Defective innate immunity in chronic obstructive pulmonary disease

Abstract

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease, comprising chronic bronchitis, small airways fibrosis and emphysema. The primary risk factor for developing COPD, in industrialised nations is cigarette smoking. Exacerbations of COPD, of which approximately half are due to bacterial infection, are associated with worsening quality of life, more rapid decline in FEV1 and increased mortality. In healthy individuals, alveolar macrophages (AM) clear inhaled bacterial pathogens from the lung by phagocytosis, resulting in sterility of the lower respiratory tract. However, COPD patients have increased bacterial colonisation of the lower airways compared to healthy smokers and non-smokers. The increased rates of bacterial colonisation in COPD, suggests that there may be a mechanistic defect in the clearance of bacteria by phagocytic cells. The main aim of this thesis was to investigate the hypothesis that defective innate immunity in COPD patients results in reduction in bacterial phagocytosis, with increased frequency of acute exacerbation, and that this defective phagocytosis may be explained by instability of microtubules. Phagocytic ability of both macrophages and neutrophils from COPD patients was compared to age-matched, non-smokers and smokers. Monocyte-derived-macrophages (MDMs) were used as a model of AM. Both MDMs and neutrophils from all subject groups displayed equivalent phagocytosis of inert beads, showing that cells from all subject groups are capable of phagocytosis. However, both MDMs and neutrophils from smokers and COPD patients showed reduced uptake of both Haemophilus influenzae (by 48%, p<0.001 and 28%, p<0.01 respectively) and Streptococcus pneumoniae (by 52%, p<0.001 and 32%, p<0.05 respectively). Whilst MDMs showed defective phagocytosis of bacteria, intracellular killing remained intact. When COPD patients were divided into those with a history of frequent (≥2/y) or infrequent exacerbations (<1/y), frequent exacerbators had significantly reduced phagocytosis of bacteria compared to infrequent exacerbators. No differences were seen when phagocytosis at baseline was compared to phagocytosis at times of exacerbation. As COPD patients appear to have defective phagocytosis, and with recent meta-analyses showing an increased risk of pneumonia with fluticasone propionate (FP), the effects of both budesonide (BUD) and FP on phagocytosis by MDMs and neutrophils from COPD patients was assessed. No differences were found in phagocytosis of bacteria by MDMs in the presence of either steroid, or in the ability of these cells to perform functions of intracellular killing. In the presence of BUD, neutrophils showed significantly improved uptake of H. influenzae (with a maximal effect of 67%, p<0.05), but neither FP nor BUD had any impact on phagocytosis of beads or S. pneumoniae. Further investigation into the mechanisms underlying defective phagocytosis revealed increased susceptibility of COPD MDMs to microtubule disruption. Associated with this finding was a reduced level of acetylated tubulin in COPD MDMs. Addition of a microtubule stabiliser increased acetylated tubulin and significantly increased bacterial phagocytosis (maximal increase of 20% and 40% in phagocytosis of Haemophilus influenzae and Streptococcus pneumoniae respectively). In contrast, neutrophils displayed no differences in acetylated tubulin and showed no improvement in phagocytosis after exposure to microtubule stabilisers, suggesting an alternative mechanistic defect in neutrophils compared to MDMs. Acetylated microtubules are deacetylated by the enzymes HDAC6 and SIRT2. Exposure to the deacetylase inhibitors, tubacin (HDAC6 inhibitor) or AGK2 (SIRT2 inhibitor), led to increases in levels of acetylation of tubulin but no improvements in phagocytosis, whilst knockdown of either HDAC6 or SIRT2 revealed a similar picture, with increased acetylation but no improvements in phagocytosis. These findings suggest that increased acetylation of tubulin alone is not sufficient to improve phagocytosis, but rather that the defect in phagocytosis is related to microtubule instability. Knockdown of C6orf134, a newly discovered tubulin acetyl-transferase, in healthy MDMs led to reductions in levels of acetylated tubulin and reduced bacterial, but not inert bead, phagocytosis, mimicking the defect seen in COPD MDMs. This suggests that alterations in activity or expression of this protein may account for the defective phagocytosis seen in COPD MDMs. Improving phagocytosis by stabilisation of microtubules may therefore lead to reduced levels of bacterial colonisation and improved exacerbation frequency in COPD patients.