NOX family NADPH oxidases: roles in innate immunity and inflammatory disease

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This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. Current research focuses on non-phagocytic NADPH oxidases (NOX1, NOX4, DUOX1, DUOX2) expressed primarily in epithelial cells, as well as NOX2 in hematopoietic cells. Deliberate reactive oxygen species (ROS) production by the epithelial enzymes relays redox signals in responses to cytokines, chemokines, growth factors, hormones, and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In addition to serving a direct microbicidal role, ROS generated by several NOX enzymes also participate in cell migration, proliferation, differentiation, senescence, apoptosis, tumor invasiveness and metastasis. In 2020, we explored functions of several NOX family NADPH oxidase components in two areas of: 1) studies on the distinct roles of NOX4 and NOX2 in the tumor microenvironment using mice with conditional expression of mutant p53 and Kras and established tumor and macrophage-like cell lines, 2) studies on genetic variants of NOX components linked to innate antimicrobial immunodeficiencies and inflammatory disease. Our previous findings showed NOX4 is induced by mutant forms of p53 in a TGF-beta and SMAD3-dependent manner in many epithelial tumors bearing TP53 hot spot mutations. Our observations support NOX4 as a critical player in tumor cell migration, invasiveness, and angiogenesis. We focused our studies this year on the function of NOX enzymes in cancer progression. We are dissecting roles for NOX4 and NOX2 in inflammatory signaling in the tumor microenvironment in cell culture models and in mice that develop pancreatic ductal adenocarcinoma (PDAC) as a consequence of conditional knock-in of TP53 and KRAS mutations (KPC mouse model). The cell culture studies demonstrated that common tumor-associated p53 mutants (R248Q and R273H) induce a pro-invasive secretome mediated by NOX4-dependent release of CCL2 and CCL5 chemokines that not only stimulate tumor cell migration, but also recruit macrophages. Macrophages, in turn, cross-talk by secreting CCL5 and TGF-beta to promote tumor cell mobilization, which is inhibited by neutralizing antibodies against these factors. Moreover, we showed CCL5-binding receptors CCR1, CCR3, CCR4, and CCR5 are upregulated in tumor cells treated with CCL5, increasing tumor cell sensitivity to the microenvironment. We are also exploring potential roles of NOX4 in hypoxia-induced tumor angiogenesis. We observed increased VEGF expression in cells expressing mutant p53 under hypoxic conditions. Furthermore, analysis of the NOX4 promoter revealed overlapping p53 and HIF-1a response elements. These observations suggest novel NOX-related inflammatory pathways with potential for therapeutic intervention. In related whole animal studies, we are investigating whether genetic deficiencies of NOX4 or NOX2 influence programs that drive cancer progression or affect overall survival in the KPC mouse model of PDAC. High NOX4 expression in pancreatic ductal epithelial cells transformed by mutant p53 appear to promote tumor cell migration and may affect metastasis beyond primary tumor sites. However, the high NOX4 observed in the fibroblast layer encapsulating the adenocarcinoma may prevent tumor cell escape or inhibit the access or recruitment of pro-inflammatory (antitumor) cells to the tumor microenvironment. In contrast, high NOX2 expression is observed within infiltrating inflammatory cells surrounding the adenocarcinoma. Thus, ongoing studies will determine whether the increased expression of two oxidases in PDAC influence the immune phenotype of tumor tissues or support metastatic disease progression. In collaboration with Drs. Roshini and Holland, we explored functional activity of a CYBA gene variant (p.S98L) detected in pediatric patient with recurrent pneumonias. Her infections with oxidant-sensitive pathogens suggested a chronic granulomatous disease (CGD) diagnosis; however, her dihydrorhodamine-123 (DHR)-based flow cytometric oxidase assays in response to phorbol ester indicated low normal respiratory burst activity. We characterized this variant in transfected NOX2 reconstituted cell models and detected 25-30% diminished NADPH oxidase activity along with enhanced proteolytic susceptibility in comparison with wild-type, consistent with structural modeling predicting deleterious functional effects of this rare variant (CADD score=20.9). The results highlight limitations of preliminary DHR-based flow cytometric assays in assessing oxidase defects and the utility of functional assays in oxidase reconstituted cell models in detecting oxidase deficiencies in atypical CGD-like patients. In collaboration with other LCIM investigators (FPS, Lionakis; IPS, Hsu and Holland), we are exploring roles of NOX enzymes in innate antifungal host defenses mechanisms. We identified several low-activity airway oxidase variants in a cohort of patients showing enhanced susceptibility to pulmonary and disseminated fungal infections; at least three rare DUOX1 missense variants predicted to be highly deleterious (CADD scores > 20) were produced in heterologous expression systems and exhibited diminished stability and a complete loss of oxidase function. In other work, we are investigating the mechanistic basis for enhanced susceptibility to Aspergillosis in lymphoma patients on chemotherapeutic agents that appear to inhibit phagocyte oxidase (NOX2) activity. Other related work is exploring the mode of action of oxidase inhibitors that suppress inflammatory myeloid cell activation and excess ROS production during acute respiratory distress syndromes (ARDS) following pulmonary viral infections with influenza A or SARS-CoV-2. Finally, collaborative efforts with the CPS (Drs. Gallin and Zarember) and NCATS aimed at screening and identification of novel NOX inhibitors are developing scaled-down systems for high-throughput cell-free and whole cell assays of several NOX isozymes. Such inhibitors offer therapeutic potential as lead compounds for treating a variety of inflammatory disease processes, including ARDS, traumatic brain injury, atherogenesis, and pathways promoting cancer progression.