Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling

Yen, Wei-Chen; Wu, Yi-Hsuan; Wu, Chih-Ching; Lin, Hsin-Ru; Stern, Arnold; Chen, Shih-Hsiang; Shu, Jwu-Ching; Tsun-Yee Chiu, Daniel

Staphylococcus aureus and Escherichia coli OP50 were incubated in Luria-Bertani (LB) media at 37°C with shaking one day before the experiment. The density of bacteria was measured at 670 nm in a DU 800 spectrophotometer after washing three times with PBS. LPS-primed THP-1 cells and PBMCs from subjects were infected with bacteria at an MOI (multiplicity of infection) of 10 and were incubated for 2 h at 37°C in a humidified atmosphere of 5% CO 2 . After being infected, the cells were washed three times with HBSS and were incubated with gentamicin (200 μg/ml) containing RPMI 1640 for 30 min to kill extracellular bacteria. After replacing the medium with fresh medium, the cells were incubated with 20 μM nigericin for 30 min to induce inflammasome activation. The cells were washed once by HBSS, followed by lysis with 1% Triton X-100. The cell lysate was serially diluted and spread onto The correlation of G6PD activity and IL-1β secretion. THP-1 cells were transfected with G6PD siRNA (0, 3.1, 6.3, 12.5, 25 or 50 nM). PMA-differentiated THP-1 were treated with LPS and nigericin, cell lysate and supernatant were collected for G6PD activity and IL-1β analysis. These data are representative of three independent experiments (n = 3, p < 0.05).