Toll-like receptor 3 ligand attenuates LPS-induced liver injury by down-regulation of toll-like receptor 4 expression on macrophages - PubMed (original) (raw)
Toll-like receptor 3 ligand attenuates LPS-induced liver injury by down-regulation of toll-like receptor 4 expression on macrophages
Wei Jiang et al. Proc Natl Acad Sci U S A. 2005.
Abstract
This study demonstrates that pretreatment with polyinosinic-polycytidylic acid (poly I:C) significantly decreased the mortality and liver injury caused by injection of lipopolysaccharide (LPS) in the presence of d-galactosamine (d-GalN) in C57BL/6 mice. Depletion of natural killer, natural killer T, and T cells did not change the protective effect of poly I:C on LPS/d-GalN-induced liver injury in vivo. However, depletion of macrophages abolished LPS/d-GalN-induced fulminant hepatitis, which could be restored by adoptive transfer of macrophages but not by transfer of poly I:C-treated macrophages. Treatment with poly I:C down-regulated the expression of the toll-like receptor 4 (TLR4) on macrophages and reduced the sensitivity of macrophages (Kupffer cells and peritoneal macrophages from C57BL/6 mice, or RAW264.7 cells) to LPS stimulation. Poly I:C pretreatment also impaired the signaling of mitogen-activated protein kinases and NF-kappaB induced by LPS in RAW264.7 cells. Blockade of TLR3 with a TLR3 antibody abolished poly I:C down-regulation of TLR4 expression and LPS stimulation of TNF-alpha production in RAW264.7 cells. Taken together, our findings suggest that activation of TLR3 by its ligand, poly I:C, induced LPS tolerance by down-regulation of TLR4 expression on macrophages.
Figures
Fig. 1.
Pretreatment with poly I:C attenuated LPS-induced acute liver injury in C57BL/6 mice. (A) Poly I:C (7.5 μg/g) was injected i.p. for 6, 12, and 24 h before coinjection with a lethal dose of LPS (5 μg/kg) and
d
-GalN (400 mg/kg). Survival rate was measured after coinjection. (B) Poly I:C was i.p.-injected into mice 6 h before LPS/
d
-GalN injection. Serum samples were collected 6 h after coinjection, and serum transaminase activity was determined. (C) Serum samples were collected at 90 min and 2 h post-LPS/
d
-GalN injection for measuring TNF-α and IL-12. Data in B and C are expressed as the mean ± SEM (n = 5–7). *, P < 0.05 in comparison with LPS/
d
-GalN alone.
Fig. 2.
Poly I:C acted on macrophages directly and down-regulated TLR4 expression on macrophages in this protective process. (A) Peritoneal macrophages (1 × 107 cells) were treated with PBS or poly I:C (50 μg/ml) for 6 h, followed by three washes with PBS, and transferred i.v. into macrophage-depleted mice by GdCl3 treatment. After transfer, mice were injected with 5 μg/kg LPS and 400 mg/kg
d
-GalN. Sera were obtained 6 h later, and ALT/AST levels were measured. Data are expressed as the mean ± SEM of three mice in each group. *, P < 0.05. (B) The mRNA level of TLR4 was detected by RT-PCR in the liver tissues of C57BL/6 mice treated with PBS or poly I:C for 6 h. I:C was the group of poly I:C stimulation. (C) TLR4 expression on the surface of Kupffer cells from C57BL/6 mice treated with poly I:C (7.5 μg/g) or PBS for 6, 12, and 24 h was determined by flow cytometry. Kupffer cells were gated on the F4/80+ population. (D) RAW264.7 cells were stimulated with poly I:C (50 μg/ml) or PBS for 6 h. TLR4 expression on the cell surface (Left) or intracellularly (Right) was examined by flow cytometry. In C and D, filled histograms represent the PBS-treated group, and the empty histograms are the poly I:C-treated group. (E) Immunohistological analysis for detecting the distribution of TLR4 in RAW264.7 cells. RAW264.7 cells stimulated with poly I:C or PBS for 6 h were treated as in Materials and Methods. Fluorescence microscope images showed the change of TLR4 expression after poly I:C stimulation.
Fig. 3.
Pretreatment with poly I:C inactivated the LPS-induced signaling pathways. (A) RAW264.7 cells (5 × 106 cells) were pretreated with either buffered medium or poly I:C (50 μg/ml) for 6 h, followed by stimulating with LPS (100 ng/ml) for the indicated time points. MAPK [p38MAPK, extracellular signal-regulated protein kinase 1/2 (ERK1/2), and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)] and inhibitor κB (I-κB) phosphorylation was determined by Western blot. (B) RAW264.7 cells (1 × 106 cells) were stimulated with poly I:C or media. After 6 h cells were washed twice and then stimulated with LPS. Supernatants were collected 24 h after secondary stimulation for TNF-α and IL-12 evaluation by ELISA. *, P < 0.05. The experiments were repeated three times with similar results.
Fig. 4.
Blockade of TLR3 prevented the down-regulation of TLR4 induced by poly I:C. RAW264.7 cells (1 × 106 cells per ml) were incubated with anti-mouse TLR3 antibody (1 μl/ml) or rat IgG for 30 min, then treated by poly I:C (50 μg/ml) for 6 h. (A) TLR4 expression was analyzed by flow cytometry, then cells were stimulated with LPS (100 ng/ml) for 24 h. Filled histograms represent the PBS-treated group, and empty histograms are the poly I:C-treated group. (B) The supernatants were collected for TNF-α evaluation by ELISA. *, P < 0.05.
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