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

点击翻译按钮获取中文摘要

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. Reactive oxygen species (ROS) production by NOX 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 direct microbicidal roles, NOX-derived ROS regulate cell migration, proliferation, differentiation, senescence, apoptosis, tumor invasiveness and metastasis. Excess NOX-derived ROS can also contribute to host tissue damage under conditions of acute or chronic inflammatory disease. In 2023, we have explored functions of several NOX family NADPH oxidase components in three areas: 1) studies on genetic variants of DUOX1 and DUOXA1 associated with defects in mucosal epithelial barrier and innate antimicrobial defense functions affecting susceptibility to infectious and inflammatory disease, 2) studies on roles of NADPH oxidases (NOX4 and NOX1) in cancer progression related to cell migration and invasiveness 3) identification and characterization of novel NOX inhibitors with therapeutic potential for treating inflammatory and metastatic disease. As whole exome sequencing (WES) data has become widely available from patients of our clinical collaborators, we have explored functional consequences of NADPH oxidase defects in mucosal innate immunity in these patients using several heterologous NOX and DUOX expression systems to recapitulate these defects in vitro. Earlier, we proposed a functional partnership of DUOX1 with lactoperoxidase in airway innate antimicrobial defense and showed mature, ciliated human bronchial cells release sufficient DUOX1-derived hydrogen peroxide to support lactoperoxidase-based killing of several oxidant-sensitive airway pathogens. In collaborative studies with investigators in the IPS/LCIM (A.P. Hsu, S.M. Holland), we characterized several DUOX1 and DUOXA1 loss-of-function variants linked to pulmonary and disseminated coccidioidomycosis, a.k.a., Valley Fever (https://doi.org/10.1172/jci.insight.159491). Several DUOX1 or DUOXA1 variant proteins showed partial or complete loss of hydrogen peroxide generating activity; some DUOX1 variants were rapidly degraded and failed to support stabilization of its subunit partner, DUOXA1, required for transit of the functional heterodimeric oxidase complex to the plasma membrane. Other patients with disseminated infections had more common loss-of-function variants in CLEC7A (encoding the fungal recognition receptor, DECTIN-1) and downstream PLCG2. PBMCs stimulated with the fungal cell wall component beta-glucan had diminished TNF-alpha release compared to healthy controls. Reconstituted cell model experiments showed that beta-glucan can trigger a Dectin1->PLCG2->DUOX1/DUOXA1->hydrogen peroxide sensing and signaling axis that is defective in a majority of the patients with disseminated coccidioidomycosis. Ongoing studies are exploring the cellular and molecular mechanistic basis for the failure to contain pulmonary fungal infections and characterizing other rare DUOX1/DUOXA1 variants seen in these patients. Our interests in NOX4 function in cancer originated with work in various epithelial tumor cell lines showing that NOX4 is induced in a TGF-beta-dependent manner in tumors bearing TP53 hot spot mutations (PMID: 22728268; PMID: 28574838). 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 (https://doi.org/10.1016/j.freeradbiomed.2023.02.012)- see report ZIA-AI-001060-16 for details. In other work with hepatoma lines, HepG2 and SNU475 cells, we showed that peroxiredoxin 6 (PRDX6), a regulator of NOX1 activity, also promotes programs of tumor cell EMT, cell migration and invasiveness (https://doi.org/10.3390/antiox12061153). Here, analysis of primary tumor databases in TCGA showed that higher expression of either NOX1 or PRDX6 predicts poorer survival in patients with hepatocellular carcinomas. Our findings provide further insight into NOX1- and NOX4-based communication in the tumor microenvironment and their potential as therapeutic targets to mitigate metastatic disease progression. In our studies aimed at characterizing candidate NOX inhibitor analogues modeled from GSK2795039, we used recombinant NOX1, NOX2, NOX3, NOX4, and NOX5 reconstituted cell models to demonstrate improved specificity of a new compound, NCATS-SM7270, for NOX2 activity (https://doi.org/10.1016/j.redox.2023.102611). In vivo experiments examining cell death in response to mild traumatic brain injury in mice showed that NOX2 deficiency protects against traumatic brain injury, whereas NOX4 deficient mice sustain a higher degree of cortical damage. Trans-cranial treatment with NCATS-SM7270 after injury prevented the NOX2-associated cell death observed in both wild-type and NOX4 deficient mice, whereas NOX2-deficient mice showed no further protection from traumatic brain injury with NCAT-SM7270 treatment. Ongoing work is characterizing related inhibitory analogues that demonstrate improved in vivo pharmacodynamics, stability, and accessibility for protecting against traumatic brain injury.