Riccardo Wysoczanski

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, p5 cells required for plate reader or FACS. This is important in precious samples such as tissue macrophages, where cell numbers isolated are often limited.

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, p5 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.

The cause of chronic inflammation in ulcerative colitis (UC) is incompletely understood. Here we tested the hypothesis that an excessive acute inflammatory response to bacteria contributes to the pathogenesis. Acute inflammatory responses were provoked in vivo in UC patients and healthy controls by intradermal inoculation with bacteria. Vascular responses were quantified by laser Doppler. Inflammatory exudates were recovered in superimposed suction blisters and cells measured by polychromatic flow cytometry, cytokines by multiplex array, and inflammatory lipids by mass spectrometry. Vascular responses in UC patients were heightened at 24h after bacterial injection (p=0·03), and remained abnormally high at 48h (p=0·0005) and this amplified response was seen in UC with Gram-positive as well as Gram-negative organisms (p=0·01). The cellular infiltrate over the injection site, composed largely of neutrophils at 4 hours a was greater in UC (p=0·002). At 48h, the increased numbers of cells in UC were composed of neutrophils (p=0·001) and CD4 lymphocytes (p=0·001). The exaggerated inflammation in UC was not a cytokine-driven phenomenon. Exaggerated onset was normalised in patients taking 5-aminosalicylates, accompanied by increased concentrations of hydroxy fatty acids 9-oxo-octadecadienoic acid (OxoODE; p=0·05) and 13-OxoODE (p=0·01) in resolving exudates. In vitro, these compounds suppressed macrophage inflammatory cytokine secretion through PPARγ (p<0·0001). Conversely, 5-aminosalicylates did not inhibit early inflammatory reactions in control participants. Acute inflammatory responses to bacteria in UC are both overly exuberant and slow to resolve. Neutrophils accumulate in excess and persist, in keeping with the pathological appearances of disease flares. These studies also provide new insight into the mechanism of 5-aminosalicylate (5ASA) drugs, which act as pro-resolution rather than indiscriminate anti-inflammatory agents by promoting formation of immunomodulatory hydroxy lipids. While production of these lipids is not defective as part of the underlying disease process, this identifies a novel mechanism of drug action harnessing pro-resolution pathways.Summary: Wysoczanski and colleagues demonstrate that the inflammatory response to injected bacteria is exaggerated and prolonged in ulcerative colitis. This disordered inflammation appears to be associated with increased secretion of PGE2. 5-aminosalicylate drugs, which are used to treat this condition, normalize inflammation and PGE2 secretion, and appear to work through PPARγ