High-content screening assay for compounds that enhance or inhibit leukocyte superoxide production to identify drugs for treating immune-mediated diseases

Abstract

Abstract Reactive oxygen species (ROS) are produced by cells to play important roles in the effector and regulatory functions of immune cells. Our previous studies revealed that ROS act as negative regulators in IL-1β-mediated inflammation. Enhanced severity of inflammation in immune-mediated arthritis was noted when leukocyte NADPH oxidase (NOX2), which mediates the production of ROS, was defective. We hence hypothesized that drugs that modulate the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. Here, we devised a high-content image assay using the dye H2DCF-DA for identifying LOPAC 1280 library compounds that may facilitate or suppress superoxide production in differentiated HL-60 (dHL-60) cells. ROS levels were measured after the cells were treated with phorbol 12-myristate 13-acetate (PMA) or menadione which may induce ROS production from NOX2 or mitochondria. From a screening of 640 compounds in a 3-point interpolate titration in cell-based imaging analysis, we identified 74 drugs that may enhance the ROS production and 72 compounds that may inhibit ROS production by PMA-stimulated-dHL-60 cells. The drugs that we identified included the agonist/antagonist of neurotransmitters and their receptors, ion channel blockers, serotonin antagonists, cell stress modulators and cell cycle inhibitors. To further clarified our hypothesis, we treated NOX2-deficient mice with menadione, which tended to increase the oxidant stress in the joint tissue. The severity of the immune-mediated arthritic joint was significantly suppressed after the treatment. This novel screening method hence may be used to identify potential drugs for treating redox-related inflammatory diseases.