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 NOX-derived reactive oxygen species (ROS) as signaling molecules in lymphoid cells through studies on inherited defects or genetic manipulation of NOX/DUOX family NADPH oxidase components. These enzymes catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide or hydrogen peroxide. These studies are exploring redox signaling roles of NOX family members in the development of the adaptive immune system and in adaptive immune responses to diverse pathogens. Several NOX family oxidases (i.e., NOX2, DUOX1, NOX5) are detected in cells of the adaptive immune system (B cells, T cells and dendritic cells) and have been associated with auto-immune and inflammatory disease processes, such as arthritis, lupus, and inflammatory bowel disease. About 40% of chronic granulomatous disease patients with NOX2 deficiencies suffer from autoimmune complications and very early onset inflammatory bowel disease. Deficiencies in other NOX isozymes have been linked to inflammatory bowel disease (NOX1 and DUOX2), although it remains unclear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. Our work in 2022 remains focused on human RAC2 mutations associated with defects in lymphopoiesis and related immunodeficiencies, as well as neutrophil dysfunction. Previously, we described several RAC2 activating mutations in patients who presented with neutrophil defects and T-cell lymphopenia (PMID: 30723080; PMID: 31071452; PMID: 33188496). RAC2 is a member of Rho-family GTPases, abundant in neutrophils serving roles in NADPH oxidase activation and cytoskeleton dynamics. It regulates neutrophil superoxide production through direct p67phox and cytochrome b558 interactions and alters actin cytoskeleton dynamics affecting cell migration and membrane trafficking. Its expression in lymphoid cells also supports lymphopoiesis. Patients with dominant, activating mutations in RAC2 (E62K, N92T, Q61R) are associated with neutrophil dysfunction (excess ROS production, reduced cell migration), lymphopenia and immunodeficiency. We have now assembled an international cohort of >50 published and unpublished patients with germline RAC2 mutations to explore the spectrum of associated phenotypes in relation to RAC2 activation status investigated in several heterologous reconstituted expression systems. Our in-vitro analysis of RAC2 effector functions included NOX2-based superoxide production, regulation of PAK1-PBD binding, activation of AKT, protein stability, sub-cellular localization, and F-actin formation. We identified both active and inactive variants associated with lymphopenia and immunodeficiency. Most RAC2 mutations are located within and near Switch regions, which assume distinct conformational states in active (GTP-bound) or inactive (GDP-bound) forms of the protein, or contact sites for binding GTPase activating protein (GAP). Functional studies demonstrated most mutations increased RAC2 activity as shown by increased pAKT and PAK1 binding, whereas superoxide production was the least sensitive indicator of RAC2 activation, particularly with unstable mutants. Confocal microscopy revealed differences in plasma membrane localization, membrane ruffling and macropinosome formation across variants. The severity of phenotype and initial clinical presentation was correlated with protein stability, with increased stability leading to neonatal presentation and decreased stability of dominant mutations having later clinical onset. Patients with dominant, active RAC2 variants with high protein stability presented in the first days of life with severe combined immunodeficiency (SCID) and absent lymphocytes. Activating variants with lower protein stability presented as combined variable immunodeficiency (CID) with lymphopenia, detectable by newborn screening but clinically presenting later with recurrent sinopulmonary infections, reduced B-cells and hypogammaglobulinemia. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity. Thus, heterologous expression and analysis of RAC2 variant function has been useful for deciphering the genetic and molecular mechanistic basis for the common underlying clinical phenotype related to lymphopenia and immunodeficiency, and has circumvented the need for obtaining fresh blood from worldwide clinical collaborators with patients with suspected RAC2 functional defects.