This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in T and B 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 possible roles of Nox-derived ROS in non-phagocytic cells. 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 as well as in autoimmunity or immunodeficiencies. 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 and B cell receptor (BCR)-stimulated B cells. We showed that TCR stimulation induces three kinetically distinct ROS generation phases in vitro. Early hydrogen peroxide 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 2014, we have focused our efforts on roles of NADPH oxidase-derived redox signals in stimulated B lymphocytes. Although Nox2 is the prevalent oxidase detected in splenic B cells, we showed Duox1 is induced more than 7-fold following anti-IgM stimulation of BCRs when combined with 3-day IL-4 treatments. We developed novel methods for detecting ROS in the vicinity of stimulated BCRs by conjugating redox-sensitive fluorescent tags to F(ab) anti-IgM ligands. These probes detect lower ROS release from BCR-stimulated splenic B cells from either Nox2- or Duox1-deficient mice when compared with wild type B cells. In related experiments we showed that stimulated B cells from Duox1-deficient mice exhibited enhanced proliferation in vitro over 3-4 days, whereas stimulated Nox2-deficient B cells proliferated at rates similar to wild type cells. These findings raise interesting questions on possible roles of Duox1 in regulating B cell development and function in vivo that will be addressed in two Duox1 knockout mouse models.
|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