Bile acid receptor activation modulates hepatic monocyte activity and improves nonalcoholic fatty liver disease - PubMed (original) (raw)

Bile acid receptor activation modulates hepatic monocyte activity and improves nonalcoholic fatty liver disease

Rachel H McMahan et al. J Biol Chem. 2013.

Abstract

Nonalcoholic fatty liver disease (NAFLD) affects a large proportion of the American population. The spectrum of disease ranges from bland steatosis without inflammation to nonalcoholic steatohepatitis and cirrhosis. Bile acids are critical regulators of hepatic lipid and glucose metabolism and signal through two major receptor pathways: farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, and TGR5, a G protein-coupled bile acid receptor (GPBAR1). Both FXR and TGR5 demonstrate pleiotropic functions, including immune modulation. To evaluate the effects of these pathways in NAFLD, we treated obese db/db mice with a dual FXR/TGR5 agonist (INT-767) for 6 weeks. Treatment with the agonist significantly improved the histological features of nonalcoholic steatohepatitis. Furthermore, treatment increased the proportion of intrahepatic monocytes with the anti-inflammatory Ly6C(low) phenotype and increased intrahepatic expression of genes expressed by alternatively activated macrophages, including CD206, Retnla, and Clec7a. In vitro treatment of monocytes with INT-767 led to decreased Ly6C expression and increased IL-10 production through a cAMP-dependent pathway. Our data indicate that FXR/TGR5 activation coordinates the immune phenotype of monocytes and macrophages, both in vitro and in vivo, identifying potential targeting strategies for treatment of NAFLD.

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Figures

FIGURE 1.

FIGURE 1.

Treatment with the FXR/TGR5 agonist INT-767 improves liver pathology in NAFLD. Obese db/db and control db/m mice were treated with INT-767 (30 mg/kg/day) for 6 weeks. A, H&E staining of livers demonstrated increased steatosis and inflammatory foci (arrows) in db/db mice and resolved liver pathology in INT-767-treated mice. B, livers from 15 mice/treatment group were scored for steatosis, inflammatory foci, and hepatocyte ballooning as described under “Experimental Procedures.” Data are expressed as the arithmetic mean ± S.E.

FIGURE 2.

FIGURE 2.

Decreased intrahepatic expression of profibrotic and proinflammatory genes in obese db/db mice treated with INT-767. Intrahepatic expression of the indicated profibrotic (A and B) and proinflammatory (C and D) genes from db/m and obese db/db mice fed INT-767 for 6 weeks was determined by quantitative PCR. Data are expressed as the arithmetic mean ± S.E. of eight mice/treatment group. p values represent unpaired t tests. ProCol1A, procollagen 1A; α_-SMA_, α-smooth muscle actin.

FIGURE 3.

FIGURE 3.

Increased intrahepatic Ly6Clow monocytes in obese db/db mice treated with INT-767. Intrahepatic monocyte populations from obese db/db mice treated with INT-767 for 6 weeks were analyzed by flow cytometry. A, the percentage of CD45+ cells in the liver that were monocytes (CD115+CD11b+Ly6G−) was increased in mice treated with INT-767. Data represent the mean ± S.E. from 16 mice/treatment group. B, upper, representative plots show Ly6C and CD11b expression on intrahepatic CD45+F4/80−Ly6G− cells from db/m and db/db mice treated with INT-767. Lower, histograms of Ly6C staining are shown for the gated populations. C, bar graphs demonstrate the percentage of total CD45+ intrahepatic cells that are Ly6Clow (left) or Ly6Chigh (right) monocytes. Data represent the mean ± S.E. from 16 mice/treatment group. D, bar graphs demonstrate the percentage of monocytes with low expression of Ly6C in the liver (left) and spleen (right). Treatment with INT-767 significantly increased the number of Ly6Clow cells in the livers of db/db mice. Data represent the mean ± S.E. from 16 mice/treatment group. E, a representative histogram plot shows increased CD11c expression on Ly6Clow (gray) compared with Ly6Chigh (black line) monocytes.

FIGURE 4.

FIGURE 4.

Decreased Ly6C expression by CD115+CD11b+ cells treated in vitro with INT-767. Bone marrow-derived monocytes from C57BL/6J mice were isolated by magnetic bead separation; treated in vitro for 48 h with the INT-767 agonist; and stained with antibodies against CD115, CD11b, Ly6G, Ly6C, and CD11c. A, representative flow cytometric plots showing down-regulation of Ly-6C on monocytes (CD115+CD11b+Ly6G−). B, bar graphs demonstrating the percentage of CD115+CD11b+Ly6G− cells from bone marrow with high expression of Ly6C. Data represent the mean ± S.E. from triplicate samples.

FIGURE 5.

FIGURE 5.

Decreased proinflammatory cytokines and enhanced IL-10 production by macrophages treated in vitro with INT-767. A, bone marrow-derived macrophages from C57BL/6 mice were stimulated with 20 ng/ml IFNγ and 100 ng/ml LPS for 24 h, and the relative expression levels of TNFα, MCP-1, and IL-1β were determined by quantitative PCR. Expression was normalized to β-actin expression, and the -fold expression over resting macrophages is shown. B, shown is IL-10 gene expression in macrophages matured in the presence of the INT-767 agonist following LPS stimulation. The bar graph represents the mean ± S.E. from two separate experiments. C, monocytes were isolated from the livers of db/db and db/m mice treated with INT-767 in vivo and cultured with 100 ng/ml LPS for 24 h. The presence of IL-10 in the supernatants was determined by ELISA. Data represent triplicate samples from two mice/treatment group.

FIGURE 6.

FIGURE 6.

Increased expression of AAM markers by monocytes following INT-767 treatment. The liver gene expression levels of the macrophage marker F4/80 (A) and the markers of alternative activation (CD206, Retnla, Clec7a and sIL-1Ra) (B) were determined in mice treated with the INT-767 agonist for 6 weeks. Expression was normalized against β-actin, and the -fold expression over livers from db/m mice was calculated. Data represent the mean ± S.E. from six mice/treatment group with the exception of sIL-1Ra, which was four mice/group. C, the percentage of intrahepatic macrophages (CD45+CD11b+F4/80+Ly6G−) expressing CD206 was determined by flow cytometry. Data represent samples from four mice/treatment group. D, bone marrow-derived macrophages from C57BL/6 mice were differentiated in the presence of INT-767 and stimulated with 100 ng/ml LPS for 24 h. The percentage of CD115+CD11b+F4/80+ cells expressing CD206 and PD-L1 was determined by flow cytometry. The bar graphs present the mean ± S.E. from two separate experiments.

FIGURE 7.

FIGURE 7.

INT-767 induces cAMP production by macrophages, and blocking cAMP inhibits cytokine production. A, bone marrow-derived macrophages from C57BL/6 mice were stimulated with 30 μ

m

INT-767 for 20 min, and cAMP production was determined by ELISA. B, macrophages were matured in the presence of the INT-767 agonist, followed by LPS stimulation for 18 h either alone or in the presence of the cAMP inhibitor MDL-2330A. IL-10 expression was determined by quantitative PCR. The bar graphs represent the mean ± S.E.

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