RT Journal Article
SR Electronic
T1 Image analysis of tissue macrophages to confirm differential phagocytosis between groups by microscopy and automated bacterial quantification
JF European Respiratory Journal
JO Eur Respir J
FD European Respiratory Society
SP PA3694
DO 10.1183/13993003.congress-2021.PA3694
VO 58
IS suppl 65
A1 Riccardo Wysoczanski
A1 Jonathan Baker
A1 Peter Fenwick
A1 Chris Dunsby
A1 Paul French
A1 Peter Barnes
A1 Louise Donnelly
YR 2021
UL http://erj.ersjournals.com/content/58/suppl_65/PA3694.abstract
AB 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.FootnotesCite this article as: European Respiratory Journal 2021; 58: Suppl. 65, PA3694.This abstract was presented at the 2021 ERS International Congress, in session “Prediction of exacerbations in patients with COPD”.This is an ERS International Congress abstract. No full-text version is available. Further material to accompany this abstract may be available at www.ers-education.org (ERS member access only).