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Abstract
Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-α. Since NF-κB is a major transcription factor that regulates genes for TLR2 and TNF-α, we examined the effect of rifampicin on the LPS-induced NF-κB activation. Rifampicin inhibited NF-κB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKK α/β activity. However, rifampicin slightly inhibited the nuclear translocation of NF-κB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-κB p65, suggesting pregnane X receptor interferes with NF-κB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-κB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-κB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.
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Fig. 1.
Rifampicin inhibits the LPS-induced expression of TLR2 mRNA, and the expression of TLR2 mRNA is dependent on the NF-κB pathway. (A, D) RAW 264.7 cells were pretreated with rifampicin at the indicated concentrations or with DMSO for 1 h and then stimulated with LPS (5 ng/ml) for 4 hours. mRNA levels of TLR2 (A) and TNF-α (D) were determined by quantitative real time RT-PCR. (B) For TLR2 mRNA stability assay, RAW 264.7 cells were pre-treated with rifampicin (50 μg/ml) and then treated with LPS (5 ng/ml) for 2 hours before adding actinomycin D1 (5 μg/ml). At designated times, the level of TLR2 mRNA was determined by quantitative real time-RT-PCR. (C) RAW 264.7 cells were pretreated with BAY 11–7085, an inhibitor of NF-κB, at the indicated concentrations or with DMSO for 1 h and then stimulated with LPS (5 ng/ml) for 4 hours. mRNA levels of TLR2 were determined by quantitative real time RT-PCR. The results are shown as the means±SD of data from at least three separate experiments, each performed with triplicate samples. ∗p<0.01 versus non-treated control cells. RIF, rifampicin.
Fig. 2.
Rifampicin inhibits the nuclear translocation of NF-κB p65 in RAW 264.7 cells. RAW 264.7 cells were pre-treated with rifampicin (50 μg/ml) or with DMSO for 1 h and then stimulated with LPS (5 ng/ml) for the indicated times. (A) The levels of phosphorylation of IKKα/β and the expression of IκBα were determined by immunoblot analyses. (B) The nuclear translocation of NF-κB subunits p50 and p65 were determined by immunoblot analyses. After treatment, nuclear and cytoplasmic extracts were prepared using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc.). Similar results were observed in three independent experiments. RIF, rifampicin.
Fig. 3.
Rifampicin suppresses LPS-induced NF-κB DNA binding activity in RAW 264.7 cells. RAW 264.7 cells were pre-treated with rifampicin (50 μg/ml) or with DMSO for 1 h and then stimulated with LPS (5 ng/ml) for 1 h. DNA binding activity of NF-κB subunits was analyzed by EMSA. After treatment, nuclear extracts were prepared using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc.). For the competition assay, a 200-fold excess of unlabeled probe was added together with the labeled probe. For the supershift assay, 1 μg of antibody against NF-κB p65 was added together with the nuclear extract. Similar results were observed in three independent experiments. RIF, rifampicin; Competitor oligo., unlabeled oligonucleotides probe for NF-κB p65.
Fig. 4.
Rifampicin activates PXR in RAW 264.7 cells. (A) PXR mRNA expression was determined by RT-PCR. (B) The expression pattern of PXR was determined by immunostaining. The cells were incubated overnight with an anti-PXR antibody (Santa Cruz Biotechnology) at 4°C. After washing with PBS, cells were incubated with the corresponding Cy3-conjugated secondary IgG at room temperature for 2 h. Nuclei were counterstained for 15 min with 10 μM Hoechst 33342 (Sigma-Aldrich Co. Ltd). (C) RAW 264.7 cells were treated with rifampicin at the indicated concentrations or with DMSO for 4 h. The expression of CYP27A1 mRNA was determined by quantitative real time RT-PCR. Similar results were observed in three independent experiments. ∗p<0.01 versus non-treated control cells. PXR, pregnane X receptor; PXR/Cy3, anti-PXR antibody/Cy3-conjugated secondary antibody; RIF, rifampicin.
Fig. 5.
Rifampicin increases the physical association of PXR with NF-κB p65 in LPS-activated RAW 264.7 cells. (A, B) RAW 264.7 cells were pre-treated with rifampicin at the indicated concentrations or with DMSO for 1 h and then stimulated with LPS (5 ng/ml) for 1 h. The physical association between PXR and NF-κB subunits was examined by a co-immunoprecipitation assay. The cell lysates were immunoprecipitated with an irrelevant rabbit IgG as a negative control and antibody against NF-κB p65, together with protein A Sepharose™ CL-4B (Amersham Biosciences, Uppsla, Sweden). The immunoprecipitates were then electrophoresed on SDS-PAGE gels, transferred onto PVDF membranes (Millipore, Bedford, MA), and immunoblotted with an antibody against PXR and NF-κB p65. Similar results were observed in three independent experiments (A). The results are shown as the means±SD of data from at least three separate experiments, each performed with triplicate samples. ∗p<0.05 versus the values for the LPS treatment only (B). RIF, rifampicin.
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