Functional responses of neutrophils to Fusobacterium nucleatum subspecies

Fusobacterium nucleatum is an opportunistic oral pathogen with five subspecies: animalis, fusiforme, nucleatum, polymorphumandvincentii. These play an important role in biofilms associated with chronic gum disease (periodontitis). Neutrophils form the first line of defence against these and other oral pathogens, employing a multitude of anti-microbial strategies, which, alongside bacterial killing, also cause collateral host-tissue damage. The immunogenic properties of F. nucleatum subspecies are poorly understood. The purpose of this study was to investigate specific neutrophil responses to all F. nucleatum subspecies.

AllF. nucleatumsubspecieswere grown planktonically overnight and inactivated using 10% neutral buffered formalin. Peripheral blood neutrophils (PBN) and neutrophil-like cells (differentiated from HL-60 cells) were challenged with inactivated bacteria (MOI 100) and production of reactive oxygen species (ROS) was quantified by Luminol/Iso-luminol enhanced chemiluminescence assays. Release of neutrophil extracellular traps (NETs) was quantified fluorescently using Sytox-green. Additionally, phagocytosis of fluorescently labelled F. nucleatum subspecies was analysed in both cell groups by flow cytometry.

In addition to differences in ROS generation by PBN and neutrophil-like cells in response to individual F. nucleatum subspecies, variations in the amount of extracellular DNA extruded during NETosis was evident. Moreover, distinct levels of phagocytic activity of both cell types will be shown.

F. nucleatum pathogenicity has been reported in oral as well as systemic diseases (colorectal cancer, cardiovascular disease). Understanding subspecies-specific effects of F. nucleatum on neutrophils will enhance our knowledge of the interactions between this bacterium and the host immune response and may help to identify new therapeutic targets.