Role of Reactive Oxygen Species in Lymphocyte Development and Function

Leto, Thomas

Abstract

This program has explored the role of reactive oxygen species (ROS) as specific signaling molecules in lymphocytes through genetic manipulation of the Nox/Duox family of NADPH oxidases. These enzymes are membrane flavocytochromes that catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide and/or hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase (Nox) containing gp91phox (Nox2). Recent discovery of multiple homologues of gp91phox (Nox1, Nox3-5, Duox1, Duox2) has opened studies on the roles of Nox-derived ROS in non-phagocytic cells. We have studied T lymphocytes as a model system because of their well-established signaling pathways and their critical roles in human health and disease. Our studies of the functions of Nox family members in lymphocytes provide opportunities to establish distinct roles of deliberate ROS generation in adaptive immune responses to diverse pathogens and their roles in autoimmunity or immunodeficiency. Although originally understood as an anti-bacterial mechanism employed by phagocytes, our research revealed that ROS intentionally generated by several NADPH oxidase family members play specific signaling roles in T cell receptor (TCR)-stimulated T cells. We showed that TCR stimulation induces endogenously three kinetically distinct ROS generation phases in vitro. Early H2O2 generation comes from Duox1, activated downstream of inositol 1,4,5 triphosphate receptor 1;one of the later responses comes from Nox2, activated downstream of the Fas receptor. Also we identified unique roles of another Nox isoform Nox4 in human and murine CD4+ T cells. Using CD4+ T cells from Nox4-deficient mutant mice, we found that Nox4 suppresses T cell receptor stimulation induced signaling. In 2013, we established the method for florescence-based live imaging of TCR-induced ROS in primary CD4+ T cells and a T cell tumor line. CD4+ T cells from Nox2 KO mice showed less ROS generation compared with WT CD4+ T cells in the live imaging study. And also we found that Duox1-derived ROS are affecting the proliferation of splenic B cells in response to various stimuli in vitro. Splenic B cells from Duox1-KO mice showed less ROS generation and enhanced proliferation in the condition of B cell receptor stimulation with cytokines in vitro. Ongoing studies of Duox1KO animal models are critical in understanding immune regulatory roles of ROS generation in B lymphocyte function in vivo.