Role of Reactive Oxygen Species in Lymphocyte Development and Function

Leto, Thomas

Abstract

This program explores the role of reactive oxygen species (ROS) as specific signaling molecules in the adaptive immune system 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. In non-phagocytic cells, Nox enzymes produce lower levels of ROS that can act as signaling molecules. We have studied T lymphocytes as a model system because of their well-established signaling function 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 TCR-stimulated T cells. We showed that TCR stimulation induces 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. In 2011, 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. These studies will allow investigations of the impact of Duox1-based signaling on immunodeficiency and autoimmune disease. In other studies, we found that TCR-stimulated CD4+ T cells from Nox4-deficient mice induced much greater amounts of IL-17 secretion compared with cells from wild-type mice. CD4+ T cells were isolated from wild type and Nox4-deficient mice by cell marker-based negative selection using magnetic beads, and CD4+ T cell subpopulations were sorted by flow cytometry. Sorted T cells were stimulated on anti-CD3/anti-CD28 antibodies-coated plates in various conditions and their cytokine production was examined. These studies identified a novel role for Nox4-derived ROS in differentiation of T helper subsets. The data suggest that ROS generation from a NADPH oxidase homologue Nox4 play regulatory roles in T cell differentiation and that Nox4-derived ROS inhibit differentiation of the inflammatory TH-17 cell lineage. Our studies suggest there are three separate signaling processes based on ROS generated from different Nox isoforms, producing different biological outcomes in TCR-stimulated T cells. The response of the immune system is multifaceted and studies in animal models are critical in understanding roles of these distinct oxidant-generating systems in host defense, adaptive immunity and related inflammatory immune processes.