacteria. In murine macrophages, B. cenocepacia delays association of the NADPH oxidase complex with macrophage vacuoles and can disrupt NADPH oxidase assembly, but these events have not been studied in human macrophages. Assembly of the NADPH oxidase requires translocation of phosphorylated p47phox, p40phox, p67phox, and Rac from the cytoplasm to the phagosomal membrane (31). B. cenocepacia specifically affects the activation of Rac in murine macrophages (30).

We undertook this study to more clearly characterize ROS production and assembly of the NADPH oxidase in human CF macrophages in response to an ROS agonist, Burkholderia and other stimuli, with hopes of identifying new targets for drug development against Burkholderia and other chronic infections in CF. We demonstrate for the first time, to our knowledge, that human CF macrophages inherently have a reduced oxidative burst in response to PMA. Ingestion of Burkholderia cenocepacia independently contributes to and worsens this defect in CF macrophages compared with non-CF macrophages. Finally, CF macrophages demonstrate decreased expression of the protein kinase C (PKC) conventional subclass leading to decreased activation of NADPH oxidase components p47phoxand p40phox in comparison with non-CF macrophages. Together, these results provide evidence for deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components and a subsequent reduction in NADPH oxidase activation in CF. These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in patients with CF.

Macrophages were infected with RFP-expressing B. cenocepacia strain k56-2, B. multivorans strain FC-445, or Pseudomonas aeruginosa PA01 for 1–2 h before treatments based on known intracellular uptake time (14, 21, 32). The B. cenocepacia strain is representative of an epidemic clinical strain from the ET12 lineage (33). Bacteria were reproducibly grown in Luria-Bertani media over 24 h. For paraformaldehyde (PFA) experiments, pelleted bacteria were resuspended in 4% PFA for 30 min and washed five times with Dulbecco’s PBS (DPBS) to remove PFA.

All human subjects were recruited as approved by the Institutional Review Board of Nationwide Children’s Hospital. All subjects underwent written consent for the procedures; that is, all adult subjects provided informed consent, and a parent or guardian of any child participant provided informed consent on their behalf along with written assent from children.

Heparinized blood was obtained from 35 CF and 35 non-CF healthy control subjects. Subjects with a history of chronic immunosuppression including chronic steroid use, CFTR modulator use, or history of transplantation were excluded. Chronic azithromycin use was allowed. Peripheral monocytes were separated from whole blood using lymphocyte separation medium (25-072-CV; Corning). Isolated monocytes were resuspended in RPMI 1640 (22400-089; Life Technologies) plus 10% human AB serum (14-490E; Lonza) and differentiated for 5 d at 37°C into monocyte-derived macrophages (MDMs) (21, 34). MDMs were isolated, placed in monolayer culture, and infected at bacterial multiplicity of infection (MOI) ranging from 2 to 10 based on prior studies on the impact of bacterial infection on ROS production (14, 21, 32). The THP-1 monocytic line was used in preliminary experiments to optimize conditions before studies with human MDMs. THP-1 cells were grown in 10% FBS (Thermo Scientific) in RPMI 1640. THP-1 cells were treated with 200 nM of PMA (Calbiochem) and 30 ng/ml GM-CSF (415-ML-050; R&D Systems) to differentiate cells into macrophages. Media were replenished with 30 ng/ml GM-CSF the next day, and the THP-1–derived macrophages were allowed to mature 5 d before experimentation. THP-1 cells were then treated with the CFTR inhibitor Inh-172 for 24 h before experimentation.

MDMs were infected at an MOI of 50 for 30 min for studies of ROS production. Supernatants were removed posttreatment, and the cells were washed twice with PBS (Fisher). The cells were lysed in lysis buffer (HEPES, MgCl, EGTA, KCL, Nonidet P-40) with protease inhibitor (10-519-978-001; Roche Applied Science). Then, 30 μg of protein was separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were immunoblotted for calreticulin (ADI-SPA-600; Enzo Life Sciences), phospo-p40phox (4311; Cell Signaling), total p40phox (ab137691; Abcam), phospho-p47phox (donated by Jamel El-Benna), total p47phox (A16636; Life Technologies), p67phox (SC-15342; Santa Cruz), Rac2 (ab2244; Abcam), gp91phox (SC-5827; Santa Cruz), p22phox (SC-20751; Santa Cruz), p-PKC α/β II (9375P; Cell Signaling), p-PKC δ (9374P; Cell Signaling), and p-PKC ζ/λ (9378P; Cell Signaling). Protein bands were detected with HRP-conjugated secondary Abs and visualized using ECL reagents (RPN2106; Life Sciences). Membrane and cytosolic fractionations were prepared via manufacturer kit instructions (78840; Thermo Fisher).

The oxidative burst was measured by a 2′,7′-dichlorofluorescein (DCF) assay (D399; Life Technologies) using relative fluorescent units (RFUs). MDMs were adhered to 96-well plates at 4E6 cells/well in duplicate for 2 h and repleted in DPBS + 10 mM HEPES + 1 mg/ml human serum albumin + 0.1% glucose. After 30-min incubation at 37°C, 10% DCF was added to the wells for 30 min at 37°C. A stimulus such as PMA was then added to macrophages, and fluorescence was measured at a 485 nm excitation wavelength and a 515 nm emission wavelength every 2 min for 2 h. In preliminary experiments, PMA was used at varying concentrations, with 200 μM determined to be the optimal concentration for DCF experiments. 1-Oleoyl-2-acetyl-sn-glycerol (OAG; Sigma) was used at a concentration of 20 μM for PKC induction.

Superoxide production was measured by a cytochrome c (CytC; Sigma) assay using relative absorbance (35). MDMs were adhered to 24-well plates at 4E6 PBMCs/ml and repleted in DPBS + 10 mM HEPES + 1 mg/ml human serum albumin + 0.1% glucose. Cells were stimulated with PMA, bacteria (MOI 10), and/or medium-only control in duplicate wells in the presence of 80 μM of CytC and 500 U/ml catalase (Sigma) at 37°C for 2 h. CytC reduction indicative of superoxide production was measured by subtracting the absorbance at 550 nm of control wells treated with 300 U/ml superoxide dismutase (SOD) (S5395; Sigma) from test well values. Unstimulated cell background values were subtracted from treated wells.

Two million MDMs were cultured on 12-mm glass coverslips in 24-well tissue culture plates and infected synchronously with bacteria at an MOI of 2–10. Nuclei were stained with the DAPI blue for imaging. MDMs were fluorescently labeled with either Phospho-p40phox (4311; Cell Signaling) or phospho-p47phox (A1171; Assay Biotech). Confocal microscopy was performed using an Axiovert 200 M inverted epifluorescence microscope equipped with the Apotome attachment for improved fluorescence resolution and an AxioCam MRM CCD camera (Carl Zeiss, Thornwood, NY). At least 100 MDMs were scored for each condition. All experiments were performed in triplicate or quadruplicate.

MDMs were resuspended at 1E7 cells/ml in HBSS with Ca2+ and MgCl2 (SH 30268.02; HyClone), 1% FBS (HyClone, SH30071.03; Thermo Scientific), and 4 mM of probenecid (P36400; Invitrogen). MDMs were treated with 1:100 anti-human CD-16 Brilliant Violet 605TM (302040; BioLegend) and 1:100 anti-human CD14-allophycocyanin-Cy7 (325620; BioLegend) to identify macrophage populations. The MDMs were incubated at 37°C for 30 min with 4 μg/ml fluorescent dye Fluo-3 AM (F1242; Life Technologies), washed twice, and resuspended in cell-loading HBSS medium at 1E7 cells/ml. MDMs were stimulated with 1 μg/ml Ionomycin (124222; Sigma-Aldrich), 1 μM platelet-activating factor (PAF) (P4904; Sigma-Aldrich), bacteria (MOI 10), or PMA (524400; Calbiochem). The intensity of intracellular Ca2+ in individual cells was assessed by flow cytometer (LSR II; BD) measuring the fluorescence emission of Fluo-3 in the FL-1 channel over 200 s. Data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

All statistical analyses were performed using GraphPad Prism software (version 6.0). Two sample t tests or Mann–Whitney U tests were used for comparisons of independent samples. Paired t tests or Wilcoxon signed-rank tests were used for within-patient comparisons. Statistical significance was defined as p < 0.05. Age- and sex-matched healthy control subjects were used in the analysis.

Human donor characteristics are summarized in Table I. More CF males (60.0%) compared with controls (34.3%) were available for sampling, and both populations were entirely white. On average, CF patients had moderate lung disease with a mean percentage of forced expiratory volume in 1 s predicted of 65.8 ± 24.7%. Most CF patients were pancreatic insufficient (83.3%) and had at least one copy of the F508del mutation (87.6%). Pseudomonas aeruginosa was the predominant respiratory pathogen isolated on the most recent respiratory cultures for the CF patients.

Table I.
Patient demographics
Non-CF (n = 35)CF (n = 35)
Mean age ± SD, y 34.7 ± 10.8 29.1 ± 10.1 
Male sex 34.3% 60.0% 
White 100.0% 100.0% 
Genotype   
 F508del homozygous N/A 54.3% 
 F508del heterozygous N/A 34.3% 
 Other N/A 11.4% 
 Pancreatic insufficiency N/A 83.3% 
 Mean FEV1 (% predicted) N/A 65.8 ± 24.7 
P. aeruginosa colonization N/A 80% 
 MRSA colonization N/A 37.1% 
Non-CF (n = 35)CF (n = 35)
Mean age ± SD, y 34.7 ± 10.8 29.1 ± 10.1 
Male sex 34.3% 60.0% 
White 100.0% 100.0% 
Genotype   
 F508del homozygous N/A 54.3% 
 F508del heterozygous N/A 34.3% 
 Other N/A 11.4% 
 Pancreatic insufficiency N/A 83.3% 
 Mean FEV1 (% predicted) N/A 65.8 ± 24.7 
P. aeruginosa colonization N/A 80% 
 MRSA colonization N/A 37.1% 

FEV1, forced expiratory volume in 1 s; MRSA, methicillin-resistant Staphylococcus aureus.

It is unclear whether CF macrophages have basal deficits in oxidative killing because some studies have suggested functional ROS responses to pathogens such as P. aeruginosa (36), whereas others have demonstrated deficient ROS production (37). Importantly, a comprehensive assessment of human CF macrophage oxidase assembly has not been performed. Therefore, we isolated human MDMs from stable CF and non-CF donors and analyzed them for their basal capacity to produce an oxidative burst in response to PMA, an analog of diacylglycerol (DAG), and a soluble, intracellular activator of the NADPH oxidase via PKC. The oxidative burst was measured by a DCF assay using RFUs of cellular oxidation through hydrogen peroxide–mediated pathways. CF MDMs demonstrated a significant decrease in the oxidative burst in response to PMA compared with non-CF MDMs (p = 0.002; Fig. 1A, 1B). This finding was then confirmed with a second specific assay of superoxide production (CytC assay). Superoxide production was measured in response to mock or PMA after 60-min stimulation by measuring the SOD inhibitable reduction of exogenous CytC. CF MDMs demonstrated a nearly 60% reduction in superoxide production in response to PMA compared with non-CF MDMs (Fig. 1C, 1D).

FIGURE 1.

CF MDMs have reduced ROS production in response to PMA. (A) CF and non-CF MDMs were treated with PMA for 30 min and assessed for ROS production using RFUs via a DCF assay. Representative assay of n = 6. (B) Summed end-point analysis of (A) experiments expressed as % ROS production at 2 h for CF MDMs relative to control non-CF MDMs in response to PMA, p = 0.002. (C) Cells were treated with PMA and/or medium-only control in triplicate wells in the presence of 500 U/ml catalase and 80 μM of CytC. CytC reduction, indicative of superoxide production, was measured after 60 min of stimulation by subtracting the absorbance at 550 nm of control wells treated with 300 U/ml SOD from test well values. Unstimulated cell background values were subtracted from test conditions, and all values were set relative to the positive control. Representative experiment of n = 3. (D) Summed end-point analysis of experiments in (C); results are expressed as percentage of non-CF PMA-stimulated cells for three independent experiments, p = 0.01. *p < 0.05, **p < 0.01.

FIGURE 1.

CF MDMs have reduced ROS production in response to PMA. (A) CF and non-CF MDMs were treated with PMA for 30 min and assessed for ROS production using RFUs via a DCF assay. Representative assay of n = 6. (B) Summed end-point analysis of (A) experiments expressed as % ROS production at 2 h for CF MDMs relative to control non-CF MDMs in response to PMA, p = 0.002. (C) Cells were treated with PMA and/or medium-only control in triplicate wells in the presence of 500 U/ml catalase and 80 μM of CytC. CytC reduction, indicative of superoxide production, was measured after 60 min of stimulation by subtracting the absorbance at 550 nm of control wells treated with 300 U/ml SOD from test well values. Unstimulated cell background values were subtracted from test conditions, and all values were set relative to the positive control. Representative experiment of n = 3. (D) Summed end-point analysis of experiments in (C); results are expressed as percentage of non-CF PMA-stimulated cells for three independent experiments, p = 0.01. *p < 0.05, **p < 0.01.

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As a result of this observed difference in ROS generation, we next examined the amounts of essential NADPH oxidase proteins in cell lysates after 30-min exposure to PMA. Membrane component p22phox along with cytosolic components Rac2, total p40phox, and total p47phox were all increased in CF in response to PMA compared with non-CF MDMs (Fig. 2). In addition, all components except total p67phox and gp91phox were increased in untreated CF MDMs compared with non-CF (Fig. 2). We therefore next measured the expression of phosphorylated p40phox and p47phox, which are required to form an activated NADPH complex. In contrast with the total protein expression, there was a marked decrease in expression of phosphorylated p47phox and p40phox in CF in response to PMA compared with non-CF MDMs (Fig. 2C, 2D). This finding was confirmed when examining the translocation of p47phox from the cytosolic to membrane-bound fraction. Expression of membrane-bound p47phox was decreased in CF compared with non-CF (Fig. 3A). In addition, phosphorylated p47phox expression was restored to non-CF levels in a subject receiving treatment with the CFTR modulator combination ivacaftor/lumacaftor (Fig. 3B). Together, these results provide evidence for a deficit in the CF macrophage oxidative burst characterized by accumulation of total NADPH complex proteins, but decreased phosphorylation of key cytosolic components required for NADPH oxidase assembly.

FIGURE 2.

Abnormal NADPH expression in CF MDMs has increased total NADPH component proteins. (A) Western blots of CF and non-CF MDM cell lysates for total amounts of NADPH components with or without 30-min treatment with PMA. Representative image of >5 experiments. (B) Densitometric analysis of ≥3 Western blots per condition in (A) normalized to the loading control calreticulin. (C) CF MDMs have decreased phosphorylation of cytosolic NADPH components. Western blots of CF and non-CF cell lysates for phosphorylated NADPH components after 30-min treatment with or without PMA. Representative image of more than five experiments. (D) Densitometric analysis of three or more Western blots per condition in (C) normalized to the loading control calreticulin.

FIGURE 2.

Abnormal NADPH expression in CF MDMs has increased total NADPH component proteins. (A) Western blots of CF and non-CF MDM cell lysates for total amounts of NADPH components with or without 30-min treatment with PMA. Representative image of >5 experiments. (B) Densitometric analysis of ≥3 Western blots per condition in (A) normalized to the loading control calreticulin. (C) CF MDMs have decreased phosphorylation of cytosolic NADPH components. Western blots of CF and non-CF cell lysates for phosphorylated NADPH components after 30-min treatment with or without PMA. Representative image of more than five experiments. (D) Densitometric analysis of three or more Western blots per condition in (C) normalized to the loading control calreticulin.

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FIGURE 3.

p47phox fails to translocate to plasma membranes in CF. (A) Western blots of CF and non-CF MDM cell lysates for phosphorylated p47phox in cytosolic and plasma membrane fractions, with or without 30-min treatment with PMA. Representative image of four experiments. (B) Western blot of CF and non-CF cell lysates for phosphorylated p47phox after 30-min treatment with or without PMA. CF subject was receiving treatment with the CFTR modulator ivacaftor/lumacaftor, n = 1.

FIGURE 3.

p47phox fails to translocate to plasma membranes in CF. (A) Western blots of CF and non-CF MDM cell lysates for phosphorylated p47phox in cytosolic and plasma membrane fractions, with or without 30-min treatment with PMA. Representative image of four experiments. (B) Western blot of CF and non-CF cell lysates for phosphorylated p47phox after 30-min treatment with or without PMA. CF subject was receiving treatment with the CFTR modulator ivacaftor/lumacaftor, n = 1.

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PKC activation is a key upstream signaling event in activation of the NADPH oxidase. In human non-CF monocytes, the PKC isoform δ (PKC-δ) is required for p47phox phosphorylation and translocation to the cell membrane to enable NADPH activation (38); however, PKC isoform activation has not been examined in CF, or specifically in human CF macrophages. There are three major classes of PKC isoforms (39). The conventional subclass is made up of PKC-α (PRKCA), PKC-β1 (PRKCB), PKC-β2 (PRKCB), and PKC-γ (PRKCG). The novel subclass consists of PKC-δ (PRKCD), PKC-δ1 (PRKD1), PKC-δ2 (PRKD2), PKC-δ3 (PRKD3), PKC-ε (PRKCE), PKC-η (PRKCH), and PKC-θ (PRKCQ). Finally, the atypical subclass consists of PKC-ι (PRKCI), PKC-ζ (PRKCZ), PK-N1 (PKN1), PK-N2 (PKN2), and PK-N3 (PKN3). Therefore, we examined representative isoforms from each class in lysates from CF and non-CF MDMs at baseline and during PMA stimulation. CF MDMs specifically demonstrated decreased expression of phospho-PKC α/β compared with non-CF (Fig. 4A, 4B), representing decreased expression of the conventional PKC subclass. There was no difference in atypical or novel isoforms (Fig. 4A, 4B). To determine the importance of the reduction in the PKC conventional isoform subclass on subsequent ROS production, we incubated CF MDMs with the PKC conventional subclass inducer OAG and measured the oxidative burst in response to PMA. CF MDMs demonstrated a significant increase in ROS production when preincubated with OAG in comparison with untreated CF MDMs (fold change 1.58 ± 0.35, p = 0.047; Fig. 4C, 4D). Addition of OAG did not increase ROS production in non-CF macrophages compared with PMA alone (data not shown).

FIGURE 4.

PKC conventional subclass expression is decreased in CF MDMs. (A) Expression of phosphorylated representatives from the three PKC subclasses (conventional: α/β, novel: δ, and atypical: ζ/λ) was determined by Western blotting of cell lysates from CF and non-CF MDMs after 30-min exposure to PMA. Representative image of three experiments. (B) Densitometric analysis of three Western blots per condition in Fig. 3A, normalized to the loading control calreticulin. (C) PKC conventional agonist OAG increases ROS production in CF. Representative image of DCF assay in CF MDMs stimulated with PMA alone, or PMA preincubated with OAG for 1 h. (D) Summed end-point analysis at 2 h of the experiments in Fig. 3B; results are expressed as a percentage of CF PMA-stimulated cells for four independent experiments, p = 0.047. *p < 0.05.

FIGURE 4.

PKC conventional subclass expression is decreased in CF MDMs. (A) Expression of phosphorylated representatives from the three PKC subclasses (conventional: α/β, novel: δ, and atypical: ζ/λ) was determined by Western blotting of cell lysates from CF and non-CF MDMs after 30-min exposure to PMA. Representative image of three experiments. (B) Densitometric analysis of three Western blots per condition in Fig. 3A, normalized to the loading control calreticulin. (C) PKC conventional agonist OAG increases ROS production in CF. Representative image of DCF assay in CF MDMs stimulated with PMA alone, or PMA preincubated with OAG for 1 h. (D) Summed end-point analysis at 2 h of the experiments in Fig. 3B; results are expressed as a percentage of CF PMA-stimulated cells for four independent experiments, p = 0.047. *p < 0.05.

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Because of the observed differences in PKC isoform expression, we next examined for differences in PKC isoform activators. The conventional class requires DAG, calcium, and phospholipid for activation, in contrast with the novel class, which requires DAG, but not calcium, and the atypical class, which requires neither DAG nor calcium (39). Therefore, we measured calcium influx in MDMs during stimulation with known calcium activators (Ionomycin, PAF) as controls compared with the addition of PMA. There was no difference in calcium production in response to Ionomycin or PAF between CF and non-CF macrophages (Fig. 5). However, there was significantly less calcium production in CF MDMs in response to PMA (p = 0.03; Fig. 5A, 5B). Taken together, these results indicate that CF macrophages have decreased activation of the oxidative burst because of decreased calcium-dependent PKC α/β activation.

FIGURE 5.

Calcium influx is decreased in CF macrophages during PMA exposure. Macrophages were stimulated with either Ionomycin, PAF, or PMA, and the increase in cytosolic Ca2+ in individual macrophages was assessed by flow cytometry measuring the fluorescence emission of Fluo-3 over 200 s. (A) Representative images of responses to experimental agents and no treatment (control) in CF and non-CF MDMs. (B) Summed analysis of calcium influx for PMA experiments in Fig. 4A. Influx calculated as the area under curve before and after PMA stimulus, n = 3, p = 0.030. Not shown are Ionomycin, p = 0.59, and PAF, p = 0.35.

FIGURE 5.

Calcium influx is decreased in CF macrophages during PMA exposure. Macrophages were stimulated with either Ionomycin, PAF, or PMA, and the increase in cytosolic Ca2+ in individual macrophages was assessed by flow cytometry measuring the fluorescence emission of Fluo-3 over 200 s. (A) Representative images of responses to experimental agents and no treatment (control) in CF and non-CF MDMs. (B) Summed analysis of calcium influx for PMA experiments in Fig. 4A. Influx calculated as the area under curve before and after PMA stimulus, n = 3, p = 0.030. Not shown are Ionomycin, p = 0.59, and PAF, p = 0.35.

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Members of the Burkholderia cepacia complex can affect NADPH oxidase assembly (30, 40) in murine models and scavenge ROS (29), and have been shown to cause increased virulence in CF through avoidance of macrophage killing (14, 21). However, it is unknown how these members affect human CF macrophage oxidative responses, or whether there are differential responses between the most predominant strains affecting patients with CF: B. cenocepacia and B. multivorans. Therefore, we next asked whether ROS responses are further suppressed in CF macrophages with the addition of Burkholderia species. Human MDMs were infected with B. cenocepacia clinical isolate strain k56-2 and B. multivorans clinical isolate strain FC-445 at MOIs between 2 and 50. CF MDMs demonstrated a significantly reduced oxidative burst in response to B. cenocepacia and, to a lesser extent, B. multivorans, compared with non-CF MDMs (B. cenocepacia: p = 0.005, B. multivorans: p = 0.008; Fig. 6A, 6B). Although Burkholderia can delay the onset of the oxidative burst in murine models (40), we observed a significant reduction in ROS production in MDMs at early time points, and this persisted over a 6-h infection (6 h; data not shown).

FIGURE 6.

Burkholderia species further reduce ROS production in CF MDMs. (A) CF MDMs have reduced ROS production as measured by the DCF assay in response to 30-min infection with B. cenocepacia (Bc) and B. multivorans (Bm) compared with non-CF; representative image of n = 6. (B) Summed end-point analysis of Fig. 5A experiments; results are expressed as a percentage of non-CF bacteria-stimulated MDMs for six independent experiments. Bc: p = 0.005, Bm: p = 0.008, n = 6. (C) CF MDMs have reduced phosphorylation of p40phox and p47phox during Bc infection. Western blots of cell lysates for phosphorylated and total NADPH components after 30-min infection with Bc. Representative image of more than three experiments. (D) Densitometric analysis of three Western blots per condition in Fig. 5C, normalized to the loading control calreticulin. **p < 0.01.

FIGURE 6.

Burkholderia species further reduce ROS production in CF MDMs. (A) CF MDMs have reduced ROS production as measured by the DCF assay in response to 30-min infection with B. cenocepacia (Bc) and B. multivorans (Bm) compared with non-CF; representative image of n = 6. (B) Summed end-point analysis of Fig. 5A experiments; results are expressed as a percentage of non-CF bacteria-stimulated MDMs for six independent experiments. Bc: p = 0.005, Bm: p = 0.008, n = 6. (C) CF MDMs have reduced phosphorylation of p40phox and p47phox during Bc infection. Western blots of cell lysates for phosphorylated and total NADPH components after 30-min infection with Bc. Representative image of more than three experiments. (D) Densitometric analysis of three Western blots per condition in Fig. 5C, normalized to the loading control calreticulin. **p < 0.01.

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Because of the previously noted differences in p40phox and p47phox phosphorylation in CF macrophages in response to PMA, we examined phosphorylation during B. cenocepacia infection. MDMs were infected with B. cenocepacia for 30 min, cell lysates collected, and Western blotting performed for total and phosphorylated p40phox and p47phox. CF MDMs demonstrated decreased phosphorylation of p40phox and p47phox and increased total p40phox and p47phox during B. cenocepacia infection in comparison with non-CF MDMs (Fig. 6C, 6D). These findings are consistent with untreated and PMA-stimulated MDMs findings as shown in Fig. 2. This finding was further assessed using confocal microscopy. Decreased colocalization of phosphorylated p40phox and p47phox with B. cenocepacia phagosomes was observed in CF MDMs in comparison with non-CF (Fig. 7). In addition, confocal microscopy demonstrated a 25.0% reduction in k56-2 colocalization with p47phox (p < 0.0001) and a 24.3% colocalization reduction of p40phox in CF MDMs compared with non-CF (p = 0.0001). Overall, we interpret the further decrease in the oxidative burst with Burkholderia in CF MDMs to indicate an additive effect with the inherent defect on ROS inhibition in these cells.

FIGURE 7.

CF MDMs have decreased colocalization with phospho-p47phox. (A) Confocal microscopy images of CF and non-CF MDMs with and without infection with B. cenocepacia for 30 min. The macrophage nucleus is stained blue with DAPI, B. cenocepacia (k56-2) is shown in red, phosphorylated p47phox is shown in green, and bacteria colocalized with p47phox are yellow in the merged image. n = 3. (B) CF MDMs have decreased colocalization with phospho-p40phox. Confocal microscopy images of CF and non-CF MDMs with and without infection with B. cenocepacia. The macrophage nucleus is stained blue with DAPI, B. cenocepacia (k56-2) is shown in red, phosphorylated p40phox is shown in green, and bacteria colocalized with p40phox are yellow in the merged image. n = 3. (C) Summed scoring of bacterial colocalization from Fig. 6A and 6B. One hundred MDMs scored per condition. p47phox: p < 0.0001, p40phox: p = 0.001. ***p < 0.001.

FIGURE 7.

CF MDMs have decreased colocalization with phospho-p47phox. (A) Confocal microscopy images of CF and non-CF MDMs with and without infection with B. cenocepacia for 30 min. The macrophage nucleus is stained blue with DAPI, B. cenocepacia (k56-2) is shown in red, phosphorylated p47phox is shown in green, and bacteria colocalized with p47phox are yellow in the merged image. n = 3. (B) CF MDMs have decreased colocalization with phospho-p40phox. Confocal microscopy images of CF and non-CF MDMs with and without infection with B. cenocepacia. The macrophage nucleus is stained blue with DAPI, B. cenocepacia (k56-2) is shown in red, phosphorylated p40phox is shown in green, and bacteria colocalized with p40phox are yellow in the merged image. n = 3. (C) Summed scoring of bacterial colocalization from Fig. 6A and 6B. One hundred MDMs scored per condition. p47phox: p < 0.0001, p40phox: p = 0.001. ***p < 0.001.

Close modal

To determine whether the reduced ROS production in response to B. cenocepacia was dependent on a live bacterial factor versus a viability-independent factor, we incubated CF MDMs with PFA-killed B. cenocepacia and production of ROS compared with that of live bacteria. There was an increased oxidative burst in response to PFA-killed B. cenocepacia in both CF and non-CF MDMs in comparison with live B. cenocepacia for both host cell types; however, CF MDMs had a significantly lower response compared with non-CF MDMs (Fig. 8A, 8B). Next, the ability of B. cenocepacia to suppress the oxidative burst in response to a secondary stimulus was tested, as might occur in a patient with CF facing multiple pathogens postinfection with B. cenocepacia. Both CF and non-CF MDMs had a reduced response to PMA when infected with B. cenocepacia 1 h before PMA stimulus (Fig. 8C, 8D). Taken together, these data suggest that a factor produced from live bacteria is at least partially responsible for B. cenocepacia’s suppression of ROS responses, which are additive in CF MDMs with basal deficits in ROS generation, but can also suppress secondary responses in non-CF MDMs.

FIGURE 8.

Impact of bacterial viability, antecedent bacterial infection, and bacterial species on ROS production. (A) Increased ROS production as measured by the DCF assay is observed in response to PFA-killed Bc in comparison with live Bc in CF (p = 0.028) and non-CF MDMs (p = 0.006). n = 3. (B) Summed end-point analysis of experiments in (A); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with B. cenocepacia. (C) Burkholderia species decrease ROS production as measured by the DCF assay in response to PMA in both CF (p = 0.05) and non-CF MDMs (p = 0.0003) when infected for 1 h before PMA exposure. n = 4. (D) Summed end-point analysis of experiments in (C); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with PMA. (E) CF and non-CF MDMs have equal ROS production in response to 30-min infection with Pseudomonas aeruginosa (Pa), as measured by the DCF assay. p = 0.70, n = 3. (F) Summed end-point analysis of experiments in (E); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with P. aeruginosa. *p < 0.05, **p < 0.01.***p < 0.001.

FIGURE 8.

Impact of bacterial viability, antecedent bacterial infection, and bacterial species on ROS production. (A) Increased ROS production as measured by the DCF assay is observed in response to PFA-killed Bc in comparison with live Bc in CF (p = 0.028) and non-CF MDMs (p = 0.006). n = 3. (B) Summed end-point analysis of experiments in (A); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with B. cenocepacia. (C) Burkholderia species decrease ROS production as measured by the DCF assay in response to PMA in both CF (p = 0.05) and non-CF MDMs (p = 0.0003) when infected for 1 h before PMA exposure. n = 4. (D) Summed end-point analysis of experiments in (C); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with PMA. (E) CF and non-CF MDMs have equal ROS production in response to 30-min infection with Pseudomonas aeruginosa (Pa), as measured by the DCF assay. p = 0.70, n = 3. (F) Summed end-point analysis of experiments in (E); results are expressed as % ROS production at 2 h of CF MDMs relative to non-CF MDMs with P. aeruginosa. *p < 0.05, **p < 0.01.***p < 0.001.

Close modal

Finally, we examined the oxidative burst in response to another common CF pathogen, P. aeruginosa, which has been previously shown to not affect the respiratory burst in CF macrophages (36). There was no difference in ROS production in response to P. aeruginosa between CF and non-CF MDMs (Fig. 8E, 8F), confirming previous findings. When combined with the B. cenocepacia and PMA data, this would suggest that CF MDMs have deficits in intracellular PKC-mediated activation of the oxidative burst in response to certain pathogenic stimuli, which may explain differential handling of pathogens in CF.

In recent years we have gained improved understanding of underlying deficits in the host immune responses of patients with CF beyond a previous emphasis on continued neutrophil overproduction in the lung (14, 4145). Despite this increased knowledge and recent advances in CF care including CFTR modulators (46, 47), patients with CF remain burdened by chronic, multidrug-resistant bacterial infections. With a continued dearth in the development of novel antimicrobials (48) combined with increasing antibiotic resistance worldwide (49, 50), it remains more critical than ever to determine how bacteria avoid host immune defenses in CF to generate new approaches to therapy. To this end, we have discovered a novel defective pathway in CF macrophages independent of pathogens involving calcium-dependent PKC activation of the oxidative burst. This deficit in macrophage oxidative killing is further exaggerated by specific bacteria such as B. cenocepacia, which may in part explain the increased prevalence of Burkholderia infections in CF.

The generation of ROS in CF has been well studied in CF neutrophils, but remains less characterized in macrophages, where intracellular bacteria may reside and avoid host defenses. Although CF neutrophils have been shown to have adequate ROS production (51), this has not been fully explored in the setting of chronic airway infections where non-CF biofilm-entrapped neutrophils demonstrate an ineffective oxidative burst (52), and CF sputum neutrophils demonstrate reduced ROS capacity compared with blood neutrophils, highlighting potential differences in activated versus basal states and differences in tissue compartments (53). Human CF MDMs have a normal ROS response to P. aeruginosa (18), which we have confirmed, but characterization of human macrophage NADPH assembly has not been performed in CF. Previously murine CF alveolar macrophages were shown to sequester gp91phox in ceramide-containing platforms, preventing the release of ROS (37). We found that accumulation of multiple NADPH components occurred in CF macrophages, but not gp91phox. This finding implicates the involvement of multiple NADPH components in human CF macrophage dysfunction, and not just specifically gp91phox, further highlighting differences from murine studies.

Importantly, despite the increased presence of NADPH components, there was decreased phosphorylation of p40phox and p47phox, resulting in defective assembly of an activated NADPH complex. Even though p40phoxphosphorylation is not required for oxidase activation by PMA in neutrophils from patients with chronic granulomatous disease where known abnormalities in NADPH assembly occur (54, 55), assembly defects in CF macrophages involving this component may be different. The phosphorylation deficit was predicated upon a decrease in calcium-dependent PKC α/β expression. This result is in contrast with non-CF monocytes where PKC-δ is required for p47phox phosphorylation (38), but correlates with a recent study showing restoration of CF macrophage microbicidal functions with OAG-mediated calcium signaling (56). In addition, conventional PKC expression is implicated in macrophage control of intracellular pathogens such as Mycobacterium tuberculosis (57), and we now demonstrate PKC to be deficient in the handling of B. cenocepacia infection in CF MDMs, lending support to the notion of dysregulated PKC control of intracellular pathogens in CF. Our findings are consistent with an early study that showed chemiluminescent defects in PKC-mediated actions in CF neutrophils (58). p47phox contains eight phosphorylation sites, and determining which of these may be involved will be the subject of further research. Importantly, deficits in p47phox phosphorylation were reversed in a patient with CF receiving treatment with the CFTR modulator combination ivacaftor/lumacaftor, demonstrating the importance of functional CFTR in NADPH assembly.

B. cenocepacia further exaggerated the reduction in ROS production seen in CF macrophages. B. cenocepacia can downregulate ROS production when grown in biofilm culture only (59), and delay association of the NADPH oxidase complex within murine macrophage vacuoles (40), but B. cenocepacia–induced ROS production in human CF macrophages has not been studied previously. In addition, we observed an early and persistent decrease in ROS production in CF macrophages rather than a time-delayed increase in ROS in macrophages, as was shown in one murine study. Furthermore, we found that Rac2 protein expression is elevated in CF macrophages independent of Burkholderia infection, emphasizing differences compared with a murine study involving Rac1 (30). However, there are multiple signaling pathways for Rac activation that will require further examination in the context of B. cenocepacia infection in humans with CF.

There have been no macrophage studies (CF or otherwise) regarding B. multivorans and the oxidative burst. B. multivorans demonstrated a decreased impact on ROS generation in CF macrophages compared with B. cenocepacia, which may explain the differences in virulence observed clinically between the two species. B. cenocepacia was able to decrease subsequent ROS production to a secondary stimulus in both CF and non-CF macrophages, highlighting a potential connection between CF and chronic granulomatous disease (60), both with defective ROS and an unusual tendency for B. cenocepacia infection compared with immunocompetent hosts.

Both CF and non-CF macrophages demonstrated increased ROS to PFA-killed B. cenocepacia, suggesting that exposed cell-wall components of B. cenocepacia are at most only partly responsible for the active subversion of macrophage ROS responses. The exact identity of a viability-associated factor in B. cenocepacia that is responsible for decreased ROS production is not known at this time, but will be an area of continued research. Exopolysaccharides from B. cenocepacia have been shown to scavenge neutrophil ROS (29), as well as the ability of B. cenocepacia to downregulate the tricarboxylic acid cycle when grown in a biofilm (59), both of which may provide future areas to target.

In summary, human CF macrophages have an inherent reduction in ROS production that is worsened by B. cenocepacia. This deficit is caused by a calcium-dependent decrease in expression of the PKC conventional subclass α/β leading to decreased activation of the NADPH oxidase in comparison with non-CF macrophages. These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in patients with CF.

We kindly thank Dr. Jamel El-Benna for providing the anti–phospho-p47 Ab. We thank Dr. Jane Burns at the CF Isolate Core at Seattle Children’s Research Institute for provision of the B. multivorans strain (National Institutes of Health Grant P30 DK089507) and Dr. Miguel Valvano for B. cenocepacia strains.

This work was supported by Clinical and Translational Science Award Grant UL1TR001070, National Institutes of Health Grants 1K08AI108792-01A1 and R21AI120013, and Cystic Fibrosis Foundation Grant KOPP14I0.

Abbreviations used in this article:

CF

cystic fibrosis

CFTR

CF transmembrane regulator

CytC

cytochrome c

DAG

diacylglycerol

DCF

2′,7′-dichlorofluorescein

DPBS

Dulbecco’s PBS

MDM

monocyte-derived macrophage

MOI

multiplicity of infection

OAG

1-oleoyl-2-acetyl-sn-glycerol

PAF

platelet-activating factor

PFA

paraformaldehyde

PKC

protein kinase C

PKC-δ

PKC isoform δ

RFU

relative fluorescent unit

ROS

reactive oxygen species

SOD

superoxide dismutase.

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The authors have no financial conflicts of interest.