Our investigations are focused on delineating immunomodulatory mechanisms of adaptive immune responses. We use the p47phox gene knockout mouse as the primary model for our ex vivo investigations. We continue to exploit this model to define cell intrinsic and induced cellular responses related to p47phoxs role as a structural protein of the phagocyte Nox-2 NADPH oxidase enzymatic complex. We also use multiple in vitro models including a novel ex vivo system we developed for differentiating Sca-1+Lin- hematopoietic progenitor cells into functional myeloid precursor, immature and mature dendritic cells. The interrelated objectives of our research focuses on distinguishing phagocyte Nox-2 NADPH oxidase p47phox derived reactive oxygen species (ROS) dependent and Nox-2 independent ROS regulation of antigen presenting cell (APC;dendritic cells, macrophages and B cells) and T cell function. Work in progress in our group over the past year can be defined by these interrelated objectives: (I) NADPH oxidase p47phox ROS dependent mediated survival, differentiation and function of cytotoxic T cells, and (II) Phagocyte NADPH oxidase p47phox-/- APC regulated T cell function. We previously reported that T cell receptor (TcR) stimulated T lymphocyte blasts generate NADPH oxidase dependent hydrogen peroxide (H2O2), and that cytokine secretion is skewed towards a T helper 1 phenotype in NADPH oxidase deficient T lymphocytes. We also reported that NADPH oxidase p47phox-/- CD8+ T cells have a previously unrecognized survival defect that is only partially corrected with exogenous pro-survival cytokines and restoration of intracellular H2O2. Ex vivo investigations resulted in our finding that NADPH oxidase p47phox-/- mice, without evidence of gross infection, develop reactive secondary lymphoid organs (SLOs) characterized by (1) LN hyperplasia with increased B cell and activated CD8+ cell division, (2) an increased accumulation of resting nave B and T lymphocytes, and (3) lower T:B cell ratios than wild type mice. Paradoxically, we also found that post-thymic CD8+ lymphocytes from p47phox-/- mouse LN and spleen undergo a rapid and profound post activation apoptosis in vitro. Using cytokine stimulation we discerned that the p47phox-/- lymphocyte apoptosis is associated in cultured p47phox-/- CD8+ lymphocytes with the induction of pro-apoptotic protein express ion, and the induction of the intrinsic death pathway. The NADPH oxidase enzyme complex is present in all professional phagocytes (macrophages, neutrophils, eosinophils) and also in other APC such as dendritic cells and B lymphocytes. Using tissue derived and ex vivo generated myeloid dendritic cells and macrophages we have begun to characterize p47phox-/- APC function, and T cell activation. Results from in vitro investigations using inactive bacteria, peptides and Toll-like receptor agonist to stimulate p47phox-/- APC reveal that p47phox-/- dendritic cells secrete more proinflammatory cytokines than normal cells following stimulation. We intend to further explore this enhancement by investigating immunological synapse interactions, and cytokine regulated pathways in p47phox-/- dendritic cells.
|Donaldson, M; Antignani, A; Milner, J et al. (2009) p47phox-deficient immune microenvironment signals dysregulate naive T-cell apoptosis. Cell Death Differ 16:125-38|