A new model measuring bacterial phagocytosis and phagolysosomal oxidisation in humans using the intradermal injection of methylene blue-labelled Escherichia coli.

Phagocytosis is an important leucocyte function, however using existing models it cannot be measured in human tissues in vivo. To address this, we characterized a new phagocytosis model using intradermal methylene blue-labelled Escherichia coli injection (MBEC). Methylene blue (MB) is a licensed human medicine and bacterial stain potentially useful for labelling E. coli that are safe for human injection. Ex vivo co-culture of leucocytes with MBEC caused MB to transfer into neutrophils and macrophages by phagocytosis. During this, a 'red shift' in MB fluorescence was shown to be caused by phagolysosomal oxidisation. Hence, MBEC co-culture could be used to measure phagocytosis and phagolysosomal oxidisation in humans, ex vivo. In healthy volunteers, inflammatory exudate sampling using suction blisters 2-24h after intradermal MBEC injection showed that tissue-acquired neutrophils and monocytes contained more MB than their circulating counterparts, whereas blood and inflamed tissue T, B and NK cells were MBlo. This was validated with spectral flow cytometry by visualizing the MB emission spectrum in tissue-acquired neutrophils. Neutrophil MB emission spectra demonstrated more 'red shift' at 24h compared to earlier time-points, in-keeping with progressive phagolysosomal MB oxidisation in neutrophils over time in vivo. This new MBEC model can therefore measure bacterial phagocytosis and phagolysosomal oxidisation in human skin, in vivo. This has a number of important research applications, for example in studying human phagocyte biology, testing novel antimicrobials, and understanding why certain groups such as males, the elderly or those with diabetes, recent surgery or malnutrition are at increased risk of bacterial infection.