Impaired Mitochondrial Microbicidal Responses in Chronic Obstructive Pulmonary Disease Macrophages

Abstract

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria.

Objectives: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated.

Methods: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage-specific expression of human induced myeloid leukemia cell differentiation protein Mcl-1 (CD68.hMcl-1) was used to model the molecular aspects of COPD.

Measurements and main results: COPD AMs had elevated levels of Mcl-1, an antiapoptotic B-cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages and human monocyte-derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected bone marrow-derived macrophages but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2.

Conclusions: Up-regulation of Mcl-1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.

Keywords: Streptococcus pneumoniae; apoptosis; mitochondrial reactive oxygen species.

Figure 1.

Induced myeloid leukemia cell differentiation protein Mcl-1 up-regulation occurs in chronic obstructive pulmonary disease (COPD). (A) Alveolar macrophages obtained from bronchoalveolar lavage of healthy control subjects or patients with COPD were mock infected (MI) or challenged with opsonized serotype 14 Streptococcus pneumoniae (S14) at the designated multiplicity of infection (MOI). Sixteen hours after challenge, the levels of Mcl-1 on alveolar macrophages were probed by western blotting. A representative blot and densitometric analysis are shown. n = 6; *P < 0.05, repeated measures one-way analysis of variance. (B and C) Lung sections from patients with COPD or healthy control subjects were double-stained with CD68 and Mcl-1. Total corrected cellular fluorescence of Mcl-1 in CD68⁺ cells was quantified. Representative images (B) and collated data (C and D) are shown (scale bars = 50 μM). In C, each point represents an individual cell (n = 74 healthy control, n = 90 COPD, from 10 donors), and in D, each point represents the median fluorescence of all cells analyzed from individual donors. For C and D, *P < 0.05, Kruskal-Wallis test. ns = nonsignificant.

Figure 2.

Chronic obstructive pulmonary disease (COPD) alveolar macrophages (AMs) have a deficiency in apoptosis-associated killing. (A) AMs were collected from healthy donors or patients with COPD and were challenged with nonopsonized (−) or opsonized (+) serotype 14 Streptococcus pneumoniae at a multiplicity of infection (MOI) of 10 for 4 hours before extracellular bacteria were killed and viable intracellular bacteria were measured. Viable bacteria in duplicate wells were measured again 3 hours later (7 h after infection). *P < 0.05, **P < 0.01 by two-way analysis of variance. (B and C) Healthy or COPD AMs were challenged with serotype 14 Streptococcus pneumoniae at an MOI of 10 for COPD cells or an MOI of 5 for healthy cells to normalize levels of bacterial internalization. Cells were analyzed for (B) nuclear fragmentation or condensation and (C) intracellular bacterial CFU at 20 hours after challenge. n = 5–6, *P < 0.05 by Student’s t test (for B) or Mann-Whitney U test (for C).

Figure 3.

Induced myeloid leukemia cell differentiation protein Mcl-1 up-regulation in alveolar macrophages (AMs) impairs bacterial clearance in the lung. (A–D) Wild-type (Wt) or CD68.hMcl-1–transgenic (Tg) mice were challenged with 10⁴ serotype 1 Streptococcus pneumoniae. At the designated time after instillation, (A) bacterial CFU in the lung homogenate, (B) bacterial CFU in the blood, (C) AM nuclear fragmentation or condensation in bronchoalveolar lavage (BAL), and (D) total polymorphonuclear leukocyte (PMN) numbers in BAL were measured. n = 4–11 mice per group from three independent experiments; *P < 0.05, ***P < 0.001, two-way analysis of variance.

Figure 4.

Induced myeloid leukemia cell differentiation protein Mcl-1 modulates generation of mitochondrial reactive oxygen species (mROS) and mROS-dependent bacterial killing. (A) Wild-type (Wt) or CD68.hMcl-1–transgenic (Tg) bone marrow–derived macrophages (BMDMs) were mock infected (MI) or challenged with opsonized serotype 2 (D39) Streptococcus pneumoniae (Spn). Twenty hours after challenge, cells were stained with MitoSOX Red and visualized by microscopy to assess mROS generation. Images are representative of three independent experiments; scale bar = 50 μm. (B) At the designated time after challenge, mROS were also assessed by flow cytometry. n = 3; *P < 0.05 for D39 Wt versus D39 Tg and ***P < 0.001 for MI Wt versus Spn Wt by two-way analysis of variance. (C) MI or D39-infected Wt BMDMs were stained with Cresyl violet to detect lysosomes (green) and MitoSOX Red (red) at 20 hours and analyzed by confocal microscopy. Colocalized signals are shown in yellow (Merge); scale bar = 5 μm. (D) Confocal fluorescence microscopy of D39 BMDMs challenged with Alexa Fluor 647–labeled bacteria (green) and stained with MitoSOX Red (red; upper panels) or endoplasmic reticulum (ER) tracker (purple; lower panels) 20 hours after bacterial challenge. Colocalized signals are shown in yellow (Merge; upper and lower panels); scale bar = 5 μm. (E) Pseudocolored structured illumination microscopic image of a monocyte-derived macrophage 16 hours after bacterial challenge with S. pneumoniae (green) and stained with MitoSOX Red (red) for mROS. Enlarged region on right shows bacteria colocalized with mROS (arrows). Scale bar = 1 μm. (F) Pearson’s correlation coefficients were calculated for the colocalization of mROS or ER with D39 or lysosomes. Data are shown as mean ± SEM (n = 4–8). (G and H) Wt or Tg BMDMs (G) or human monocyte-derived macrophages (H) were challenged with D39 in the presence or absence (vehicle) of mitoTEMPO (mT). Sixteen hours after challenge, intracellular CFU were assessed. n = 5 (for G) and n = 8 (for H); *P < 0.05 and **P < 0.01 by repeated measures two-way analysis of variance (for G) or Wilcoxon matched-pairs signed-rank test (for H). MFI = mean fluorescence intensity.

Figure 5.

Induced myeloid leukemia cell differentiation protein Mcl-1 modulates mitochondrial responses leading to mitochondrial reactive oxygen species (mROS) generation. (A–F) Wild-type (Wt) or CD68.hMcl-1–transgenic (Tg) bone marrow–derived macrophages (BMDMs) were mock infected (MI) or challenged with opsonized serotype 2 (D39) Streptococcus pneumoniae (Spn) for 4 hours before extracellular acidification (ECAR) (A), and parameters related to oxidative phosphorylation were measured kinetically using oligomycin (Oligo), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone (Rot), or antimycin A (AntiA) at the indicated concentrations. Using the kinetic data (B), basal oxygen consumption rate (OCR) (C), maximum respiration capacity (D), ATP-linked OCR (E), and proton leak (F) were calculated. n = 6 per group; *P < 0.05, **P < 0.01 by two-way analysis of variance (ANOVA). (G) MI Wt and Tg BMDMs were stained with MitoSOX Red to measure baseline mROS production. (H and I) Wt or Tg BMDMs (H) or human monocyte-derived macrophages (I) were MI or challenged with D39 in the presence of the pan-caspase inhibitor zVAD or control zFA. At 20 hours after challenge, cells were stained for mROS and caspase 3/7 activity. MitoSOX Red staining was assessed for the whole-cell populations (histograms) showing forward scatter (FSC-H) versus caspase 3/7. Representative plots are shown, with collated data graphed. n = 4; *P < 0.01 by two-way ANOVA (for H) or one-way ANOVA (for I). MFI = mean fluorescence intensity; zFA = benzyl N-[1-[(4-fluoro-3-oxobutan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl]carbamate; zVAD = carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone.

Figure 6.

Chronic obstructive pulmonary disease (COPD) alveolar macrophages (AMs) fail to increase mitochondrial reactive oxygen species (mROS) production after pneumococcal challenge. (A and B) AMs obtained from bronchoalveolar lavage (BAL) of healthy control subjects or patients with COPD were mock infected (MI) or challenged with opsonized serotype 14 Streptococcus pneumoniae (S14), at a multiplicity of infection (MOI) of 10 for COPD cells or an MOI of 5 for healthy cells. AMs were left unstained (US) or stained with MitoSOX Red, and mean fluorescence intensity (MFI) was recorded at 16 hours as a measure of mROS, with representative plots shown and collated data graphed (A), and intracellular bacterial CFU were estimated in the presence or absence (vehicle) of mitoTEMPO (mT) (B) at 20 hours. Both n = 6; *P < 0.05, paired Student’s t test (A) or Wilcoxon signed-rank test (B). (C) AMs from BAL of healthy control subjects or patients with COPD were MI or challenged with S14 at the designated MOI. At 16 hours after challenge, the levels of superoxide dismutase 2 (SOD2) in AMs were probed by western blotting. Representative blot and densitometric analysis are shown. n = 4. (D) The ratio of mROS to SOD2 induced by bacterial challenge was calculated for healthy and COPD AMs using the samples in C. n = 4; *P < 0.05, Student’s t test. (E and F) Healthy or COPD AMs were MI or challenged with S14 in the presence (+) or absence (−) of rotenone to induce mROS. AMs were assessed for (E) intracellular bacterial CFU and (F) nuclear fragmentation or condensation 20 hours after challenge. n = 3; *P < 0.05, Wilcoxon signed-rank test.