Study on the nuclear translocation mechanism in the inhibition of nuclear factor-ΚB activation in bacterial lipoprotein-tolerant THP-1 cells

Abstract

To approach the nuclear factor-ΚB (NF-ΚB) nuclear translocation mechanism in bacterial lipoprotein (BLP) tolerance. Human monocytic THP-1 cells were first pretreated with 10, 100, 1 000 ng/ml BLP for 20 hours to induce BLP tolerance. Then THP-1 cells without BLP pretreatment (control group) or with BLP pretreatment (tolerance group) were stimulated with 0, 10, 100, 1 000 ng/ml BLP again for 6 hours. The tumor necrosis factor-α (TNF-α) content in culture medium was measured by enzyme linked immunosorbent assay (ELISA) in order to determine the most suitable BLP pretreatment and stimulation concentration. Western blotting was used to detect the protein level, nuclear translocation and phosphorylation of NF-ΚB p50 and p65 in the cells of control and tolerance groups treated with respective conditions for 0, 0.5, 1, 2 and 6 hours. In control group BLP stimulation (10, 100, 1 000 ng/ml) could induce THP-1 activation and TNF-α production (pg/ml: 184.86±32.51, 3 215.88±167.09, 6 042.96±245.37) in a dose-dependent manner. In tolerance group, 100 ng/ml BLP pretreatment resulted in almost complete inhibition of TNF-α production as induced by 101 000 ng/ml BLP stimulation. Therefore, 100 ng/ml BLP pretreatment and 1 000 ng/ml stimulation were selected for following cell treatment. Western blotting analysis showed that there was an increase of p50 protein level in BLP-tolerant cells comparing with control group (0 hour: 542.9±15.6 vs. 272.8±13.2, 0.5 hour: 558.0±16.9 vs. 236.4±11.8, 1 hour: 524.7±17.5 vs. 211.6±9.8, 2 hours: 584.9±15.6 vs. 222.4±12.3, all P<0.01), whereas the p65 protein level was similar between the two groups. BLP stimulation also induced the nuclear translocation of p50 and p65 in control group (1-hour p50: 344.2±13.6 vs. 79.0±5.2, p65: 78.4±4.5 vs. 0, both P<0.05), but not in tolerance group. In addition, the phosphorylation of p65 at serine 536 was induced after BLP stimulation in control THP-1 cells (0.5 hour: 0.67±0.08 vs. 0.04±0.01, 1 hour: 0.71±0.11 vs. 0.04±0.01, both P<0.05), but this change was not detected in BLP-tolerant cells. It was found that in BLP-tolerant cells, the expression of inhibitory subunit p50 was increased and the nuclear translocation and phosphorylation of p65 with trans-activation ability was inhibited. These changes are likely responsible for the reduced gene expression of NF-ΚB dependent genes in BLP-tolerant cells.