Genetic and epigenetic characterization of the sphingosine-1-phosphate signalling system in macrophages in chronic obstructive pulmonary disease

Abstract

Alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic bronchial epithelial cells (a process termed ‘efferocytosis’) and bacteria. These defects may contribute to COPD pathogenesis in several ways. Secondary necrosis of uncleared apoptotic material may result in chronic airways inflammation and perpetuation of COPD disease. A reduced alveolar macrophage phagocytic host response to bacteria, especially non-typeable H influenzae (NTHi), may contribute to neutrophilic inflammation and NTHi colonization of the lower airway. However, the exact mechanism that leads to the phagocytic dysfunction is still unknown. The sphingosine 1-phosphate (S1P) signalling system is known to regulate macrophage function. Experiments described in Chapter 2 of the thesis therefore applied a novel approach of measuring all S1P signalling system components in alveolar macrophages from COPD patients and healthy controls. Several components of the S1P system, in particular relative mRNA levels for sphingosine kinases SPHK1 and S1P receptor S1PR5, were dysregulated in COPD and were strongly correlated with efferocytosis, suggesting a potential link to the defective alveolar macrophage phagocytic ability in COPD. Oxidative stress and inflammation have been shown to contribute to many COPD characteristics, such as uncontrolled activation of cell signalling pathways, increased airway epithelial cell apoptosis, and defective alveolar macrophage phagocytic ability. Chapter 3 describes the effect of two models of oxidative stress and inflammation, cigarette smoke (potential oxidative conditions) and lipopolysaccharide (LPS) (potential inflammatory conditions) on components of S1P signalling and on efferocytosis and phagocytosis of NTHi, using a human macrophage cell line in vitro. Cigarette smoke and LPS increased the mRNA expression of SPHK1 and S1PR5 in macrophages, extending the results in Chapter 2 and further supporting the potential link between the S1P signalling system and macrophage phagocytic ability. Cigarette smoke decreased the capacity of macrophages to phagocytose apoptotic cells and bacteria. However, LPS reduced phagocytosis of bacteria only. Treatment option for oxidative stress is anti-oxidants and thymoquinone (TQ) is anti-oxidant/anti-inflammatory agent that has been shown to modulate macrophage inflammatory responses and has successfully been trialled in human clinical studies. Chapter 3 further reports that TQ per se had a pro-phagocytic effect on macrophage phagocytic ability. TQ also rescued macrophages from the negative effects of cigarette smoke, and to lesser extent LPS, on macrophage efferocytosis and the mRNA expression respectively. In addition, TQ demonstrated a pro-survival effect on bronchial epithelial cells treated with cigarette smoke. The effects on relative mRNA expression of SPHK1 and S1PR5 in the cell line were mirrored using acutely isolated alveolar macrophages from COPD patients. COPD patients are at increased risk for developing lung cancer and there is strong evidence that pulmonary macrophage dysfunction plays an important role in the pathogenesis of both diseases. DNA methylation has been shown to be modified in COPD and lung cancer. However, it unknown whether the change in mRNA expression of the S1P system (Chapter 2) are controlled by epigenetic modifications such as DNA methylation, and whether DNA methylation regulates macrophage efferocytosis. Data presented in Chapter 4 connect epigenetic modulation, mRNA expression and macrophage function. The results indicate that DNA methylation potentially regulates macrophage efferocytosis and is negatively correlated with the mRNA expression of S1P system components, in particular the S1PR5 receptor, suggesting epigenetic for COPD such as anti-oxidants or epigenome modifying agents.