RT Journal Article
SR Electronic
T1 Analysis of defective phagocytosis in COPD using super-resolution microscopy and automated bacterial quantification
JF European Respiratory Journal
JO Eur Respir J
FD European Respiratory Society
SP 1058
DO 10.1183/13993003.congress-2020.1058
VO 56
IS suppl 64
A1 Riccardo Wysoczanski
A1 Jonathan R Baker
A1 Peter Fenwick
A1 Edwin Garcia
A1 Sunil Kumar
A1 Mark A A Neil
A1 Christopher Dunsby
A1 Paul M W French
A1 Peter J Barnes
A1 Louise E Donnelly
YR 2020
UL http://erj.ersjournals.com/content/56/suppl_64/1058.abstract
AB 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.FootnotesCite this article as: European Respiratory Journal 2020; 56: Suppl. 64, 1058.This abstract was presented at the 2020 ERS International Congress, in session “Respiratory viruses in the "pre COVID-19" era”.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).