S52 Association of microtubule instability with defective phagocytosis in COPD

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Abstract

Acute exacerbations of COPD are the commonest cause of acute medical admissions in the UK and ∼50% are associated with bacterial infection. Alveolar macrophages (AM) normally clear inhaled bacteria but defective phagocytosis may lead to chronic colonisation and increased exacerbations. Monocyte-derived macrophages (MDM), used to model AM, were obtained from COPD, smoking and healthy subjects. MDM phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae (HI) or Streptococcus pneumoniae (SP) was measured by fluorimetry. MDM derived from all subjects showed equivalent ability to phagocytose beads, however, COPD and smoker MDM showed significantly reduced phagocytosis of bacteria. Phagocytosis of HI was reduced by 28% and 48% in COPD and smoker MDM respectively, compared to healthy, while SP phagocytosis was reduced by 32% and 52% in COPD and smoker MDM respectively, compared to healthy (Abstract S52 table 1). Having identified defective bacterial phagocytosis in smoker and COPD MDM, the next step was to elucidate the underlying mechanism. Cytoskeletal rearrangement was investigated, with COPD MDM showing significantly reduced phagocytosis of bacteria in comparison to healthy after pre-incubation with nocodazole (microtubule disruptor). Microtubules are involved in membrane trafficking of the phagolysosome and microtubule stability is necessary for effective phagocytosis. Tubulin is acetylated to form stable microtubules and is deacetylated by HDAC6 and Sirt2. COPD MDM showed reduced levels of acetylated tubulin compared to healthy MDM. Pre-incubation with epothilone B (10 nm) a microtubule stabiliser, improved HI phagocytosis in COPD MDM by 20% (p<0.05) and SP phagocytosis in smoker MDM by 40%. Levels of acetylated tubulin increased on exposure to bacteria alone in healthy and smoker MDM but not in COPD MDM. Pre-incubation with epothilone B was associated with significantly increased levels of acetylated tubulin in COPD cells. No significant differences were seen in the expression of HDAC6 or Sirt2 in COPD compared to healthy cells. MDM from smoking and COPD subjects show reduced phagocytosis of common respiratory bacterial pathogens. Acetylation of microtubules appears to be reduced in COPD, whereas, increasing tubulin acetylation is associated with improvements in phagocytosis, which may allow for targeted development of future therapies to treat colonisation and prevent exacerbations of COPD.

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