Temporal phosphoproteomic programmes set a redox-gated rheostat on neutrophil phagocytosis

Authors:
Preprint
Publisher website
Google Scholar
Abstract
Neutrophils are essential frontline immune cells in the defence against bacterial pathogens such as Staphylococcus aureus , but their killing machinery also causes host tissue damage. How neutrophils regulate these processes remains incompletely understood. Here, we profiled the proteome and phosphoproteome of human neutrophils across a two-hour exposure to S. aureus , alongside five simultaneous functional readouts, before investigating how an initial encounter modulates the response to a second. The phosphoproteomic response resolved into qualitatively distinct early and late windows: the early landscape drives particle uptake, while by the late window this machinery is deactivated, with a broadly supressed kinome, and cells geared towards inflammatory output. Correlating protein phosphorylation and functional kinetics — an approach we term KINETIC — implicates putative mediators of key neutrophil antimicrobial functions. A genome-wide CRISPR knockout screen in neutrophil-like PLB-985 cells, sorted on phagocytic load, recovered canonical engulfment machinery among the strongest facilitators of phagocytosis, additionally identifying the lysosomal Ragulator-mTORC1 nutrient sensing pathway and HOPS complex corresponding to the KINETIC-identified pathways. Conversely, the NRF2 repressor KEAP1 was the strongest phagocytic brake, positioning oxidative burden as a regulator of phagocytic appetite. Neutrophils in the late window exhibited impaired phagocytosis and phagosomal acidification on S.aureus re-challenge. Underlying these functional deficits, cells in the late window were refractory across multiomic layers: a second S. aureus meal altered no phosphoprotein, protein, or transcript, whereas the chemoattractants PAF and fMLP evoked a largely consistent phosphoproteomic programme, delineating the phenotype as receptor-specific rather than a global loss of signalling capacity. Consistent with de-repressing the antioxidant response via genetic loss of KEAP1 increasing phagocytosis, reducing ROS production via inhibition of the NADPH oxidase resulted in a partial rescue of phagocytosis of the re-exposed S.aureus . Together, these findings describe a ROS-gated phagocytic rheostat, enabling neutrophils to meter their phagocytic appetite according to their exposure history, likely limiting uptake of pathogens they lack the capacity to effectively digest. Inappropriate activation of this rheostat under conditions of physiological stress or severe illness may underpin the defects in phagocytosis identified in such patients.
Funding Information
  • National Institute of Academic Anaesthesia (WKRO-2018-0051)

This publication has 55 references indexed in Scilit: