Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia

Abstract

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 p47 ^phox and p40 ^phox 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.

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 1A experiments expressed as %ROS production at 2 h for CF MDMs relative to control non-CF MDMs in response to PMA, P value = 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 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 1C experiments, results are expressed as percentage of non-CF PMA-stimulated cells for three independent experiments, P value = 0.01.

Figure 2. Abnormal NADPH expression in CF

CF MDMs have increased total NADPH component proteins. A) Western blots of CF and non-CF MDM cell lysates for total amounts of NADPH components with/without 30 min treatment with PMA. Representative image of > 5 experiments. B) Densitometric analysis of ≥ 3 Western blots per condition in 2A, 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/without PMA. Representative image of > 5 experiments. D) Densitometric analysis of ≥ 3 Western blots per condition in 2C, normalized to the loading control calreticulin.

Figure 3. p47^phox fails to translocate to plasma membranes in CF

A) Western blots of CF and non-CF MDM cell lysates for phosphorylated p47^phox in cytosolic and plasma membrane fractions, with/without 30 min treatment with PMA. Representative image of 4 experiments. B) Western blot of CF and non-CF cell lysates for phosphorylated p47^phox after 30 min treatment with/without PMA. CF subject was receiving treatment with the CFTR modulator ivacaftor/lumacaftor, n=1.

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 3 experiments. B) Densitometric analysis of 3 Western blots per condition in 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 pre-incubated with OAG for 1 h. D) Summed end-point analysis at 2 h of the experiments in 3B; results are expressed as a percentage of CF PMA-stimulated cells for four independent experiments, P= 0.047.

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 4A. Influx calculated as the area under curve before and after PMA stimulus, n=3, P value = 0.030. Not shown are Ionomycin P value = 0.59 and PAF P value =0.35.

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 Burkholderia cenocepacia (Bc) and Burkholderia multivorans (Bm) compared to non-CF, representative image of n=6. B) Summed end-point analysis of 5A experiments, results are expressed as a percentage of non-CF bacteria-stimulated MDMs for six independent experiments. Bc P value =0.005, Bm P value =0.008, n=6. C) CF MDMs have reduced phosphorylation of p40^phox and p47^phox during Bc infection. Western blots of cell lysates for phosphorylated and total NADPH components after 30 min infection with Bc. Representative image of > 3 experiments. D) Densitometric analysis of 3 Western blots per condition in 5C, normalized to the loading control calreticulin.

Figure 7. CF MDMs have decreased co-localization with phospho-p47^phox

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 p47^phox is shown in green, and bacteria co-localized with p47^phox are yellow in the merged image. N=3. B) CF MDMs have decreased co-localization with phospho-p40^phox. 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 p40^phox is shown in green, and bacteria co-localized with p40^phox are yellow in the merged image. N=3. C) Summed scoring of bacterial co-localization from 6A and 6B. 100 MDMs scored per condition. p47^phox P value < 0.0001, p40^phox P value = 0.001.

Figure 8. Impact of bacterial viability, antecedent bacterial infection, and bacterial spp. on ROS production

A) Increased ROS production as measured by the DCF assay is observed in response to paraformaldehyde killed Bc in comparison to live Bc in CF (P value = 0.028) and non-CF MDM s (P value = 0.006) n=3. B) Summed end-point analysis of 8A experiments, results are expressed as % ROS production at 2h 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 value = 0.05) and non-CF MDMs (P value = 0.0003) when infected for 1 h prior to PMA exposure. n=4. D) Summed end-point analysis of 8C experiments, results are expressed as % ROS production at 2h 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 value = 0.70, n=3. F) Summed end-point analysis of 8E experiments, results are expressed as % ROS production at 2h of CF MDMs relative to non-CF MDMs with P. aeruginosa.