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. 2012 Sep;61(9):1323-9.
doi: 10.1136/gutjnl-2011-301857. Epub 2012 Mar 17.

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease

Wing-Kin Syn  1 , Kolade M Agboola, Marzena Swiderska, Gregory A Michelotti, Evaggelia Liaskou, Herbert Pang, Guanhua Xie, George Philips, Isaac S Chan, Gamze F Karaca, Thiago de Almeida Pereira, Yuping Chen, Zhiyong Mi, Paul C Kuo, Steve S Choi, Cynthia D Guy, Manal F Abdelmalek, Anna Mae Diehl
Affiliations

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease

Wing-Kin Syn et al. Gut. 2012 Sep.

Abstract

Objective: Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells.

Design: The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans.

Results: When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18-/- and CD1d-/- mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity.

Conclusion: Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

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Conflict of interest statement

Conflict of Interest:

All authors declare no conflict of interest

Figures

Figure 1
Figure 1. NKT-deficient mice develop less fibrosing-NASH after methionine-choline deficient (MCD) diet
Wild-type (WT), Jα18−/− and CD1d−/− mice were fed control chow (n=10 per strain) or MCD diet (n=10 per strain) for 8 weeks, and then sacrificed. (A) Representative hematoxylin & eosin and (B) Sirius red staining after MCD diet. (C) Sirius red quantification by morphometric analysis; results are expressed as fold change relative to WT control chow-fed mice, and graphed as mean ± SEM. (D) Hepatic hydroxyproline content at the end of the treatment period. *P<0.05 vs. control chow-fed mice.
Figure 2
Figure 2. NKT-deficient mice exhibit attenuated fibrogenic response compared with WT mice
Mice were fed control chow and MCD diets as described in Figure legend 1. (A) αSMA immunoreactivity and (B) αSMA morphometry. Sections from 4 animals were used and 15 randomly selected, 40× fields chosen for analysis by the Metaview software. Small inserts in (A) show representative staining from chow-fed mice. Whole liver tissues were harvested for RNA analysis by QRT-PCR. (C) αSMA mRNA, (D) Collagen Iα1 mRNA, and (E) ALT IU/L. Results are expressed as fold change relative to WT control chow-fed mice and graphed as mean ± SEM. *P<0.05 vs. control chow-fed mice
Figure 3
Figure 3. NKT-deficient mice harbor less liver and plasma OPN than WT mice after diet-induced non-alcoholic steatohepatitis (NASH)
Mice were fed control chow or MCD diet for 8 weeks and then sacrificed as described in Figure legend 1. Matched plasma was collected for OPN ELISA. (A–C) Representative OPN immunostaining after control chow or MCD diets in WT and Jα18−/− mice (final magnification 200X), and quantification by morphometry. Sections from 10 animals were used per group and 10 randomly selected, 200X fields chosen for analysis by the Metaview software. Results are expressed as fold change (% positive staining) relative to WT control mice, and graphed as mean ± SEM. (D) Plasma OPN. Plasma from 10 animals per group were used for OPN ELISA, and displayed as pg/ml. Each sample was run in duplicate. *P<0.05 vs. WT control mice
Figure 4
Figure 4. αGalactosylceramide (αGC)-activated liver NKT cell associated OPN promotes primary hepatic stellate cell (HSC) activation
Primary liver mononuclear cells (LMNC) (1 × 105) from WT mice were cultured in complete NKT media, and treated with vehicle (V) or αGC (to activate NKT cells) for 24 hours. Conditioned medium (CM) was collected for OPN ELISA; 603B mouse cholangiocyte-CM was used as a positive control. (A) OPN protein was quantified by ELISA. Each LMNC-CM sample was run in duplicate and expressed as pg/ml. (B) LMNC were isolated from WT mice that were fed control chow (n=4) or MCD diet (n=4) for 8 weeks, and OPN mRNA expression was analyzed by QRTPCR. Results are expressed as fold change relative to control chow derived LMNC. (C–D) Mouse primary HSC were cultured with CM from αGC-activated WT LMNC (αGC-CM) + OPN aptamers or sham aptamers for 48 hours; HSC expression of collagen I α1(C) and opn (D) mRNA were assessed by QRTPCR. Results are expressed as fold change relative to HSC that were treated with CM from vehicle-treated WT LMNC (V-CM). (E) OPN was quantified by ELISA in CM from LMNC that were harvested from WT, Jα18−/− or CD1d−/− mice and treated with either vehicle (V) or αGC to activate NKT cells. Results are expressed as fold change relative to vehicle (V)-treated WT LMNC. Mouse primary HSC were cultured for 48 h in CM from LMNC that were harvested from WT, Jα18−/−, and CD1d−/− mice and treated with vehicle or αGC to activate NKT cells. HSC expression of collagen I α1 mRNA was assessed by QRT PCR (F). Results are expressed as fold change relative to HSC that were cultured with CM from respective vehicle-activated LMNC. Mean ± SEM of duplicate experiments are graphed. *P<0.05
Figure 5
Figure 5. αGalactosylceramide (αGC)-activated liver NKT cell associated Hedgehog ligand contributes to hepatic stellate cell (HSC) activation
Primary cultures of mouse HSC were cultured for 48 h with conditioned media (CM) from vehicle-treated (V-CM) or αGC-activated (αGC-CM) mouse primary liver mononuclear cells (LMNC) (1 × 105) + Hh neutralizing antibody (5E1), a Hh signaling antagonist (GDC) or control vehicle (DMSO, dimethyl sulfoxide). QRTPCR analysis was used to quantify effects on expression of (A) Collagen I α1 and (B) OPN mRNA. Results are expressed as fold change relative to HSC that were cultured in CM from vehicle-treated LMNC (V-CM). Mean ± SEM of duplicate experiments are graphed. *P<0.05.
Figure 6
Figure 6. NASH progression in humans is accompanied by upregulation of liver and plasma OPN
(A–B) Coded liver sections from 10 patients with early and advanced NASH fibrosis were stained for OPN, and OPN expression was quantified by computer-assisted morphometry. (A) Representative photomicrographs from patients with early and advanced NASH fibrosis. (B) Quantitative analysis of liver OPN-positive cells in all patients. Numbers of OPN-positive cells are expressed as percentage of stained cells per high-powered field. (C) Plasma was collected from individuals with early or advanced NASH (n=25/group) and plasma OPN measured by ELISA. Plasma samples were run in duplicate. Mean ± SEM are graphed; * p<0.001 vs early fibrosis.
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