A subset of neutrophil phagosomes is characterised by pulses of Class I PI3K activity

Class I PI3kinases coordinate the delivery of microbicidal effectors to the phagosome by forming phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). However, the dynamics of PIP3 in neutrophils during a live bacterial tissue infection are unknown. We have therefore developed an in vivo, live zebrafish infection model that enables visualisation of dynamic changes in Class 1 PI3kinases (PI3K) signalling on neutrophil phagosomes in real-time. We have identified that on approximately 12% of neutrophil phagosomes PHAkt-eGFP, a reporter for Class 1 PI3K signalling, repeatedly fades and re-recruits in pulsatile bursts. This phenomenon occurred on phagosomes containing live and dead bacteria as well as beads, and was dependent on the activity of the Class 1 PI3K isoform, PI3kinase γ. Detailed imaging suggested that pulsing phagosomes represent neutrophils transiently reopening and reclosing phagosomes, a conclusion supported by observations that a subset of phagosomes in human neutrophils rapidly accumulate dye from the extracellular space. Therefore, we propose that some neutrophil phagosomes remain unsealed and are consequently able to exchange contents with the extracellular environment, with implications for phagosome fate and communication with surrounding cells.