Nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases

Abstract

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1β (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1β was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

Keywords: azithromycin; chronic lung disease; chronic obstructive pulmonary disease; nonantibiotic macrolides; nontypeable H. influenzae.

Fig. 1.

Dose-response experiments showing effects of various concentrations of GS-459755 (GS4) and GS-560660 (GS5) on phagocytic capacity of THP-1 macrophages. Phagocytosis of nontypeable Haemophilus influenzae (A) and efferocytosis of apoptotic bronchial epithelial cells (B). GS-459755 and GS-560660 were used at concentrations of 0.5–20 µg/ml. Azithromycin (Az; 0.5 µg/ml) was used as a positive control; n = 3 experiments from separate donors performed in triplicate. C, control. *P < 0.05 vs. control unless otherwise indicated. Box plots present the median and 25th and 75th percentiles (solid box), with the 10th and 90th percentiles shown by whiskers outside of the box. Data analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 2.

Effect of macrolides ± cigarette smoke extract (CSE) on the phagocytic capacity of primary human alveolar macrophages. Phagocytosis of nontypeable Haemophilus influenzae (A) and efferocytosis of apoptotic bronchial epithelial cells (B). GS-459755 (G4) and GS-560660 (G5) were used at concentrations of 0.5 and 1 µg/ml; n = 5 experiments performed in triplicate. *P < 0.05 vs. control; †P < 0.05 vs. CSE, unless otherwise indicated. Data presented as box plots, as described in Fig. 1, and analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 3.

Effect of macrolides ± cigarette smoke extract (CSE) on phagocytic capacity of THP-1 macrophages. Phagocytosis of nontypeable Haemophilus influenzae (A) and efferocytosis of apoptotic bronchial epithelial cells (B). GS-459755 (G4) and GS-560660 (G5) were used at concentrations of 0.5 and 1 µg/ml; n = 5 experiments performed in triplicate. *P < 0.05 vs. control; †P < 0.05 vs. CSE unless otherwise indicated. Data presented as box plots, as described in Fig. 1, and analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 4.

Effect of macrolides on THP-1 expression of receptors involved in recognition of apoptotic cells and/or nontypeable Haemophilus influenzae. Azithromycin (Az), GS-459755 (GS4), and GS-560660 (GS5) were used at concentrations of 0.5 µg/ml; n = 5 experiments performed in triplicate. No significant changes were observed. Data presented as box plots, as described in Fig. 1, and analyzed using Friedman and Wilcoxon signed-rank tests. MFI, mean fluorescence intensity.

Fig. 5.

Effect of macrolides on THP-1 macrophage viability. Cells were treated with GS-459755 (GS4) and GS-560660 (GS5) and then stained with the apoptotic marker Annexin V (A) or the cell death marker Sytox Green (B) and analyzed using flow cytometry; n = 5 experiments performed in triplicate. Data presented as box plots, as described in Fig. 1, and analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 6.

Effect of macrolides on IL-1β secretion by THP-1 macrophages. Azithromycin (Az), GS-459755 (G4), and GS-560660 (G5) were used at a concentration of 0.5 µg/ml and IL-1β secretion measured using a cytometric bead array assay; n = 5 experiments performed in triplicate. *P < 0.05 vs. control. Data presented as box plots, as described in Fig. 1, and analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 7.

Effect of macrolides ± cigarette smoke extract (CSE) on cleaved IL-1β in THP-1 macrophages. Azithromycin (Az), GS-459755 (G4), and GS-560660 (G5) were used at a concentration of 0.5 µg/ml and IL-1β measured using immunofluorescence/confocal microscopy. Serial images (n = 10 per well) were captured from 5 experiments. *P < 0.05 vs. control; †P < 0.05 vs. CSE unless otherwise indicated. Data analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 8.

Effect of macrolides ± cigarette smoke extract (CSE) on NLRP3 in THP-1 macrophages. Azithromycin (Az), GS-459755 (G4), and GS-560660 (G5) were used at a concentration of 0.5 µg/ml and NLRP3 measured using immunofluorescence/confocal microscopy. Serial images (n = 10 per well) were captured from 5 experiments. *P < 0.05 vs. control (no CSE); †P < 0.05 vs. CSE unless otherwise indicated. Data analyzed using Friedman and Wilcoxon signed-rank tests.

Fig. 9.

Colocalization of NLRP3 and cleaved IL-1β (IL-1b) in cigarette smoke (CS) extract-exposed THP-1 macrophages. Immunofluorescence staining showing colocalization (yellow) of NLRP3 (red) and cleaved IL-1β (green) in cigarette smoke extract-exposed THP-1 macrophages. DAPI, 4′,6-diamidino-2-phenylindole.