Fig. 4

Bone particles promote inflammation via NLRP3 and IκB-ζ activity. a Primary chondrocytes were cultured with BP for 24 h and Nlrp3 gene expression was measured (***P = 0.000 9). Bars represent mean ± SEM from n = 4 independent experiments. b Immunoblotting was performed for Nlrp3 protein using chondrocytes lysates under BP treatment time course, with actin loading used as control. The representative image is shown. c Medial and lateral knee articular cartilage was isolated from patients undergoing TKA with medial compartment OA. Gene expression analysis was performed for NLRP3. Bars represent mean ± S.D. for n = 6 samples. d, e Cells were treated with IL-1β or BP for 24 h and IL-1β gene expression was measured. Bars are mean ± SD from three separate experiments. (*P = 0.024 8). f Cells were retrovirally transduced with GFP or IKK2ca, then treated with bone particles for 24 h. Supernatant was collected and ELISA was performed for IL-1β levels (GFP vs IKK2ca + BP **P = 0.001 8, n = 4 replicates). g, h Chondrocytes isolated from WT or Nlrp3-deficient mice and treated with BP for 24 h. Il-6 and Mmp13 gene expression measured by qPCR (IL-6: WT + BP vs Nlrp3^−/− + BP ****P < 0.000 1, Mmp13: WT + BP vs Nlrp3^−/− + BP ^****P < 0.000 1). Bars are mean ± SD for n = 4 replicates. i Primary chondrocytes were treated with BP for 24 h. Nfkbiz gene expression was measured by qPCR (*P = 0.018 7). Bars are mean ± SEM for n = 4 independent experiments. j Chondrocytes were exposed to IL-1β and/or bone particles. Lysates were collected and Immunoblotting was performed for IκB-ζ. Representative immunoblot is shown. k–n Nfkbiz flox chondrocytes were transduced with adeno-GFP (WT) or adeno-cre (Nfkbiz^−/−). Cells were then treated with BP ± IL-1β. Gene expression of Il-6, Mmp13, Nlrp3 and Rankl was measured by qPCR (IL-6: GFP + BP vs Nfkbiz^−/− + BP **P = 0.002 9, GFP⁺ BP + IL-1β vs Nfkbiz^−/− + BP + IL-1β ****P < 0.000 1. Mmp13: GFP + BP vs Nfkbiz^−/− + BP ****P < 0.000 1, GFP + BP + IL-1β vs Nfkbiz^−/− + BP + IL-1β ****P < 0.000 1. Nlrp3: GFP + BP vs Nfkbiz^−/− + BP ****P < 0.000 1, GFP + BP + IL-1β vs Nfkbiz^−/− + BP⁺ IL-1β ****P < 0.000 1. Rankl: GFP + BP vs Nfkbiz^−/− + BP ^****P < 0.000 1, GFP + BP + IL-1β vs Nfkbiz^−/− + BP + IL-1β ****P < 0.000 1). Bars represent mean ± S.D. from one representative experiment out of three