Role of Reactive Oxygen Species in Lymphocyte Development and Function

National Institute Of Allergy And Infectious Diseases

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Abstract

This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in B and T lymphocytes through genetic manipulation of NOX/DUOX family NADPH oxidases. These enzymes catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide or 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., CYBB or NOX2). Although originally understood as an oxidant-dependent microbial killing mechanism deployed by phagocytes, our research revealed that ROS intentionally generated by other NOX homologues 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 immunodeficiencies and autoimmunity. Several NOX family oxidases have been associated with auto-immune inflammatory disease-like processes, such as arthritis, lupus and inflammatory bowel disease. About 40-50% of chronic granulomatous disease patients with NOX2 deficiencies suffer from autoimmune complications and very early onset inflammatory bowel disease (VEOIBD). Deficiencies in several other NOX isozymes have been linked to inflammatory bowel disease, although it is remains clear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. In 2019, we completed studies on the involvement of Duox1 in B lymphocyte responses to IL-4 and B cell receptor stimulation. We showed that Duox1 is induced in murine B lymphocytes treated with IL-4 in vitro and that co-stimulation through B cell receptors (BCR) generates a ROS signal attributable to Duox1. Interestingly, stimulated Duox1-deficient B cells proliferate in vitro more rapidly than wild type or NOX2-deficient B cells, which was correlated with enhanced expression of several BCR signaling intermediates (Akt, BCAP and RGS16). Furthermore, we demonstrated that the hydrogen peroxide scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of wild type CD19+ B cells in vitro. In contrast, NOX2-deficient B cells showed enhanced apoptosis and diminished IgM, IgG1 and IgG2a production relative to wild type or DUOX1-/- cells. These observations were correlated with whole animal immunization findings: the Duox1 knockout mice immunized with T cell-dependent (TD) or -independent (TI) antigens produce normal serum Ig levels, but immunization with the TI antigen, nitrophenyl-lipopolysaccharide (NP-LPS), resulted in more numerous and enlarged splenic reactive (Ki67 and peanut antigen positive) germinal centers in Duox1 knockout mice when compared with wild type or NOX2-deficient mice. Our results suggest Duox1 may have a role in limiting early proliferative responses to BCR stimulation, particularly with pathogens that avoid T cell recognition. Thus, the ROS generated by distinct NOX isozymes have different effects on the fate and function of stimulated lymphoid cell populations. Future work should explore roles for Duox1 in generation of long-lived plasma cells or in infection models where Th2-based immunity prevails.