An investigation of the Rubicon/LC3 associated phagocytosis (LAP) dysregulation as a therapeutic target in chronic obstructive pulmonary diseases (COPD) and in response to cigarette smoke exposure

Abstract

Phagocytic clearance of bacteria and apoptotic cells (a process termed efferocytosis) in COPD is critical to protect against microbial infection and lung tissue injury. The compromised phagocytic capacity of alveolar macrophages in COPD allows bacterial colonisation and is postulated to contribute to the disease severity. However, the precise mechanisms that lead to the macrophage phagocytic dysfunction in COPD remains incompletely understood. LC3-associated phagocytosis (LAP) is a recently discovered cellular event characterised as a critical regulator of effective processing of ingested microbes and apoptotic cells by macrophages. Defective LAP impairs the clearance of pathogens and apoptotic cells by macrophages. Therefore, experiments described in Chapter 2 used novel approaches to measure components of the LAP signalling system including TIM-4, Rubicon, LC3, Atg5, NOX2 in blood monocyte derived macrophages, differentiated THP-1 macrophages and lung tissues of mice exposed to cigarette smoke extract (CSE). Further, the expression of the LAP specific regulator, Rubicon, was examined in bronchoalveolar lavage (BAL)-derived macrophages from COPD patients and healthy controls. The findings of this study showed for the first time that Rubicon/LAP is dysregulated in COPD as a result of CSE exposure. Moreover, the study found that Rubicon inhibition correlated with a defective efferocytosis capacity of alveolar macrophages, confirming a link between LAP inhibition and defective efferocytosis in COPD. Furthermore, this report characterises LAP as a potential therapeutic target for potentiating macrophage efferocytic function in COPD. Modulation of Rubicon/LAP requires a better understanding of the processes that lead to Rubicon/LAP inhibition in COPD. Hence, Chapter 3 of this thesis addressed the mechanisms of Rubicon inhibition by CSE. It was noted that CSE shortens the half-life of Rubicon protein but does not have significant effects on Rubicon mRNA levels. This led to the hypothesis that a protein degradation pathway may contribute to the reduction in Rubicon in macrophages exposed to the factors in cigarette smoke. Further observation that Rubicon degradation could be attenuated by anti-proteases and lysosomal enzyme inhibitors confirmed this hypothesis and demonstrated that lysosomal enzymes may mediate the Rubicon degradation. Moreover, alterations in autophagy or proteasomes did not have significant effects on Rubicon suggesting that Rubicon downregulation by CSE is independent of autophagy or proteasomes.