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. 2014 Nov 5;9(11):e112327.
doi: 10.1371/journal.pone.0112327. eCollection 2014.

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

Alexander Wehr  1 , Christer Baeck  1 , Florian Ulmer  2 , Nikolaus Gassler  3 , Kanishka Hittatiya  4 , Tom Luedde  1 , Ulf Peter Neumann  2 , Christian Trautwein  1 , Frank Tacke  1
Affiliations

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

Alexander Wehr et al. PLoS One. .

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl4)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hepatic macrophage infiltration is increased in liver of patients with non-alcoholic-steatohepatitis and accompanied by slightly enhanced CXCR6 expression.
Liver biopsies of patients with NAFLD (n = 20) and healthy controls (n = 3) were analyzed. (A–B) Hematoxylin-Eosin stainings (H&E) of liver paraffin sections demonstrated steatosis development in patients with NAFLD compared to healthy controls. NAFLD severity was scored according to the NAS-Kleiner-Score. (C–D) CD68 immunohistochemical stainings showed a significant increase of intrahepatic macrophages in livers of NAFLD patients compared to healthy controls. (E) Hepatic macrophages (CD68 positive) significantly correlated with the NAS. (F) Patients with NAFLD developed fibrosis, as evidenced by collagen deposition (blue) in Ladewig staining of liver biopsies. (G) High CXCR6 expression correlated with elevated serum ALT levels. P-values and correlation coefficients are given in the figure. All data are expressed as mean ± SD. *p<0.05, **p<0.005, ***p<0.001.
Figure 2
Figure 2. Anti-CXCL16 antibody sufficiently blocks early hepatic NKT cell accumulation upon acute liver injury.
(A) B6-mice received a singular intraperitoneal application of either αCXCL16 or BSA (unspecific protein control) 4 hours before injection of carbon tetrachloride (CCl4). Mice were sacrificed 6 hours after CCl4 application. (B–C) As evidenced by H&E stainings and serum ALT activity, αCXCL16 treated mice showed reduced toxic liver damage in vivo compared to BSA treated mice. (D–E) Flow cytometric analysis of intrahepatic leukocytes (representative FACS plots in D, statistical analyses in E) showed a significant decrease of hepatic NKT cells in mice that received αCXCL16 compared to controls. Other CXCR6 expressing cells like CD4 T- and NK cells were not altered with respect to their migration behavior into the injured liver upon αCXCL16 injection. All data are expressed as mean ± SD from three independent experiments, summarizing n = 6 animals per group. *p<0.05, **p<0.005, ***p<0.001.
Figure 3
Figure 3. Therapeutic administration of αCXCL16 inhibits macrophage infiltration and hepatic inflammation in experimental steatohepatitis.
(A) B6-mice were subjected to a methionine-choline deficient (MCD) diet over 6 weeks. During the last three weeks of progressing steatohepatitis, mice were treated with αCXCL16 or BSA i.p. once per week. (B–D) F4/80 immunohistochemical staining of liver tissue (B) and flow cytometric analysis of intrahepatic leukocytes (representative plots, C; statistical analysis, D) showed a significant reduction of infiltrating CD11b+F4/80+ macrophages in αCXCL16 treated mice compared to controls. (E) Pro-inflammatory cytokines like TNFα and MCP-1 were slightly decreased in liver tissue of mice that received weekly αCXCL16 injections compared to controls during MCD diet. All data are expressed as mean ± SD from three independent experiments, summarizing n = 6 animals per group. *p<0.05, **p<0.005, ***p<0.001.
Figure 4
Figure 4. Therapeutic administration of αCXCL16 attenuates steatosis development in experimental steatohepatitis.
(A) Liver histology showed no altered fibrosis development, but a clear reduction of lipid accumulation in injured livers of αCXCL16 treated mice compared to controls as evidenced by H&E- and Sirius red stainings. (B–C) Serum ALT activity and hydroxyproline content were not affected by the administration of αCXCL16. (D–E) Intrahepatic triglyceride content and periportal lipid droplets were significant reduced in mice that received weekly αCXCL16 injections compared to controls during MCD diet. (F) Fatty degeneration score was significantly decreased in αCXCL16 treated mice compared to controls. All data are expressed as mean ± SD from three independent experiments, summarizing n = 6 animals per group. *p<0.05, **p<0.005, ***p<0.001.
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This work was supported by the German Research Foundation (DFG Ta434/2-1 to F.T., DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.