Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-kappa B and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22(P)(hox), the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.