Image analysis of tissue macrophages to confirm differential phagocytosis between groups by microscopy and automated bacterial quantification

Background: COPD macrophages display reduced phagocytosis of bacteria, yet the mechanism is unknown. Current methods of assessing phagocytosis such as plate reader fluorimetry and flow cytometry require thousands of cells and do not quantify internalised bacteria by single cells. Super-resolved microscopy (SRM) allows quantification of bacteria and could be used to elucidate molecular defects in cells undergoing phagocytosis. Aim: Image analysis of phagocytosis in single tissue macrophages (TM) to compare responses between healthy, ex-smoker and COPD subjects. Method: TM from non-smokers (n=4), ex-smokers (n=4) and COPD patients (n=3) were incubated with fluorescent Haemophilus influenzae (HI) for up to 60 min. Cells were imaged live by deconvolved widefield (WF) microscopy and bacteria quantified using MicrobeJ. Comparative SRM images were acquired in fixed samples by stochastic optical reconstruction microscopy. Results: WF microscopy of TM from ExSm and COPD patients showed reduced phagocytosis at 60 min compared to NS (HI/cell, NS = 12.22 ± 0.77, ExSm = 7.77 ± 0.91, COPD = 4.93 ± 0.52, p<0.01). The percentage of TM that phagocytosed was not significant between groups (NS: 91.1±4.2%, ExSm: 79.4±3.2%, COPD: 80.6±2.7%, p=0.11). STORM images identified significantly more bacteria than WF (4-fold) and deconvolved WF (2-fold). Conclusions: Using SRM and new analysis software, differences in phagocytosis between NS, ExSm and COPD was shown by analysis of 100 cells, significantly fewer than the 10⁵ cells required for plate reader or FACS. This is important in precious samples such as tissue macrophages, where cell numbers isolated are often limited.