Collins, George B; (2024) Characterising a human in vivo model of neutrophil phagocytosis using intradermal methylene blue-labelled Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

Text George Collins PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 March 2025. Download (34MB)

Abstract

Phagocytosis is important in defending against infection but cannot currently be measured in human tissues in vivo. This could improve our understanding of phagocyte biology. The current investigation therefore set out to measure bacterial phagocytosis in human tissues in vivo using the intradermal injection of Escherichia coli labelled with methylene blue (MB), a bacterial stain safe for human injection. E. coli were labelled with MB and co-cultured with leucocytes ex vivo. Phagocytosis of MB-labelled E. coli (MBEC) caused MB to accumulate in neutrophils and monocytes. Suction blisters were then used to sample infiltrating leucocytes after intradermal MBEC injection. Blister neutrophils and monocytes contained MB, but circulating leucocytes and blister T, B and NK cells did not. Neutrophils infiltrated before monocytes and were more phagocytic. Bacterial clearance was complete 7-9h after injection, evidenced by a decrease in blister fluid endotoxin levels and increase in MB in blister phagocytes. Blood and blister neutrophils separated into mature and immature populations, and at the end of bacterial clearance skewed towards a mature expression profile. Compared to immature neutrophils, mature neutrophils phagocytosed less at the cell-to-cell level but more as a population owing to their higher abundance. This temporal separation of mature and immature neutrophils suggested mature neutrophils were more involved in inflammation resolution. This was a supported by the emergence of an MBloCD62Lhi blister neutrophil population at the end of bacterial clearance recognised to be an aged T cell suppressing phenotype. By measuring phagocytosis in human tissues for the first time, this investigation was therefore the first to identify this population as non-phagocytic and present during the resolution of healthy immune responses. More broadly, this investigation suggests mature and immature neutrophils have different roles in the defence against bacterial infection, with immature neutrophils more involved in inflammation onset, and mature neutrophils in inflammation resolution.

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