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 the development and function of the adaptive immune system 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. Several NOX family oxidases (i.e., NOX2, NOX4, NOX5, DUOX1) 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 including 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. In mice, deficiency of NOX4, a constitutive ROS generator, results in pro-inflammatory phenotypes and altered immune polarization in myeloid and lymphoid cell lineages. In 2023, we have completed a large multi-center study of the effects of RAC2 mutations associated with lymphopenia, immunodeficiencies and neutrophil dysfunction (under review). Mutations in RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, have been associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later onset atypical SCID (PMID: 30723080, 31071452, 33188496). We investigated a cohort of 54 patients with RAC2 mutations (23 previously reported) from 37 families. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n=5), infantile LAD-like disease (n=5), or atypical SCID (n=44). Disease severity correlated to RAC2 activity: Constitutively active RAS-like mutations caused neonatal SCID, dominant negative mutations displayed LAD-like disease, activating mutations developed atypical SCID. Significant T- and B-lymphopenia with low immunoglobulins are seen in most patients, while myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration and visible neutrophil macropinosomes. Among the 42 atypical SCID patients with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants were identified and characterized. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, PAK1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosome formation. Also, altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function. Various effector functions were differentially impacted depending on the locations of mutations. Most mutants produced elevated superoxide, whereas mutations unable to support superoxide formation were associated with bacterial infections. Thus, RAC2 mutations result in immune dysfunction causing a spectrum of disease ranging from early-onset severe combined to later onset combined immunodeficiencies depending on RAC2 activity. In another long-term project, we have studied NOX4 functions in cancer using lung, breast, liver, and pancreatic tumor cell lines and showed NOX4 is induced in a TGF-beta-dependent manner in tumors bearing TP53 hot spot mutations (PMID: 22728268; PMID: 28574838). An in-depth pan-cancer informatics analysis of primary human tumors of 23 cancer types in The Cancer Genome Atlas (TCGA) validated our in vitro observations demonstrating roles of NOX4 in promoting programs of cancer progression in tumors with TP53 mutations (PMID: 33557266). Thus, it appears that TP53 mutations switch NOX4 from acting as a protective oxidase predicting a favorable prognosis to one with deleterious effects on cancer progression and clinical outcome in late-stage cancers. In this last year, related follow-up cell culture experiments explored NOX4-dependent chemotactic and inflammatory signaling between macrophages and tumor cells as a model of the tumor microenvironment (TME). We found the secretome from p53-null H1299 lung epithelial cells stably expressing mutant p53 proteins (R248Q or R273H) promotes the migration and invasion of naive H1299, as well as chemotactic recruitment of THP-1 monocytes. These effects were diminished when the secreting tumor cells were transfected with dominant negative NOX4 (P437H). Immunoblot-based cytokine array analysis revealed tumor cell secretion of CCL5 was mutant p53 and NOX4-dependent, promoting autocrine and paracrine-mediated cell migration and invasion, whereas neutralization of CCL5 reduced autocrine-mediated H1299 cell mobility. We also showed through neutralization of CCL5 and TGF-beta released by M2-polarized macrophages that these signals participate in this TME crosstalk by promoting H1299 cell migration and invasion (https://doi.org/10.1016/j.freeradbiomed.2023.02.012). Current studies are examining the impact of NOX4 and NOX2 gene disruptions on the development and immunophenotype of pancreatic ductal adenocarcinoma using the 'KPC' mouse model created by pancreatic-targeted knock-in of oncogenic KRAS and TP53 mutations (KRAS-G12D; TP53-R172H). In KPC tumors, we detect 10-15-fold enhanced expression of NOX2 and NOX4 in distinct tumor cell types: NOX2 is detected primarily in tumor-infiltrating macrophages, whereas NOX4 accumulates in high TP53 expressing tumor cells and in fibroblasts encapsulating the tumor. Our work until now has indicated that NOX4 promotes tumor growth and pro-metastatic programs in mutant TP53 tumors based on a Pan-cancer TCGA analysis, whereas NOX2 is Th1-induced and likely exerts anti-tumor effects. Several observations indicate that NOX4 deficiency (knockout) in mice enhances susceptibility to inflammatory processes (https://doi.org/10.1016/j.redox.2023.102611) and promotes a bias toward Th1 and M1-based immunity, suggesting this constitutively active ROS generator supports anti-inflammatory processes and cancer progression. Ongoing studies using the 'KPC' mouse model are assessing whether NOX4 in tumor associated fibroblasts and tumor cells can affect 1) access of immune cells to the tumor microenvironment, 2) immune polarization of lymphoid and myeloid cells detected within the tumor microenvironment, or 3) escape of tumor cells from primary tumor sites (metastasis).