Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid

Abstract

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRalpha). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver ( approximately 60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with approximately 50% reduction of PXR RNA and nuclear protein levels of RXRalpha. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.

Fig. 1

Regulation of DME RNA levels in mouse liver following LTA treatment. Mice were IP-injected with 6 mg/kg LTA and livers were harvested from 0 to 16 hours (n=6-8 per group). RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described in Materials and methods. RNA levels of phase I (A) and phase II (B) enzymes were determined. All data were presented as ± SD and standardized for cyclophilin RNA levels. Expression in 0h LTA-injected mice was set to 1, fold change after LTA treatment from 1-16h was compared to the 0h LTA-injected controls. The asterisks indicate significant difference (p < 0.05).

Fig. 2

Regulation of transporter RNA levels in mouse liver following LTA treatment. Mice were IP-injected with 6 mg/kg LTA and livers were harvested at various time-points (n=6 per group). RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described in Materials and methods. All data were presented as ± SD and standardized for cyclophilin RNA levels. Expression in 0h LTA-injected mice was set to 1, fold change after LTA treatment from 1-16h was compared to the 0h LTA-injected controls. The asterisks indicate significant difference (p < 0.05).

Fig. 3

Regulation of NR expression in mouse liver by LTA. Mice were IP-injected with 6 mg/kg LTA and livers were harvested at various time-points (n=6 per group). RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described in Materials and methods. All data were presented as ± SD and standardized for cyclophilin RNA levels. Expression in 0h LTA-injected mice was set to 1, fold change after LTA treatment from 1-16h was compared to the 0h LTA-injected controls. The asterisks indicate significant difference (p < 0.05) (A). Nuclear extracts were prepared from livers from saline and LTA -injected mouse livers RXRα protein levels were measured by western blotting (B).

Fig. 4

Regulation of cytokine expression by LTA. Mice were IP-injected with 6 mg/kg LTA and livers were harvested at various time-points (n=6 per group). RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described previously in Materials and methods (A). Serum cytokine levels were measured by enzyme-linked immunosorbent assay (n=6 per group). Error bars denote S.D. (B).

Fig. 5

Regulation of TLR2 expression by LTA. Mice were IP-injected with 6 mg/kg LTA and livers were harvested at various time-points (n=6 per group). RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described previously in Materials and methods (A). TLR2 expression was analyzed by western blot analysis of membrane fractions prepared from livers of mice injected with LTA at various time-points (B).

Fig. 6

Regulation of cell-signaling pathways by LTA. Whole cell extracts were prepared from the livers of saline and LTA-injected mice, and the samples were analyzed by immunoblotting. Phosphorylation of JNK (P-JNK) and degradation IκBα of were measured as markers of JNK and NF-κB activation, respectively.

Fig. 7

Role of Kupffer cells. Mice were given a single dose of GdCl₃ intravenously (10 mg/kg) or the vehicle, saline, followed by IP injection of LTA (6 mg/kg) 24h after GdCl₃ treatment. Livers were harvested and RNA and protein analysis was done as described previously in Materials and methods. RNA levels of cytokines were measured after 24h of GdCl₃ treatment, followed by LTA treatment for 1h (A). DMEs and nuclear receptors were measured after 4h of LTA treatment (B), and TLR2 RNA levels were measured after 2h of LTA treatment (C). Error bars denote S.D. *p < 0.05 between saline and LTA-treated samples (without GdCl₃ treatment); # p < 0.05, comparing LTA injections with and without GdCl₃ pre-treatment.