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. Our data suggest that the different ROS generation phases require receptor-receptor transactivation processes involving different activation mechanisms and locations. In terms of cytokine production, Nox2-derived late-phase ROS inhibit Th1 and augment Th2 cytokine production, whereas early-phase ROS from Duox1 augment both Th1 and Th2 cytokine production. We have developed complete and lymphoid cell-targeted (conditional) Duox1-deficient mouse models in order to examine our hypothesis on the role of Duox1 as a positive regulator of TCR function within whole animals. In 2012, we identified selective inhibition of TCR-induced STAT5 phosphorylation as a potential mechanism for skewed T helper differentiation in Nox2KO mice. Also we identified unique roles of another Nox isoform Nox4 in human and murine CD4+ T cells. Using CD4+ T cell from Nox4-deficient mutant mice, we found that Nox4 suppresses T cell receptor stimulation induced signaling. We observed that knockdown of Nox4 in human CD4+ T cell line produced the same effects. Our results suggest that Nox2, Nox4, and Duox1 produce ROS as signaling molecules to regulate different signaling pathways in TCR-stimulated CD4+ T cells. The studies of animal models in which these isoforms are genetically manipulated are critical in understanding immune regulatory roles of three distinct oxidant-generating systems in host defense.