NDP52 inhibition rescues cigarette smoke mediated impairment of autophagy in macrophages

Abstract

Introduction: Autophagy is a process that maintains intracellular homeostasis by degrading unwanted proteins, protein aggregates, dysfunctional organelles and pathogens. The role of autophagy in the pathogenesis of COPD is not well understood. NDP52 is an autophagic adaptor protein which binds intracellular pathogens for autophagy-mediated clearance and facilitates autophagosome maturation. Method: The expression of p62, LC3 and NDP52 were measured in lung homogenates from COPD patients (n=24), smokers (n=11) and healthy volunteers (n=4) by Western blot. U937-derived macrophages were treated with cigarette smoke extract (CSE) and autophagic flux was evaluated. Furthermore, the role of NDP52 in autophagy was evaluated by knockdown using siRNA. Results: NDP52 was increased in GOLD4 COPD patients. CSE (30%) increased the level of NDP52, LC3, p62 oligomers and p62 in U937-derived macrophages. The LC3 turnover assay and long-lived protein degradation assay revealed that CSE impaired the late phase of autophagic flux. Knockdown of NDP52 reduced LC3-2 and p62 in the presence and absence of CSE. Furthermore the LC3 turnover assay revealed that while LC3-2 was reduced by NDP52 knockdown, the autophagic flux was functional. Conclusion: Cigarette smoke increases expression of autophagic markers and impairs autophagic flux in macrophages and may be the cause of the increased NDP52 expression observed in COPD patient lungs. Knockdown of NDP52 restored the flux in CSE exposed macrophages and resulted in a reduction of autophagic makers to basal levels.