Analysis of defective phagocytosis in COPD using super-resolution microscopy and automated bacterial quantification

Background: Phagocytosis of bacteria, fungal spores and apoptotic cells is reduced in macrophages from COPD patients, but the mechanism remains unknown. The use of super-resolved microscopy (SRM) may allow investigation of specific molecular defects in single cells undergoing phagocytosis using fewer cells than conventional methods such as flow cytometry and plate reader fluorimetry. Aim: Develop a methodology to assess phagocytosis of bacteria in single macrophages and compare responses between cells from healthy and COPD subjects. Method: Monocyte-derived macrophages (MDM) from non-smokers (NS, n=5) and COPD patients (n=3) were incubated with fluorescently-labelled Haemophilus influenzae (HI) for up to 60 min. Cells stained for actin or tubulin with silicon rhodamine dyes were imaged live by widefield (WF) microscopy, deconvolved with Huygens software and bacteria quantified using MicrobeJ. Comparative SRM images were acquired in fixed samples by stochastic optical reconstruction microscopy (STORM). Results: WF microscopy of MDM from COPD patients showed reduced phagocytosis compared to NS at 60 min (HI/cell, NS = 4.92 ± 0.47, COPD = 1.73 ± 0.31, p<0.01). The percentage of MDM that did not phagocytose was not significant between groups (NS: 33.5±8.3%, COPD: 61.1±7.7%, p=0.143). STORM images identified significantly more bacteria than WF (4-fold) and deconvolved WF (2-fold). Conclusions: Analysis of 100 cells revealed significant differences in phagocytosis between NS and COPD with fewer than the 10⁵ cells required for plate reader or FACS. SRM and new analysis software allows phagocytosis in live cells to be determined in detail using fewer cells than other methods.