[ODP-135] Lipid A up-regulates caspase-11 expression via RIPK3 activation
Purpose: Caspase-11 is a receptor of LPS and participates in LPS-induced IL-1β production independently of Toll-like receptor 4. In this study, we investigated whether GSK’872, an inhibitor of RIPK3, suppressed lipid A-augmented caspase-11 expression and IL-1β release by J774.1 cells.
Method: The mouse macrophage-like J774.1 cells (3 × 106 cells/dish or 2 × 105 cells/well) cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2. After overnight, the cells were washed and treated with or without 100 ng/ml lipid A for 24 h. For inhibition assays, J774.1 cells were pretreated with 10 μM GSK'872 for 1 h prior to the addition of lipid A. The expression of caspase-11 was examined by Western blotting. Caspase-8 activation was analyzed by flow cytometry.
Results: (1) GSK'872 inhibited lipid A-augmented caspase-11 expression by J774.1 cells. (2) However, GSK'872 up-regulated lipid A-induced IL-1β release by the cells. (3) GSK'872 also up-regulated lipid A-induced caspase-8 activation by J774.1 cells. (4) GSK'872 down-regulated lipid A-induced IFN-β production.
Discussion: These results suggest that RIPK3 activation mediates lipid A-induced production of IFN-β by enhancing expression of caspase-11. However, inhibition of RIPK3 might augment lipid A-induced IL-1β release via promoting caspase-8 activation by J774.1 cells.
Method: The mouse macrophage-like J774.1 cells (3 × 106 cells/dish or 2 × 105 cells/well) cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2. After overnight, the cells were washed and treated with or without 100 ng/ml lipid A for 24 h. For inhibition assays, J774.1 cells were pretreated with 10 μM GSK'872 for 1 h prior to the addition of lipid A. The expression of caspase-11 was examined by Western blotting. Caspase-8 activation was analyzed by flow cytometry.
Results: (1) GSK'872 inhibited lipid A-augmented caspase-11 expression by J774.1 cells. (2) However, GSK'872 up-regulated lipid A-induced IL-1β release by the cells. (3) GSK'872 also up-regulated lipid A-induced caspase-8 activation by J774.1 cells. (4) GSK'872 down-regulated lipid A-induced IFN-β production.
Discussion: These results suggest that RIPK3 activation mediates lipid A-induced production of IFN-β by enhancing expression of caspase-11. However, inhibition of RIPK3 might augment lipid A-induced IL-1β release via promoting caspase-8 activation by J774.1 cells.