Inhibition of sphingosine 1-phosphate signaling ameliorates murine nonalcoholic steatohepatitis - PubMed (original) (raw)

Inhibition of sphingosine 1-phosphate signaling ameliorates murine nonalcoholic steatohepatitis

Amy S Mauer et al. Am J Physiol Gastrointest Liver Physiol. 2017.

Abstract

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disorder, wherein proinflammatory lipids, such as ceramide and its derivative sphingosine 1-phosphate (S1P), contribute to macrophage-associated liver inflammation. For example, we have previously demonstrated a role for S1P in steatotic hepatocyte-derived S1P-enriched extracellular vesicles in macrophage chemotaxis in vitro. Therefore, we hypothesized that FTY720, an S1P antagonist, would ameliorate NASH by inhibiting proinflammatory monocyte chemotaxis. To test our hypothesis, NASH was established in C57BL/6 male mice by feeding a diet high in fructose, saturated fat, and cholesterol for 22 wk. Then mice received daily intraperitoneal injections of FTY720 for 2 wk before analysis of liver injury, inflammation, and fibrosis. FTY720-treated mice with NASH demonstrated improved liver histology with a significant reduction in hepatocyte ballooning and inflammatory foci. Hepatomegaly was reversed, and liver triglycerides were reduced following FTY720 administration to mice with NASH. Correspondingly, serum ALT levels, hepatic inflammatory macrophage accumulation, and the expression of Ly6C in recruited myeloid cells was reduced in FTY720-treated mice. Hepatic collagen accumulation and expression of α-smooth muscle actin were significantly lowered as well. Body composition, energy consumption and utilization, and hepatic sphingolipid composition remained unchanged following FTY720 administration. FTY720 ameliorates murine nonalcoholic steatohepatitis. Reduction in liver injury and inflammation is associated with a reduction in hepatic macrophage accumulation, likely due to dampened recruitment of circulating myeloid cells into the liver. Nonalcoholic steatohepatitis may be a novel indication for the therapeutic use of FTY720.NEW & NOTEWORTHY There are no approved pharmacologic therapies for nonalcoholic steatohepatitis (NASH), the leading cause of chronic liver disease worldwide. This study describes the use of FTY720, a novel small molecule, for the amelioration of NASH in a mouse model. We demonstrate that 2-wk administration of FTY720 to mice with NASH led to a reduction in liver injury, inflammation, and fibrosis. These data provide a preclinical rationale for studying this drug in human NASH.

Keywords: FTY720; Kupffer cells; fatty liver; macrophage obesity.

Copyright © 2017 the American Physiological Society.

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Figures

Fig. 1.

Fig. 1.

FTY720 treatment decreases liver injury. A: representative hematoxylin-and-eosin-stained liver photomicrographs from chow-fed mice treated with either saline or FTY720 or FFC-fed mice treated with either saline or FTY720 are shown. Scale bar: 100 μm. B: representative CARS photomicrographs from chow-fed mice treated with either saline or FTY720 or FFC-fed mice treated with either saline or FTY720 are shown. Scale bar: 100 μm. C: components of NAFLD activity score consisting of inflammatory foci, fibrosis, and hepatocyte ballooning for FFC-fed saline treated (n = 5) and FFC-fed FTY720-treated mice (n = 6) are shown. *P < 0.05. D: quantification of CARS-positive area from chow-fed mice (n = 4) treated with either saline or FTY720 or FFC-fed mice (n = 5) treated with either saline or FTY720. *P < 0.05, ***P < 0.001. E: liver triglyceride (TG) content measured biochemically in all four groups, *P < 0.05, **P < 0.01, #P < 0.05. Serum ALT (F), initial body weight (G), final body weight (H), liver mass (I), and relative liver mass (J)for the four experimental groups are shown. *P < 0.05, **P < 0.01, ns, not significant.

Fig. 2.

Fig. 2.

FFC-feeding increases hepatic sphingolipids, which remain unchanged with FTY720 administration. Liver sphinganine (A), C16:0 ceramide (B), C16 dihydroceramide (C), C18:0 ceramide (D), C24:0 ceramide (E), C24:1 ceramide (F), sphingosine (G), and sphingosine 1-phosphate (S1P; H) were measured by tandem mass spectroscopy and normalized to protein content. Liver expression of ceramide synthases, Lass2 (I), Lass4 (J), and Lass6 (K) was measured by quantitative PCR (qPCR). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Fig. 3.

Metabolic parameters remain unchanged in FTY720 treated FFC-fed mice. Food intake (A), lean mass (B), fat mass (C), resting energy expenditure (D), activity energy expenditure (E), total energy expenditure (F) respiratory exchange ratio (G), metabolic rate (H), heat production (I), activity (J), ambulation (H), and rearing (L) were measured in saline-treated FFC-fed and FTY720-treated mice (n = 5 each). Metabolic parameters were measured for 24 h each under fed and fasted conditions. ns, not significant.

Fig. 4.

Fig. 4.

Macrophage accumulation in reduced in FTY720-treated FFC-fed mice. A: representative images from Mac-2 immunohistochemistry are shown, scale bar 100 μM. B: quantification of Mac-2-positive area. C: liver mRNA expression of Mac-2 from each of the four groups is shown. *P < 0.05, **P < 0.01, ***P < 0.001. Monocyte chemotactic protein 1 (Mcp-1) (D) and macrophage inflammatory protein 1α (Mip1α) (E) mRNA expression in livers from each of the four experimental groups is shown, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Fig. 5.

Proinflammatory monocyte-derived macrophages are reduced in FFC-fed FTY720-treated mice. A: representative images from Ly6C immunohistochemistry are shown (scale bar: 100 μM). B: quantification of liver Ly6C mRNA expression. C: liver C-C chemokine receptor 2 (CCR2) mRNA expression in each of the four experimental groups is shown, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Fig. 6.

Liver fibrosis is reduced in FTY720-treated FFC-fed mice. A: representative images from Picrosirius red-stained liver sections from saline-treated and FTY720-treated FFC-fed mice (left; scale bar: 100 μM). Quantification of Picrosirius red-positive area from all four experimental groups is shown on the right. B: representative images from immunohistochemistry for α-smooth muscle actin (αSMA) from saline-treated and FTY720-treated FFC-fed mice liver sections (left; scale bar: 100 μM). Quantification of αSMA-positive area from all four experimental groups is shown on the right. C: representative images from immunohistochemistry for collagen 1(Col1) from saline-treated and FTY720-treated FFC-fed mice liver sections (left; scale bar: 100 μM). Quantification of αSMA-positive area from all four experimental groups is shown on the right. Chow-fed saline-treated and FTY720-treated mice had only minimal normal levels of positivity for Picrosirius red, αSMA, and Col1; therefore, their photomicrographs were omitted. They were included in the quantitate morphometry. Liver mRNA expression of αSMA (D), collagen 1α1 (Col1α1) (E), osteopontin (F), tissue inhibitor of metalloproteinase 1 (Timp1) (G), and matrix metalloproteinase 13 (MMP13) (H) from each of the four groups is shown. Col1α1 and MMP13 mRNAs were not detected in chow-fed mice. #P < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Fig. 7.

FTY720 does not directly alter hepatic stellate cell or macrophage activation. The hepatic stellate cell line (LX-2) was treated with 10 ng/ml TGFβ for 1 h followed by 250 nM FTY720 for 4 h. The expression of markers of hepatic stellate cell activation α-smooth muscle actin (αSMA) (A), Col1α1 (B), connective tissue growth factor (CTGF; C), platelet-derived growth factor-bb (PDGF-BB) (D), and proinflammatory genes interleukin 1 beta (IL1β) (E) and monocyte chemotactic protein 1 (MCP-1; F) was measured by quantitative PCR. BMDMs were treated with 50 ng/ml LPS for 1 h followed by 250 nM FTY720 for 4 h. The expression of proinflammatory genes Mcp-1 (G), and IL-1β (H), and the profibrotic gene platelet-derived growth factor-bb (PDGF-BB) (I) were measured by qPCR. αSMA, Col1α1, and CTGF were not detected in BMDM. J: cell death in LX-2 cells stimulated with TGFβ and treated with FTY720 as above. K: cell death in BMDM stimulated with LPS and treated with FTY720 as above. L: cell death in primary mouse hepatocytes treated with 250 nM FTY720 for 16 h. *P < 0.05.

Fig. 8.

Fig. 8.

FFC-feeding increases circulating extracellular vesicles (EVs) and their sphingolipid content. A: circulating EVs were measured in 200 μl of plasma obtained from each experimental group. EVs were quantified by nanoparticle tracking analysis. *P < 0.05, **P < 0.01, ***P < 0.001. B: circulating EV S1P content and C16:0 ceramide content was calculated as the product of the EV particle concentration and S1P (ng/EV) and C16:0 (ng/EV), respectively. *P < 0.05.

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