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 deficiencies 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 adaptive immune responses to diverse pathogens, as well as in the development of the adaptive immune system. 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 (VEOIBD). Deficiencies in several other NOX isozymes have been linked to inflammatory bowel disease, although it remains unclear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. Our current efforts are focused on RAC2 mutations associated with defects in lymphopoiesis, as well as myeloid cell dysfunction. In 2021, we have continued our investigations on the effects of a variety of human RAC2 mutations linked to defects in lymphopoiesis and neutrophil dysfunction. RAC2 expression in humans is restricted to myeloid and lymphoid cell lineages. RAC2 has long been recognized as a critical regulator of neutrophil NADPH oxidase activation and related microbicidal functions and has distinct roles in regulation of the actin-based cytoskeleton affecting neutrophil migration. Its role in lymphopoiesis was later recognized when patients with null or inactivating mutations in RAC2 were first reported. Thus far, we have described in detail three distinct heterozygous RAC2 variants (p.E62K, p.N92T, and p.Q61R) detected in patients with lymphopenia and neutrophil dysfunction that all appear to act through dominant activating pathways. Other inactivating and null RAC2 variants were also identified that are associated with a range of lymphoid and myeloid disease severities. Whether activating or inactivating variants, they are all manifested in the similar disease phenotypes, reflecting the importance of RAC2 cycling between active and inactive states to support the dynamic cytoskeletal changes needed for cell migration and membrane trafficking. We developed several independent assays of RAC2 activation status in heterologous cell culture expression systems: 1) detection of superoxide release in NOX2 reconstituted cells, 2) detection of AKT phosphorylation by Western blotting, 3) detection of RAC-p21 activated kinase (PAK1) binding, and 4) confocal microscopy-based assays of membrane ruffling and macropinosome formation due the enhanced formation of F-actin filaments. These transfected cell approaches recapitulate the effects of these RAC2 activating variants observed in patients neutrophils, thus circumventing the need for obtaining fresh blood from worldwide clinical collaborators whose patients have presented with suspected RAC2-related defects. The activating mutations show varying degrees of accumulation of RAC2 with F-actin along membrane ruffles and nascent macropinosomes, whereas the inactive variants show distinct subcellular distribution patterns. By comparing the yields and stabilities of these recombinant RAC2 proteins, we could correlate RAC2 protein expression levels to disease severity (i.e., SCID, CVID, CID), as well as inheritance patterns (dominant vs recessive). All the non-synonymous RAC2 variants characterized to date have been located on the same face of the 3-dimensional structure of RAC2 at the sites for interaction with its effectors between Switch1 and Switch2 regions. These studies have expanded our understanding of the cellular and molecular basis of RAC2-related immunodeficiencies.