Tom Speight1 ; Ian Dransfield2; Mark Bradley1; ; Kevin Dhaliwal1;

AcknowledgementsWe would like to thank Engineering and Physical Sciences Research Council (EPSRC, UnitedKingdom) Interdisciplinary Research Collaboration grant EP/K03197X/1 and the College of MedicineandVeterinary Medicine, University of Edinburgh, for funding this work.

Macrophages play an essential role in the human immune system. The activity of alveolar macrophages is particularly important: these cells arethe first to respond to particles we inhale into our lungs. A defining role of alveolar macrophages is to phagocytose (ingest) foreign particlesand any immune cells that are undergoing cell death. A loss of phagocytosis can contribute to a number of lung diseases, such as ChronicObstructive Pulmonary Disease (COPD)1. Although there appear to be pharmaceutical techniques to restore alveolar macrophagephagocytosis in these diseases, there remains a challenge to truly profile human alveolar macrophage activity while they are still in their nativelung environment of the alveolar space2.

Alveolar Macrophage Activity: A medical and research challenge

Optical Profiling of Human Macrophage Activity

References1 Hodge, S. et al. (2007) Smoking alters alveolar macrophage recognition and phagocytic ability:Implications in chronic obstructive pulmonary disease. American Journal of Respiratory Cell and Molecular Biology 37: 748–755.2 Harvey, C. J. et al. (2011) Targeting Nrf2 Signalling Improves Bacterial Clearance by Alveolar Macrophages in Patients with COPD and in a Mouse Model. Science Translational Medicine 3: 78.

• Develop and characterise a model of human macrophages• Develop an optical based assay to quantify the phagocytic activity• Use novel imaging methods to reliably profile phagocytosis• Apply Proteus technology to the imaging of macrophages in vivo

Culturing Human Monocytes Into Macrophages

B)

C)

A)

A) Monocytes are purified from whole blood before being cultured

B) Cell morphology changes from monocytes to macrophages during cultureC) Expression of monocyte/macrophage markers before and after culture (n=4 ±SEM)

Quantifying Phagocytic Activity

A) Red-labelled macrophages are gated using Flow Cytometry

B) Example plots quantifying macrophage phagocytosis of green labelled targetsC) Quantification of macrophage phagocytosis of dying neutrophils or live bacteria, with and without phagocytic inhibitor Cytochalasin D (n=5 ±SEM)

Project Aims

Monocytes Day 0

Macrophages Day 6

FSC

CellTrace

Red

Neutrophils+ Cytochalasin D

Neutrophils Bacteria

A) C)

B)

FSC

CellTrackerGreen

Imaging Macrophage Phagocytosis

0 min 15 min 30 min 45 min

Developing imaging methods will allow us to understand the kinetics of phagocytosisin different macrophage populations. The above time series taken with a confocalmicroscope shows how we can track a red labelled macrophage phagocytosing agreen labelled dying neutrophil. The neutrophil has been labelled with a pH-activateddye, pHrodo. The dye emits fluorescence when there is a drop in pH, which is whatoccurs inside the macrophage following phagocytosis.

The Proteus imaging system, Versicolour, is a widefield fibre-based system capable ofimaging the alveolar spaces of the lung in real time. One application of thistechnology can be to profile human alveolar macrophages in situ. The above imagesshow initial work of imaging red labelled macrophages in a dish with pHrodo Greenlabelled dying neutrophils. We can infer that green signal represents phagocytosis, asthe signal is blocked using an inhibitor of phagocytosis (Cytochalasin D).

1EPSRC IRC Hub in Optical Molecular Sensing & Imaging, MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK2MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK

Application to Proteus Technology

Without Cytochalasin D With Cytochalasin D

Future Work

• Apply primary patient cells to experiments• Characterise imaging of macrophages using Versicolour in a

human lung model• Explore more optical techniques to profile macrophage activity

(multispectral imaging, fluorescence lifetime imaging)• Develop optical imaging probes (see Gavin Birch, Proteus

Poster) to label macrophages