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. 2015 May 15;290(20):12804-11.
doi: 10.1074/jbc.M114.635763. Epub 2015 Apr 6.

Hepatic serum amyloid A1 aggravates T cell-mediated hepatitis by inducing chemokines via Toll-like receptor 2 in mice

Young Rae Ji  1 , Hei Jung Kim  1 , Ki Beom Bae  1 , Sanggyu Lee  1 , Myoung Ok Kim  2 , Zae Young Ryoo  3
Affiliations

Hepatic serum amyloid A1 aggravates T cell-mediated hepatitis by inducing chemokines via Toll-like receptor 2 in mice

Young Rae Ji et al. J Biol Chem. .

Abstract

Serum amyloid A is a proinflammatory molecule that induces leukocyte infiltration and promotes neutrophil adhesion to endothelial cells under inflammatory conditions. The aim of this study was to examine whether Saa1 aggravates T cell-mediated hepatitis by inducing chemokines in a liver-specific, Saa1-overexpressing, transgenic (TG) mouse model. We generated TG mice in which Saa1 was overexpressed specifically in liver tissue. The chemokines monocyte chemotactic protein 1 (MCP1), MIP1α, MIP1β, interferon γ-induced protein 10 (IP-10), and eotaxin were induced in Saa1 TG mice. After concanavalin A treatment, Saa1 expression was higher in Saa1 TG mice than in WT mice. More severe liver injury, increased hepatocyte apoptosis, and higher levels of hepatic enzymes were observed in Saa1 TG mice than in WT mice. Liver infiltration of CD4(+) T cells and macrophages increased after inducing hepatitis. Activation of T cells was higher in Saa1 TG mice than in WT mice, and the populations of Th17 cells and regulatory T cells were altered by overexpressing Saa1 in TG mice. Secretion of various cytokines, such as interferon γ, tumor necrosis factor α, and interleukin 6, increased in Saa1 TG mice. Injecting a Toll-like receptor 2 (TLR2) antagonist in vivo inhibited chemokine expression and IκBα phosphorylation and showed that the induction of chemokines by Saa1 was dependent on TLR2. Hepatic Saa1 accelerated T cell-mediated hepatitis by inducing chemokine production and activating T cells by TLR2. Therefore, Saa1 might be a novel inflammatory factor that acts as a chemokine modulator in hepatitis.

Keywords: Saa1; T cell activation; Toll like receptor; chemokine; concanavalin A; cytokine; inflammation; liver injury; transgenic mice.

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Figures

FIGURE 1.
FIGURE 1.
Saa1-overexpressing transgenic mice were generated using a liver-specific promoter and expressed various chemokines. A, we used an albumin enhancer and promoter to induce liver-specific expression of Saa1. B, to identify potential transgenic mice, total genomic DNA, extracted from tail biopsy specimens from 6-week-old pups, was screened with PCR using the primer pair albumin-F and Saa1-R. P, positive control (vector used for template); N, negative control (no vector). C, Saa1 protein was detected in the livers of WT and Saa1 TG mice by Western blot analysis. β-actin was used as a loading control. D, the mRNA expression of the chemokines MCP1 (CCL2), MIP1α (CCL3), MIP1β (CCL4), Rantes (CCL5), IP-10 (CXCL10), and eotaxin (CCL11) in the livers of WT and Saa1 TG mice was detected by real-time PCR. β-actin was used as a control for normalization. *, p < 0.05; **, p < 0.01 versus WT control. WT, C57BL/6 wild-type mice; TG, Saa1-overexpressing transgenic mice.
FIGURE 2.
FIGURE 2.
Overexpression of Saa1 promotes liver injury and apoptosis of hepatocytes in T cell-mediated hepatitis. A, Saa1 was detected in the livers of WT and Saa1 TG mice 24 h after injection with ConA. β-actin was used as a loading control. B, liver specimens were stained from WT and Saa1 TG mice collected 24 h after ConA injection for histological analysis. C, liver specimens were stained from WT and Saa1 TG mice collected 24 h after ConA injection using the TUNEL assay. **, p < 0.01 versus WT control.
FIGURE 3.
FIGURE 3.
Saa1 regulates liver enzyme expression and the secretion of proinflammatory cytokines in ConA-induced hepatitis. A, the levels of AST and ALT were measured from serum 0, 6, and 24 h after ConA injection. B, the inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-10 in the serum of the WT and Saa1 TG mice were measured by ELISA at 0, 6, and 24 h after ConA injection. ***, p < 0.001 versus WT control.
FIGURE 4.
FIGURE 4.
Overexpression of Saa1 increases the percentage of CD4+ T cells and macrophages among mononuclear cells in the liver. T cell (CD4+ and CD8+ cells, A and B), B cell (B220+ cells, C), and macrophage (F4/80+CD11b+ cells, D) populations were counted in mononuclear cells isolated from the perfused livers of WT mice and Saa1 TG mice 24 h after ConA injection. *, p < 0.05; **, p < 0.01 versus WT control.
FIGURE 5.
FIGURE 5.
Saa1 modulates T cell activation with regulation of Th17 and Treg cells. A and B, the expression of CD69 and CD25 was assessed by FACS analysis from the livers and spleens of WT and Saa1 TG mice 24 h after ConA injection. C and D, the expression of IL-17 and FoxP3 was assessed by FACS analysis from the livers and spleens of WT and Saa1 TG mice 24 h after ConA injection. MNC, mononuclear cell. *, p < 0.05; **, p < 0.01 versus WT control.
FIGURE 6.
FIGURE 6.
Induction of chemokines by Saa1 is regulated by TLR2-dependent NF-κB signaling. A, the expression of MIP1α (CCL3) and eotaxin (CCL11) mRNA was detected in the perfused liver of Saa1 TG mice treated with or without 3 mg/kg CU-CPT22 (a TLR2 antagonist) for 16 h. B, phosphorylation of IκBα in the perfused liver of Saa1 TG mice treated with or without 3 mg/kg CU-CPT22 for 2 h was detected by Western blot analysis. *, p < 0.05 versus TG control.
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