The effect of cigarette smoke extract on macrophages

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder which causes permanent airway obstruction, and is one of the leading causes of mortality and morbidity around the world. It is characterised by irreversible obstruction of the airways, the development of chronic infections, exaggerated inflammatory responses leading to bronchitis and emphysema, and a progressive decline in respiratory function. Whilst many risk factors contribute to disease occurrence, cases commonly occur due to oxidative insults, particularly cigarette smoke, which can trigger an immune response and cause inflammation. 15-20 % of smokers are diagnosed with COPD, and the mechanisms underlying the development of COPD need to be investigated further. Macrophages play a crucial role in the establishment of the immune response to pathogens and irritants within the body, and they play an important role in the progress and outcome of many diseases, including COPD. Current evidence suggests that irritants activate epithelial cells and macrophages (especially alveolar macrophages), which recruit other inflammatory cells to the lungs. Some studies have suggested macrophages can cause a deleterious effect in COPD due to an increased number of alveolar macrophages with an M2 profile, which can result in increased tissue damage and fibrosis (Vlahos & Bozinovski 2014). As the relative roles of M1 and M2 macrophages are not fully understood in terms of COPD progression and severity, macrophages may be involved in both fighting and inducing the disease (Vlahos & Bozinovski 2014). The hypothesis of this thesis was that studying the effect of cigarette smoke on macrophage function may promote understanding of the importance of macrophages in the pathogenesis of COPD. The initial experiments with the THP-1 cell line showed that these cells were not a suitable choice for investigating how macrophages are affected by cigarette smoke extract (CSE) or nicotine, therefore, peripheral blood mononuclear cells (PBMCs) were chosen as a replacement. This thesis has established a procedure to generate macrophages from cluster of differentiation-14 (CD14+) monocytes isolated by positive selection from PBMCs and stimulated to mature using macrophage colony stimulating factor (M-CSF). It has also shown the novel use of hexamethyldisilane (HMDS) for coating tissue culture plates, which facilitated the recovery of high numbers of cells with no effect on the expression of cell surface markers. The investigation into the effects of different combinations of CSE, nicotine, polyinosinic-polycytidylic acid (Poly-IC) and lipopolysaccharide (LPS) on the expression of CD cell surface markers showed there was significant suppression of CD14 expression by nicotine with and without Poly-IC, and significant suppression of CD206 after treatment with nicotine only, Poly-IC, or LPS, when compared to the controls. To further investigate the effects of these treatments on macrophages, the expression of markers that are key regulators of macrophage function and associate with either the M1 or the M2 macrophage phenotype were investigated in monocyte-derived macrophages by reverse-phase protein microarray (RPPA). The initial analysis of the RPPA data for the individual signalling molecules did not show any clearly significant changes associated with CSE or nicotine treatment, therefore further analysis was carried out following subtraction of all the relevant blank values from the corresponding values for the signalling molecules expression. The results from this chapter show that RPPA detected differences in the markers selected: there were significant differences after 2 h treatment, with mainly overall down-regulation of the signalling molecules by treatment with CSE. Lesser effects were observed after 24 or 48 hours of treatment, suggesting some re-equilibration of the signalling molecule expression following the acute changes. However, the effects of nicotine on global expression of the signalling molecules was less consistent than that of CSE. To complement the RPPA results, changes in the expression of interleukin-10 (IL-10) and tumour necrosis factor alpha (TNF-α) were investigated by ELISA. The results showed mainly suppression of IL-10 production by nicotine, whereas both CSE and nicotine enhanced production of TNF-. Overall, the most consistent findings from the RPPA data and the ELISA data are that CSE induced overall down-regulation of the signalling molecules at 2 hours, and that CSE up-regulated TNF- production at 24 and 48 hours. Since the majority of the signalling molecules analysed were M2-related, and TNF- is predominantly produced by M1 macrophages, these findings support the idea that CSE suppresses M2 function and favours M1 activation. Although the effects of CSE on the signalling molecules ameliorated by 24 hours of treatment, the early effects observed at 2 hours of treatment might trigger the intra-cellular processes resulting in TNF- production at 24-48 hours. With respect to cytokine production, nicotine again up-regulated TNF- production in certain conditions, and down-regulated IL-10 production. As with CSE, this is also consistent with nicotine promoting a general shift towards M1 function. This study indicates that CSE predominantly promotes M1 macrophage function. However, some of the results with CSE, and still more so with nicotine, also provide evidence for the promotion of M2 function in some conditions. These in vitro observations are consistent with in vivo findings that smoking differentially promotes M1 and M2 macrophage function in different lung compartments, or the generation of macrophages with a mixed M1/M2 phenotype