Bacterial load and inflammatory response in sputum of alpha-1 antitrypsin deficiency patients with COPD

Abstract

Background: Airway inflammation may drive the progression of chronic obstructive pulmonary disease (COPD) associated with alpha-1 antitrypsin deficiency (AATD), but the relationship between airway microbiota and inflammation has not been investigated.

Methods: We studied 21 non-treated AATD (AATD-noT) patients, 20 AATD-COPD patients under augmentation therapy (AATD-AT), 20 cigarette smoke-associated COPD patients, 20 control healthy smokers (CS) and 21 non-smokers (CON) with normal lung function. We quantified sputum inflammatory cells and inflammatory markers (IL-27, CCL3, CCL5, CXCL8, LTB₄, MPO) by ELISA, total bacterial load (16S) and pathogenic bacteria by qRT-PCR.

Results: AATD-AT patients were younger but had similar spirometric and DLCO values compared to cigarette smoke-associated COPD, despite a lower burden of smoking history. Compared to cigarette smoke-associated COPD, AATD-noT and AATD-AT patients had lower sputum neutrophil levels (p=0.0446, p=0.0135), total bacterial load (16S) (p=0.0081, p=0.0223), M. catarrhalis (p=0.0115, p=0.0127) and S. pneumoniae (p=0.0013, p=0.0001). Sputum IL-27 was significantly elevated in CS and cigarette smoke-associated COPD. AATD-AT, but not AATD-noT patients, had IL-27 sputum levels (pg/ml) significantly lower than COPD (p=0.0297) and these positively correlated with FEV₁% predicted values (r=0.578, p=0.0307).

Conclusions: Compared to cigarette smoke-associated COPD, AATD-AT (COPD) patients have a distinct airway inflammatory and microbiological profile. The decreased sputum bacterial load and IL-27 levels in AATD-AT patients suggests that augmentation therapy play a role in these changes.

Keywords: COPD; alpha-1 antitrypsin deficiency; chronic airway inflammation; respiratory disability; sputum.

Figure 1

Total inflammatory cells (cells/ml) (A), and percentages of neutrophils (B), eosinophils (C), lymphocytes (D) and macrophages (E) in cytospin preparations from control non-smokers (CON, n=21), control smokers (CS, n=20), cigarette smoke-associated COPD (n=20), α1-anti trypsin deficiency not in therapy (AATD-noT, n=21) and AATD patients on augmentation therapy (AATD-AT, n=20). The total number of inflammatory cells, and the percentage of eosinophils, macrophages and lymphocytes did not differ (K. Wallis: p=0.6777, p=0.0508 and p=0.0560, p=0.0955, respectively) in the five groups studied. The percentage of neutrophils (K. Wallis: p=0.0137) was significantly higher in cigarette smoke-associated COPD compared to CNS (p=0.0015) and AATD-AT (p=0.0135) (B). Cigarette smoke-associated COPD also differed from AATD-NoT (p=0.0446). Data are presented as median and interquartile range. Statistical analysis between groups: Mann-Whitney U test, reported only when the Kruskal-Wallis (KW) test resulted significant. Abbreviations: CNS, control non-smokers; CS, control smokers; COPD, chronic obstructive pulmonary disease; α1ATDnoT, α1-anti trypsin deficiency patients not in therapy; α1ATDA.T., α1-anti trypsin deficiency patients on augmentation therapy.

Figure 2

Chemokines and cytokines quantified (pg/ml) in the sputum supernatants of control non-smokers (CON, n=16), control smokers (CS, n=16), cigarette smoke-associated chronic obstructive pulmonary disease (COPD, n=16), α1-anti trypsin deficiency not in therapy (AATD-noT, n=16) and AATD in augmentation therapy (AATD-AT, n=16). Data are expressed as median and interquartile range. The Kruskal-Wallis (KW) test was used for multiple comparisons followed by the Mann-Whitney U-test for comparison between groups. CCL3, CCL5, LTB4 and MPO levels were similar in all five patient groups (A-D). CXCL8 (IL-8) was increased in CS, cigarette smoke-associated COPD and AATD-AT in comparison to CON, even though the difference did not reach statistical significance at the Kruskal Wallis test (E). IL-27 was significantly higher in cigarette smoke-associated COPD and in CS compared to CON. Cigarette smoke-associated COPD also showed higher values compared to AATD-AT (F). Abbreviations: CCL3, chemokine (CC motif) ligand 3; CCL5, chemokine (CC motif) ligand 5; IL, interleukin; LTB4, leukotriene-B4; MPO, myeloperoxidase.

Figure 3

Total and specific bacterial load (copies/ml) quantified in the sputum of control non-smokers (CON, n=21), control smokers (CS, n=20), cigarette smoke-associated chronic obstructive pulmonary disease (COPD, n=20), α1-anti trypsin deficiency not in therapy (AATD-noT, n=21) and AATD in augmentation therapy (AATD-AT, n=20). Data are expressed as median and interquartile range. The Kruskal-Wallis (KW) test was used for multiple comparisons followed by the Mann-Whitney U-test for comparison between groups. The total bacterial load was significantly increased in cigarette smoke-associated COPD compared to CS, AATD-noT and AATD-AT. (A) M. catarrhalis was increased in cigarette smoke-associated COPD compared to CON, AATD-noT and AATD-AT. CS also had higher values compared to CON. (B) S. pneumoniae was significantly increased in cigarette smoke-associated COPD compared to CON, CS, AATD-noT and AATD-AT; CS also had higher values compared to CON and AATD-AT (C); P. aeruginosa (D); H. influenzae (E) and S. aureus (F) did not differ significantly at the Kruskal-Wallis test. Abbreviations: CNS, control non-smokers; CS, control smokers; COPD, chronic obstructive pulmonary disease; α1ATDnoT, α1-anti trypsin deficiency patients not in therapy; α1ATDA.T., α1-anti trypsin deficiency patients on augmentation therapy.

Figure 4

Relative proportions of P. aeruginosa, H. influenzae, M. catarrhalis, S. aureus and S. pneumoniae in sputum of control non-smokers (CON), control smokers (CS), cigarette smoke-associated chronic obstructive pulmonary diseased (COPD) patients, α1-anti trypsin deficiency not in therapy (AATD-noT) and AATD in augmentation therapy (AATD-AT) patients. The percentage of M. catarrhalis and S. pneumoniae was higher in cigarette smoke-associated COPD compared to CON. Interestingly, augmentation therapy in COPD patients with AATD significantly reduced the percentage of M. catarrhalis and S. pneumoniae to values close to those observed in CON subjects. Abbreviations: CNS, control non-smokers; CS, control smokers; COPD, chronic obstructive pulmonary disease; α1ATDnoT, α1-anti trypsin deficiency patients not in therapy; α1ATDA.T., α1-anti trypsin deficiency patients on augmentation therapy.