Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells

Jérémy Joly,Dirk Roos,Dominik Wrona,Ulrich Siler,Paul Verkuijlen,Sophie Dupré-Crochet,Sandrine Lecart,Oliver Nüsse,Elodie Hudik,Janine Reichenbach

doi:10.3389/fcell.2020.608600

Abstract

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.

Highlights

The phagocytic NADPH oxidase produces reactive oxygen species (ROS) that are crucial for killing pathogens during phagocytosis
We first investigated whether the level of NOX2 in the phagosomal membrane increases during phagocytosis in PLB985 cells
The NOX2 increase could be due to lateral diffusion of NOX2 protein in the plasma membrane, which might accumulate in the phagosomal cup, or to fusion of NOX2containing organelles with the phagosomes

Summary

INTRODUCTION

The phagocytic NADPH oxidase produces reactive oxygen species (ROS) that are crucial for killing pathogens during phagocytosis. Immunofluorescence studies indicated that PLB-985 cells, as well as primary human neutrophils, contained early and recycling endosomes positive for NOX2. During phagocytosis, these endosomes were in close contact with the phagosome suggesting that they could contribute to the phagosomal gain in NOX2. The number of clusters increased during phagocytosis in PLB-985 WT cells but not in PLB-985 NCF1 GT cells that lack a functional oxidase due to the absence of p47phox (Wrona et al, 2017) These data suggest that the presence of p47phox and/or NADPH oxidase activity triggers a positive feedback with the recruitment of NOX2 positive vesicles

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT