Differentiation of macrophages with cigarette smoke extract alters bacterial phagocytosis

<b>Background:</b> Defective macrophage phagocytosis of bacterial <i>spp</i>. is a pathophysiological feature of COPD and may increase susceptibility to colonisation and infective exacerbation. These, in turn, lead to accelerated lung function decline and associated mortality. Oxidative stress is important in COPD pathogenesis and may be responsible for this macrophage dysfunction. Hence, the role of chronic reactive oxygen species (ROS) exposure during monocyte derived macrophage (MDM) differentiation was investigated. <b>Methods:</b> PBMC isolated from non-smoking volunteers were differentiated over 12d with GM-CSF. Cigarette smoke extract (CSE) or (ROS generating) hypoxanthine/xanthine oxidase (HX/XO) were frequently added during differentiation. ROS production was confirmed by dichlorodihydrofluorescein (DCF). MDM were incubated for 4h with fluorescently labelled inert beads, heat-killed <i>Haemophilus influenzae</i> (<i>HI</i>) or <i>Streptococcus pneumoniae</i> (<i>SP</i>) and phagocytosis measured using fluorometry or flow cytometry. Cell viability was measured via MTT assay or live/dead fluorophore. <b>Results:</b> Both CSE and HX/XO generated ROS over the time-course. CSE differentiated MDM demonstrated reduced phagocytosis of both <i>HI</i> (control 2.30±0.40 <i>vs.</i> CSE 1.40±0.21 RFUx10³ [n=10, p&lt;0.01]) and <i>SP</i> (control 5.50±0.43 <i>vs.</i> CSE 3.71±0.46 [p&lt;0.01]). Phagocytosis of inert beads was unaffected. Internalisation was confirmed using pHrodo® labelled bacteria (<i>HI</i> - control 51.3±4.9 <i>vs.</i> CSE 36.2±9.0 % viable cells [n=4] and <i>SP</i> - control 51.1±4.6 <i>vs.</i> CSE 32.2±4.5). In contrast, HX/XO had no significant effect on phagocytosis. <b>Conclusion:</b> Oxidative stress alone does not drive defective phagocytosis in MDM but cigarette smoke extract reduced these responses.