Differential role of Toll-interleukin 1 receptor domain-containing adaptor protein in Toll-like receptor 2-mediated regulation of gene expression of hepatic cytokines and drug-metabolizing enzymes

Abstract

Pharmacological activities of drugs are impaired during inflammation because of reduced expression of hepatic drug-metabolizing enzyme genes (DMEs) and their regulatory nuclear receptors (NRs): pregnane X receptor (PXR), constitutive androstane receptor (CAR), and retinoid X receptor (RXRα). We have shown that a component of Gram-positive bacteria, lipoteichoic acid (LTA) induces proinflammatory cytokines and reduces gene expression of hepatic DMEs and NRs. LTA is a Toll-like receptor 2 (TLR2) ligand, which initiates signaling by recruitment of Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) to the cytoplasmic TIR domain of TLR2. To determine the role of TIRAP in TLR2-mediated regulation of DME genes, TLR2(+/+), TLR2(-/-), TIRAP(+/+), and TIRAP(-/-) mice were given LTA injections. RNA levels of the DMEs (Cyp3a11, Cyp2b10, and sulfoaminotransferase), xenobiotic NRs (PXR and CAR), and nuclear protein levels of the central NR RXRα were reduced ∼ 50 to 60% in LTA-treated TLR2(+/+) but not in TLR2(-/-) mice. Induction of hepatic cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6), c-Jun NH(2)-terminal kinase, and nuclear factor-κΒ was blocked in TLR2(-/-) mice. As expected, expression of hepatic DMEs and NRs was reduced by LTA in TIRAP(+/+) but not in TIRAP(-/-) mice. Of interest, cytokine RNA levels were induced in the livers of both the TIRAP(+/+) and TIRAP(-/-) mice, whereas LTA-mediated induction of serum cytokines was attenuated in TIRAP(-/-) mice. LTA-mediated down-regulation of DME genes was attenuated in hepatocytes from TLR2(-/-) or TIRAP(-/-) mice and in small interfering RNA-treated hepatocytes. Thus, the effect of TLR2 on DME genes in hepatocytes was mediated by TIRAP, whereas TIRAP was not involved in mediating the effects of TLR2 on cytokine expression in the liver.

Fig. 1.

Inflammation-associated cell-signaling pathways in the liver. Activation of TLR2 on Kupffer cells (KCs) recruits the adaptor proteins and kinases to induce proinflammatory cytokines (IL-1β, TNFα, and IL-6). These cytokines bind to specific receptors (IL-1R, IL-6R, and TNF-R) on hepatocytes to alter gene expression in the hepatocytes.

Fig. 2.

Regulation of DME gene expression by TLR2 and TIRAP in vivo. TLR2(+/+) and TLR2(−/−) mice (A) and TIRAP(+/+) and TIRAP(−/−) mice (B) were given intraperitoneal injections of 6 mg/kg LTA or saline (SAL), and livers were harvested after 8 h. n = 6/group. RNA was isolated from the livers and analyzed by TaqMan real-time PCR, with primers and probes specific for Cyp3a11, Cyp2b10, and Sultn. All data were presented as ±S.D. and standardized for cyclophilin RNA levels. Expression in saline-injected mice was set to 1; fold change after LTA treatment was compared with the saline controls. *, significant difference (p < 0.05).

Fig. 3.

Regulation of NR expression by TLR2 and TIRAP. TLR2(+/+) and TLR2(−/−) mice (A) and TIRAP(+/+) and TIRAP(−/−) mice (B) were given intraperitoneal injections of 6 mg/kg LTA or saline (Sal), and livers were harvested after 8 h. RNA was isolated from the livers (n = 6/group) and analyzed by TaqMan real-time PCR as described above. *, significant difference (p < 0.05). Nuclear extracts were prepared from saline and LTA-injected mouse livers, and RXRα protein levels were measured by Western blotting. Quantification of the blots by densitometry was done after RXRα levels were normalized over RARα. Data are representative of four to five independent experiments. Error bars denote S.D.

Fig. 4.

Regulation of hepatic cytokine gene expression by TLR2 and TIRAP. TLR2(+/+) and TLR2(−/−) mice (A) and TIRAP(+/+) and TIRAP(−/−) mice (B) were given intraperitoneal injections of 6 mg/kg LTA or saline (Sal), and livers were harvested after 1 h. n = 6/group. RNA was isolated from the livers and analyzed by TaqMan real-time PCR as described above. *, significant difference (p < 0.05).

Fig. 5.

Regulation of cell-signaling pathways by TLR2 and TIRAP. TLR2(+/+) and TLR2(−/−) mice (A) and TIRAP(+/+) and TIRAP(−/−) mice (B) were given intraperitoneal injections of 6 mg/kg LTA or saline, and livers were harvested after 1 h. 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) (top panel) and degradation of ΙκΒα (bottom panel) were measured as markers of JNK and NF-κΒ activation, respectively. Quantification of the blots by densitometry was done after normalizing P-JNK levels over total JNK and IκΒα levels over β-actin. Data are representative of four to five independent experiments. Error bars denote S.D.

Fig. 6.

Induction of serum cytokine levels is regulated by TIRAP. TIRAP(+/+) and TIRAP(−/−) mice were given intraperitoneal injections of 6 mg/kg LTA or saline, mice were sacrificed after 1 h, and blood was collected. Serum IL-1β, IL-6, and TNFα levels were determined by enzyme-linked immunosorbent assay. n = 6/group. Error bars denote S.D.

Fig. 7.

Regulation of DME gene expression by LTA in hepatocytes treated with TLR2 or TIRAP siRNA. Primary hepatocytes from C57BL/6 mice were preincubated with siRNA for 24 h, followed by saline or LTA treatment (1 μg/ml) for 8 h. A, RNA was isolated, and real-time PCR was performed as described under Materials and Methods. n = 6/group. Error bars denote S.D. B, total cell lysates were analyzed by immunoblotting to determine the inhibition of TLR2 or TIRAP by their respective siRNAs.

Fig. 8.

Regulation of DME gene expression by LTA in hepatocytes isolated from TLR2(−/−) or TIRAP(−/−) mice. Primary hepatocytes from TLR2(−/−) or TIRAP(−/−) and their corresponding wild-type mice were treated with saline or LTA (1 μg/ml) for 8 h. RNA was isolated, and real-time PCR was performed as described under Materials and Methods. n = 6/group. Error bars denote S.D.