S51 Hungry hungry macrophages: how multiple prey affects macrophage phagocytosis

Background Macrophage phagocytosis is a fundamental process for maintaining tissue homeostasis and controlling infection by removing invading pathogens as well as dying cells. This is especially important in the respiratory tract, where alveolar macrophages patrol the airways, removing inhaled bacteria and mounting an immune response when infection takes hold. Studies looking at macrophage phagocytosis usually consider one prey, but this may not be a true representation of what happens in the lungs, where macrophages are faced with multiple types of bacteria, as well as apoptotic cells which require removal. Methods Monocyte-derived macrophages from healthy donors were incubated for 4 hours with fluorescently labelled apoptotic neutrophils (AC), non-typable Haemophilus influenzae (NTHI), and Streptococcus pneumoniae (SP) either alone or in combination, and phagocytosis measured by flow cytometry, with cytokine output measured by ELISA. Results The percent of macrophages that phagocytosed a single prey ranged from 12% with AC, to 56% for NTHI, or 74% for SP. When macrophages were exposed to two prey - a bacteria (NTHI/SP) and an AC, four populations emerged – cells that had eaten only AC (4%/3%), cells that had eaten only bacteria (23%/39%), cells that had eaten AC and bacteria (16%/11%) and cells that had eaten nothing (57%/47%). When macrophages were exposed to both bacteria (NTHI/SP), four populations emerged – cells that had eaten HI only (6%), cells that had eaten SP only (14%), cells that had eaten both HI and SP (51%) or cells that hate eaten nothing (29%). When all three prey were combined, macrophages ate a combination of all types, with 19% consuming all three prey. Phagocytosis of bacteria induced the release of CXCL-8, IL-6 and TNFa, which was inhibited by uptake of ACs. Conclusion Macrophages are capable of consuming multiple prey at once. Uptake of an apoptotic cell alongside bacteria inhibited pro-inflammatory cytokine release, thus limiting inflammation. The effects of phagocytosis of multiple prey on macrophage phenotype and stress responses are unknown, but this experiment may be a more physiological model of the lungs and should be considered to understand the impact of macrophages in lung disease.