Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4 + T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)-stimulated production of hydrogen peroxide (H 2 O 2 ) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of ζ chain-associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca 2+ ), and activation of extracellular signal-regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain-containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3ζ chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H 2 O 2 that acts in a positive feedback loop to enhance and sustain further TCR signaling.

Aims: Down Syndrome (DS), a genetic disease caused by a triplication of chromosome 21, is characterized by increased markers of oxidative stress. In addition to cognitive defects, patients with DS also display hematologic disorders and increased incidence of infections and leukemia. Using the Ts65Dn mouse model of DS, the goal of this study was to examine hematopoietic stem and lymphoid progenitor cell function in DS. Results: Analysis of hematopoietic progenitor populations showed that Ts65Dn mice possessed fewer functional hematopoietic stem cells and a significantly decreased percentage of bone marrow lymphoid progenitors. Increased reactive oxygen species and markers of oxidative stress were detected in hematopoietic stem cell populations and were associated with a loss of quiescence. Bone marrow progenitor populations expressed diminished levels of the IL-7Ra chain, which was associated with decreased proliferation and increased apoptosis. Modulating oxidative stress in vitro suggested that oxidative stress selectively leads to decreased IL-7Ra expression, and inhibits the survival of IL-7Ra-expressing hematopoietic progenitors, potentially linking increased reactive oxygen species and immunopathology. Innovation: The study results identify a link between oxidative stress and diminished IL-7Ra expression and function. Further, the data suggest that this decrease in IL-7Ra is associated with defective hematopoietic development in Down Syndrome. Conclusion: The data suggest that hematopoietic stem and lymphoid progenitor cell defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signaling may alter hematologic development in Ts65Dn mice. Antioxid. Redox Signal. 15, 2083Signal. 15, -2094

Summary Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contrast to this innate immune deficit, CGD patients and animal models display predisposition to autoimmune disease and enhanced response to H. pylori and influenza infection. These data imply an altered, perhaps augmented, adaptive immune response in CGD. Previous data demonstrated functional NOX2 expression in T cells, and the goal was to determine if NOX2-deficient T cells are inherently altered in their responses. Activation of purified naive CD4+ T cells from NOX2-deficient mice led to augmented IFN-γ and diminished IL-4 production and an increased ratio of expression of the TH1-specific transcription factor T-bet versus the TH2-specfic transcription factor GATA-3, consistent with a TH1 skewing of naïve T cells. Selective inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Exposure to anti-oxidants inhibited, while pro-oxidants augmented TH2 cytokine secretion and STAT5 phosphorylation, supporting the redox dependence of these signaling changes. These data suggest that TCR-induced ROS generation from NOX2 activation can regulate the adaptive immune response in a T cell inherent fashion, and propose a possible role for redox signaling in T helper differentiation.

SummaryIn addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses. Reflecting this deficient thymic function, there were fewer naive T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation, suggesting a senescent phenotype. In contrast, B-cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to lipopolysaccharide. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor interleukin-7Ra, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased interleukin-7Ra expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signalling may alter lymphocyte development in Ts65Dn mice.

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In addition to this innate immune dysfunction, CGD patients or animal models display an altered, perhaps augmented, adaptive immune response. It has been previously shown that T cells express the phagocyte oxidase and also been proposed that NOX2 expression alters APC function. The goal of this study was to determine if the differences in adaptive responses are inherent to T cells, or if other cells create an environment that promotes T helper polarization. Anti-CD3 activation of purified naive T cells from NOX2-deficient mice led to augmented IFNγ and IL-17 secretion. NOX2-deficient antigen presenting cells (APC) showed selective increases in GM-CSF, IL-6, TNF and MCP-1 induced by LPS challenge in vitro. APC function was measured using OVA-specific OT-II T cells, and oxidase deficient APC induced more IFNγ and IL-17 upon antigen stimulation. Adoptive transfer of OT-II T cells into wild type or NOX2(-/-) hosts followed by immunization with OVA in the presence of either CFA or Alum revealed altered cytokine profiles of T cells after restimulation in vitro. These results suggest roles for the phagocyte NADPH oxidase in shaping the adaptive and innate immune responses in both a T cell and APC dependent fashion.

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In seeming contradiction, immune responses to agents such as H. pylori and influenza imply an altered or augmented adaptive immune response in CGD. We have shown NOX2 expression in T cells, and oxidase deficient T cells demonstrate Th1 skewed cytokine secretion upon in vitro stimulation. The goal of this study was to determine if these differences are T cell inherent, or if other cells promote T helper polarization. Activation of purified naive CD4+ T cells from NOX2-deficient mice led to augmented IFN-γ and diminished IL-4 production, consistent with a Th1 skewing of naïve T cells. This was supported by enhanced expression of the transcription factor T-bet and lower levels of GATA-3. Similar to NOX2-deficient cells, Th2-biased BALB/c T cells treated with the antioxidant N-acetyl cysteine produced increased IFN-γ and decreased IL-4 as compared to untreated controls. Inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Stimulation of TCR-transgenic, OT-II T cells in vitro with NOX2-deficient splenic APC produced similar amounts of IL-4, IL-17, and IFN-γ as compared to those incubated with wild type APC, supporting the conclusion that NOX2 can regulate the adaptive immune response in a T cell inherent fashion.