This program explores the roles of reactive oxygen species (ROS), such as H2O2 and superoxide anion, as specific regulators of signaling in the adaptive immune system. Receptor stimulation induces generation of ROS as signaling molecules, which control receptor function in several cell types. Oxidants such as H2O2 have been linked to lymphocyte activation, but the molecular mechanisms behind this observation are unclear. T cell receptor (TCR)-induced signaling is regulated by deliberate receptor-mediated ROS production, although the source(s) of ROS and their mechanism of action remain unknown. 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 family members produce lower levels of ROS that can act as signaling molecules. Our studies of the functions of Nox family members in the lymphocyte provide opportunities to establish distinct roles of deliberate ROS generation in adaptive immune responses to diverse pathogens and investigate possible links to autoimmunity or immunodeficiency. Our early studies showed that T cells exhibit two phases of H2O2 production upon TCR stimulation, and that the later phase involves a phagocytic Nox2/p47phox-based NADPH oxidase through TCR-Fas receptor transactivation. However, it remained unclear how TCR stimulation triggers early H2O2 generation and how this works as a second messenger in TCR signaling. We identified functional expression of calcium dependent, non-phagocytic NADPH oxidases, Duox1 and Duox2, in cultured and primary human CD4+ T cells. Duox1, not Duox2, is responsible for early TCR-stimulated ROS generation. Duox1 activation depends on TCR-induced activation of tyrosine kinase signaling pathways. Both calcium release from intracellular stores and possibly phosphorylation are necessary for Duox1-dependent H2O2 generation. Duox1-derived H2O2 positively regulates TCR signaling and cytokine production. Our previous studies showed that Nox2-deficient murine CD4+ T cells have a TH1-skewed cytokine profile due to enhanced ERK activation. Duox1 showed a very different role in TCR signaling. Knockdown of Duox1 in human CD4+ T cells selectively inhibits H2O2 generation, tyrosine phosphorylation of proximal signaling molecules and activation of ERK, suggesting a positive role in TCR signal transduction. Knockdown of Duox1 led to a decrease in cytokine production of human CD4+ T blasts without any obvious skewing to a TH1- or TH2-type profile. Proximal TCR signaling molecules are considered to be in a dynamic equilibrium between phosphorylated and dephosphorylated states. Our study supports a scheme of TCR signal amplification through a positive feedback loop involving Duox1-mediated H2O2 production. Upon TCR stimulation, the protein tyrosine phosphatase SHP-2 associates with proximal TCR signaling molecules and negatively regulates ZAP-70 through dephosphorylation of pY319. Concomitantly, TCR-dependent signaling leads to local production of H2O2 through Duox1 and oxidation of the active site cysteine of SHP-2. Duox1-mediated inactivation of SHP-2 thereby diminishes its negative regulatory activity and further enhances TCR signaling through the Lck-TCR-ZAP-70 pathway. In human CD4+ T cells, Duox1 knockdown also impaired TCR-stimulated sustained calcium influx, activation of NFAT, and cytokine production.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2010
Total Cost
$194,144
Indirect Cost
City
State
Country
Zip Code
Kang, Ju-Gyeong; Amar, Marcelo J; Remaley, Alan T et al. (2011) Zinc finger protein tristetraprolin interacts with CCL3 mRNA and regulates tissue inflammation. J Immunol 187:2696-701
Kwon, Jaeyul; Shatynski, Kristen E; Chen, Haiyan et al. (2010) The nonphagocytic NADPH oxidase Duox1 mediates a positive feedback loop during T cell receptor signaling. Sci Signal 3:ra59