Randomised, double-blind, placebo-controlled trial with azithromycin selects for anti-inflammatory microbial metabolites in the emphysematous lung

Abstract

Introduction: Azithromycin (AZM) reduces pulmonary inflammation and exacerbations in patients with COPD having emphysema. The antimicrobial effects of AZM on the lower airway microbiome are not known and may contribute to its beneficial effects. Here we tested whether AZM treatment affects the lung microbiome and bacterial metabolites that might contribute to changes in levels of inflammatory cytokines in the airways.

Methods: 20 smokers (current or ex-smokers) with emphysema were randomised to receive AZM 250 mg or placebo daily for 8 weeks. Bronchoalveolar lavage (BAL) was performed at baseline and after treatment. Measurements performed in acellular BAL fluid included 16S rRNA gene sequences and quantity; 39 cytokines, chemokines and growth factors and 119 identified metabolites. The response to lipopolysaccharide (LPS) by alveolar macrophages after ex-vivo treatment with AZM or bacterial metabolites was assessed.

Results: Compared with placebo, AZM did not alter bacterial burden but reduced α-diversity, decreasing 11 low abundance taxa, none of which are classical pulmonary pathogens. Compared with placebo, AZM treatment led to reduced in-vivo levels of chemokine (C-X-C) ligand 1 (CXCL1), tumour necrosis factor (TNF)-α, interleukin (IL)-13 and IL-12p40 in BAL, but increased bacterial metabolites including glycolic acid, indol-3-acetate and linoleic acid. Glycolic acid and indol-3-acetate, but not AZM, blunted ex-vivo LPS-induced alveolar macrophage generation of CXCL1, TNF-α, IL-13 and IL-12p40.

Conclusion: AZM treatment altered both lung microbiota and metabolome, affecting anti-inflammatory bacterial metabolites that may contribute to its therapeutic effects.

Trial registration number: NCT02557958.

Keywords: Bronchoscopy; COPD ÀÜ Mechanisms.

Figure 1

CONsolidated Standards of Reporting Trials diagram showing the selection of subjects from Early Detection Research Network (EDRN) cohort. Subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) at baseline. After bronchoscopy, subjects were randomised to either placebo or azithromycin for 8 weeks. Post-treatment bronchoscopy was performed at the end of the treatment period. BAL was used to evaluate microbiome (16S), metabolome and inflammation. NYU, New York University.

Figure 2

Graphs showing baseline microbiome. (A) Total bacterial 16S rRNA gene was measured by real-time quantitative PCR of 16S rRNA using universal primers. (B) Rarefaction curves of phylogenetic α-diversity showed no significant differences between the placebo and azithromycin (AZM) group at baseline. (C) β-Diversity of baseline bronchoalveolar lavage (BAL) samples was distributed in two distinct clusters. Similar to online supplementary figure S1, Nonmetric Multidimensional Scaling (NMDS) analysis was performed where grey square samples represent samples from subjects assigned to the placebo group and grey triangle samples represent samples from subjects assigned to the AZM group. Similar to online supplementary figure S1, samples clustered in two distinct groups driven by taxa characteristic for background (pneumotype_BPT), represented in green, or by taxa characteristic for supraglottic (pneumotype_SPT), represented in red. Equal number of samples from placebo and AZM groups was present in both clusters (six in pneumotype_BPT and four in pneumotype_SPT from each group). BALF, BAL fluid.

Figure 3

Effects of azithromycin (AZM) on the lower airway microbiome are shown. (A) Bacterial load assessed by quantitative PCR of 16S rRNA gene from samples obtained post-treatment is shown. (B) Comparison of α-diversity (based on Shannon's diversity index where sequences were rarified at 1000 reads per sample) before and after treatment is shown. (C) Change in β-diversity before and after placebo or AZM had been evaluated using Procrustes analysis. Random permutations had been done using Monte Carlo simulation. BALF, bronchoalveolar lavage fluid.

Figure 4

Evaluation of change in taxonomic composition after placebo or azithromycin (AZM) treatment is shown. (A) Linear discriminant analysis (LDA) effect size (LEfSe) is calculated comparing 16S data at baseline and after 8 weeks of placebo/AZM. No taxonomic differences are noted in placebo group. However, there had been several consistent taxonomic changes in the AZM group as evident by differences in colour of cladogram (red increased post-AZM treatment and green decreased post-AZM). (B) LDA effect size of taxa is found to be differentially enriched (LDA>2) pre-AZM and post-AZM treatment and its correspondent relative abundances pre-AZM and post-AZM are plotted as a bar graph.

Figure 5

Azithromycin (AZM) treatment increased bacterially produced metabolites and decreased inflammatory mediators in the lower airways. (A) Metabolites were measured by gas chromatography-mass spectrometry. Among metabolites shown to change post treatment with AZM (and not placebo), Of the 14 metabolites, 4 were identified as potential microbial metabolites. Comparisons between metabolites levels pre-treatment versus post-treatment had been based on Wilcoxon rank-sum test. (B) Concentrated bronchoalveolar lavage fluid (BALF) was used to measure cytokines with Luminex. Paired comparisons between pre-treatment versus post-treatment had been based on the Wilcoxon rank-sum test. CXCL1, chemokine (C-X-C) ligand 1; IL, interleukin; TNF, tumour necrosis factor.

Figure 6

Graphs showing evaluation of the effects of azithromycin (AZM), glycolic acid and indole-3-acetate on ex-vivo cytokine production during TLR4 stimulation. (A) Alveolar macrophages obtained during baseline bronchoscopy from 12 enrolled subjects were cultured in media alone (MA), were exposed to lipopolysaccharide (LPS) 40 ng or LPS+AZM (10 μg/mL). Compared with MA, LPS stimulation yielded significant increase in tumour necrosis factor (TNF)-α, interleukin (IL)-12p40, IL-13 and chemokine (C-X-C) ligand 1 (CXCL1) (p value <0.01 for all comparisons). AZM did not significantly decreased the concentration of these cytokines during LPS stimulation. (B and C) Alveolar macrophages from eight subjects from a similar cohort were cultured in MA, LPS 40 ng, LPS+glycolic acid (2 nM) or LPS+indole-3-acetate (2 nM). Compared with MA, LPS stimulation yielded significant increase in TNF-α, IL-12p40, IL-13 and CXCL1 (p value <0.01 for all comparisons). Both glycolic acid and indole-3-acetate significantly decreased the concentration of these cytokines during LPS stimulation. Each pair is an individual's bronchoalveolar lavage alveolar macrophage preparation. Paired comparisons are based on Wilcoxon rank-sum test. TLR4, Toll-like receptor 4.