This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in B and T lymphocytes through genetic manipulation of the Nox/Duox family of NADPH oxidases. These enzymes catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide and hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase containing gp91phox (a.k.a., Nox2). Although originally understood as an anti-bacterial mechanism deployed by phagocytes, our research has revealed that ROS intentionally generated by several Nox family members play specific signaling roles in B cell receptor (BCR)-stimulated B cells and T cell receptor (TCR)-stimulated T cells. Our studies in lymphocytes are exploring roles of Nox family members in adaptive immune responses to diverse pathogens as well as in autoimmunity or immunodeficiencies. In 2016, we completed work characterizing NADPH oxidase-derived redox signals in stimulated B lymphocytes in studies that have uncovered distinct roles for two oxidases: Nox2 and Duox1 (manuscript submitted for publication). We confirmed Nox2 is the prevalent oxidase detected in human and mouse B cells and showed that it is the major source of extracellular hydrogen peroxide detected in the vicinity of the BCR in freshly isolated murine CD19+ splenic B cells. In contrast, we found that 3-day treatment of splenic B cells with interleukin-4 (IL-4) in combination with anti-IgM caused enhanced Duox1 expression. Hydrogen peroxide release from Duox1-/- splenic B cells was greatly reduced in comparison to that detected from wild type and Nox2-/- cells at 3-days pos-ttreatment. In contrast, assays of intracellular superoxide detected less production in Nox2 -/- cells in comparison with wild type or Duox1 -/- cells. Thus, both oxidases show late-phase responses to IL-4/BCR co-stimulation but produce different types of ROS (hydrogen peroxide versus superoxide) within distinct cellular compartments. Co-stimulation of Nox2- or Duox1-deficient cells appears to have distinct functional consequences at 3-4 days: Nox2-/- cells show enhanced apoptosis and diminished IgM, IgG1 and IgG2a production relative to wild type or Duox1-/- cells. Duox1-/- cells exhibit enhanced proliferation, which was correlated with enhanced expression of BCR signaling intermediates, BCAP, Akt and RGS16 signaling. Therefore, Duox1 and Nox2 appear to regulate distinct functions in primary CD19+ B cell responses under the influence of IL-4. The effects of Duox1 deficiency on stimulated cell proliferation were mimicked by treatment of wild type cells with the hydrogen peroxide scavenger, catalase, suggesting ex vivo redox manipulations could have clinical applications. Future work will examine consequences of immunization and infection in Duox1 -/- and Nox2-/- mice, versus wild type, to confirm the importance of both NADPH oxidases as immune regulators in whole animals.
| 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 |
