NTHi killing activity is reduced in COPD patients and is associated with a differential microbiome

Abstract

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by airway obstruction and inflammation. Non-typeable Haemophilus influenzae (NTHi) lung infections are common in COPD, promoting frequent exacerbations and accelerated lung function decline. The relationship with immune responses and NTHi are poorly understood. Herein, we comprehensively characterized the respiratory microbiome and mycobiome of patients while investigating microbial dynamics and host immune changes attributable to NTHi killing activity. Mild-to-moderate COPD patients encompassing frequent and infrequent exacerbators and healthy volunteers (HV) were enrolled. Microbial composition, proteomics and NTHi killing activity was analyzed using bronchoalveolar lavage fluid (BALF). In addition, antigen-antibody titers in sera to COPD pathogens were determined using a multiplex assay. Differential abundance analysis revealed an enrichment of Actinobacteria and Bacteroidetes in the BALF of COPD and HV subjects respectively. Significant differences in the IgA titer response were observed against NTHi antigens in COPD vs. HV. Notably, there was also significantly greater killing activity against NTHi in BALF from COPD vs. HV subjects (OR = 5.64; 95% CI = 1.75-20.20; p = 0.001). Stratification of COPD patients by NTHi killing activity identified unique microbial and protein signatures wherein Firmicutes, Actinobacteria and haptoglobin were enriched in patients with killing activity. We report that differences in host immune responses and NTHi-killing activity are associated with microbiome changes in mild-to-moderate COPD. This is suggestive of a potential link between the respiratory microbiome and immune activity against NTHi in the context of COPD pathogenesis even at this disease stage.

Keywords: COPD; Exacerbations; Microbiome; Non-typeable Haemophilus influenzae.

Fig. 1

Characterizing the microbial landscape. A Comparison of alpha-diversity (measured using the Inverse Simpson index) in sputum (n = 30) vs. BALF (n = 65 patients, 130 samples) samples. p-value by Wilcoxon ranksum test. B Ordination of beta-diversity (Bray–curtis) distances by principal coordinate analysis (p = 0.001). p-value by ANOSIM 999 permutations. Taxonomic landscape at the phylum level in C BALF (n = 65 patients, 130 samples) and D sputum (n = 27 patients). Taxonomic landscape at the order level in E BALF (n = 65 patients, 130 samples) and F sputum (n = 27 patients)

Fig. 2

Differential abundance comparisons. A Taxonomic cladogram and B LDA score plot by LEfSe (LDA score minimum = 2.0, p = 0.05) displaying differential taxa in COPD patients in A BAL (P; n = 30 patients, 60 samples) vs. healthy volunteers (HV; n = 35 patients, 70 samples), B sputum (P; n = 17) vs. healthy volunteers (HV; n = 10)

Fig. 3

Serum immunological response to NTHi. A Unsupervised hierarchical clustering heatmap of antibody titers to NTHi antigens. Significantly different antibody titers are marked with an asterix. Comparison of the IgA titers (log₂) of B P6, C Protein D and D Protein F by subject group. p-value by Kruskal–Wallis test. HV-NS n = 15; HV-ES n = 20; P-IE n = 14; P-FE n = 12. Antigen titers are z-scaled

Fig. 4

NTHi killing activity in BALF. A Contingency plot of killing activity between COPD patients and healthy volunteers. B Taxonomic landscape at the family level by killing activity (with killing activity: n = 22 patients, 44 samples; no killing activity: n = 7 patients, 14 samples). C LEfSe LDA score plot by NTHi killing activity (LDA score minimum = 2.0, p = 0.05; with killing activity: n = 22 patients, 44 samples; no killing activity: n = 7 patients, 14 samples). D A bubble plot depicting top and bottom 20 differentially expressed proteins ranked by the log fold-changes (with killing activity: n = 19 patients, 33 samples; No killing activity: n = 7 patients, 13 samples). E Comparison of Haptoglobin protein expression by killing activity (with killing activity: n = 19 patients, 33 samples; no killing activity: n = 7 patients, 13 samples)