NKT associated Hedgehog and Osteopontin drive fibrogenesis in nonalcoholic fatty liver disease

Abstract

Immune responses are important in dictating nonalcoholic 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 harbor large numbers of NKT cells. Here, we evaluated the hypothesis that activated NKT cells drive fibrogenesis during NASH 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. When fed methionine choline deficient (MCD) diets for 8 weeks, WT mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Jα18−/− and CD1d−/− mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNC) from MCD diet-fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated hepatic stellate cells (HSC) to become myofibroblasts (MF); neutralizing these factors abrogated the fibrogenic actions of WT LMNC. LMNC from NKT cell deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSC. 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. In 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.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of hepatic dysfunction. Compared to individuals with simple hepatic steatosis, individuals with steatosis plus hepatocyte injury, cell death and inflammatory changes (dubbed nonalcoholic steatohepatitis, NASH) are more likely to develop progressive liver fibrosis, cirrhosis and cancer (1, 2). Although the mechanisms which drive disease progression remain obscure, recent studies suggest that cells of the innate immune system, such as Kupffer cells, natural killer T (NKT), and NK cells, may participate (3–6).

The liver contains a large number of NKT cells, which provide intravascular immune surveillance (7, 8). NKT cells respond to glycolipid antigens presented by MHC class 1-like molecule, CD1d, and when activated, secrete both Th1 (pro-inflammatory / anti-fibrotic) and Th2 (anti-inflammatory / pro-fibrotic) cytokines (8–11). In the hepatitis B virus-transgenic mouse (12), NKT-derived IL4 and IL13 were reported to be responsible for the fibrogenic outcomes. Conversely, CD1d knockout (CD1d−/−) mice, which lack NKT cells, were protected from xenobiotic-induced hepatic inflammation, injury and fibrosis (13). These findings complement human studies showing NKT accumulation in primary biliary cirrhosis (14, 15) and chronic viral hepatitis C (16).

The role of NKT cells in NAFLD progression is only beginning to emerge. NKT cells are generally depleted in fatty livers (17–19), but appear to accumulate in livers with NASH-related fibrosis (19, 20). Recently, we reported that Hedgehog (Hh)-mediated accumulation of natural killer T (NKT) cells contributes to development and progression of NASH in mice and humans (21). Mice with an overly active Hh pathway express higher levels of the NKT cell chemokine, CXCL16, the adhesion molecule VCAM-1, and IL15, a factor that promotes NKT viability (22). These mice accumulate more hepatic NKT cells, and develop worse NASH fibrosis. Consistent with this, we found that individuals with NASH-cirrhosis harbored 4 fold more NKT cells than those with healthy livers.

In addition to secreting the classical cytokines, IL4 and IL13, NKT cells can also secrete osteopontin (OPN), a cytokine and matricellular protein that exacerbates Concavalin A induced hepatitis (23), and promotes NAFLD progression (24). Recently, we showed that NKT cells also secrete the fetal morphogen, Sonic Hh (Shh) (25). Shh induces the transition of quiescent hepatic stellate cells (HSC) into activated, collagen-producing myofibroblasts (26), and amplifies the ‘repair-associated inflammatory response’ (27). Intriguingly, inhibition of the Hh pathway in vivo leads to the amelioration of fibrosis in mice with cholestatic liver injury (28).

In this study, we evaluated the hypothesis that fibrosis progression in NASH is NKT cell-dependent. We fed mice that were genetically deficient in NKT cells (Jα18−/− and CD1d−/−) methionine choline deficient (MCD) diets to induce NASH, evaluated if NKT depletion protected against NASH fibrosis, and assessed if NKT cell-mediated fibrogenesis required OPN and Hh. Findings in mice and cultured HSC were then corroborated by evaluating similar endpoints in patients with NASH.

Methods

A) Animal experiments

Mice

Wild type (WT) (Jackson Laboratories, Bar Harbor, ME), CD1d-deficient (CD1d−/−), and Jα18-deficient (Jα18−/−) mice (RIKKEN, Japan) of similar backgrounds (B6) were fed methionine-choline deficient (MCD) diet or control chow for 8 weeks. Animal care and procedures were approved by the Duke University Institutional Animal Care and Use Committee as set forth in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.

Analysis of Liver Architecture, Morphometry and Immunohistochemistry

Formalin-fixed paraffin embedded (FFPE)-livers were analyzed. H&E and picrosirius red staining with quantification by morphometric analysis were performed as previously described (24). Primary antibodies used: Osteopontin (OPN) (R&D, Minneapolis, MN, USA, AF808; 1:40), α Smooth muscle actin (αSMA) (Abcam, Cambridge, MA, USA, 5694; 1: 800). Polymer-HRP anti-rabbit (Dako; Carpinteria, CA, USA, K4003) and anti-goat (Santa Cruz; Santa Cruz, California, USA, sc-2768; 1:250) were used as secondary antibodies. Antigens were demonstrated by diaminobenzidine (DAB) (DAKO). Omitting primary antibodies eliminated staining, demonstrating specificity. For OPN and αSMA quantification, 15 randomly selected, 40× fields (excluding the major bile duct in each portal tract from consideration) were analyzed with the MetaView software.

ALT determination

Serum alanine aminotransferase (ALT) was measured using kits commercially available from Biotron Diagnostics (66-D; Hemet, CA) according to the manufacturers’ instructions.

RNA extraction and mRNA quantification

Total liver RNA extraction and mRNA quantification by Real time RT-PCR were performed as published (24). Details of primer sequences are in Supplemental Table 1.

Hydroxyproline Assay

Hydroxylproline content in whole liver specimens was quantified colorimetrically as previously described (29).

Effect of primary liver NKT cells on activation of primary HSC

Primary liver mononuclear cells (LMNC) were isolated as previously described (25), and cultured in complete NKT media (30), with or without the NKT cell ligand, αGalactosylceramide (αGC) (100 ng/ml; Axxora, San Diego, California, USA, Cat no 306027, CA), for 24 hours. αGC specifically activates iNKT cells (21, 31). αGC-activated LMNC conditioned medium (LMNC-CM) were then added to primary murine hepatic stellate cells (HSC), in the presence or absence of the Hh neutralizing antibody, 5E1 (10ug/ml; Iowa Hybridoma Bank), the Smoothened antagonist (inhibitor of Hh signaling) GDC (GDC-0449; Selleck, Houston), or vehicle for 48 hours and RNA was harvested for QRTPCR. Experiments were performed in duplicate wells and repeated twice.

In separate experiments, LMNC-CM was added to primary murine HSC with either sham-aptamers (OPN-R3-2; 100nmol/l) or OPN-aptamers (OPN-R3; 100nmol/l) (both synthesized by Dharmacon, Lafayette, CO) (24) for 48 hours, and RNA harvested as above. This concentration of OPN aptamer has been shown to inhibit adhesion, migration and invasion in the MDA-MB-231 breast cancer cell line (which highly expresses OPN and is a standard tool for evaluating OPN actions) (32).

ELISA assays for plasma OPN

Peripheral blood was collected at the time of sacrifice, and plasma obtained and stored at −80°C till analysis. Plasma OPN was measured using the commercially available OPN ELISA kit (R&D; Minneapolis, MN, USA, DY441) and in accordance with the manufacturer’s instructions. All samples were run in duplicate, and expressed as pg/ml. *P<0.05 vs. WT control mice.

B) Human subjects

Immunohistochemistry

FFPE liver sections of de-identified subjects with biopsy-proven NASH-related early and advanced fibrosis (n=10/group) from the Departments of Pathology at Duke University were studied in accordance with NIH and Institutional guidelines for human subject research.

Primary antibodies used were: OPN (R&D, AF1433, 1:40); Indian Hedgehog, Inh (Abcam, Ab39634, 1: 1000), Sonic Hedgehog, Shh (Epitomics; Burlingame, CA, USA, 1843-1, 1:7500); Gli2 (Genway; San Diego, CA, USA, 18-732; 1:4500), CD57 (NeoMarkers, Fremont, CA, USA, MS136, CA, USA). Polymer-HRP anti-mouse (Dako; K4001) was used as secondary antibodies. Antigens were demonstrated by diaminobenzidine (DAB) (DAKO). Omitting primary antibodies eliminated staining, demonstrating specificity.

Plasma OPN measurements

Peripheral blood was taken from patients with early (n=25) or advanced NASH (n=25) at the time of liver biopsies, and plasma stored at −80°C till analysis. Plasma OPN was measured using the commercially available OPN ELISA kit (R&D; DY1433) and in accordance with the manufacturer’s instructions. All samples were run in duplicate, and expressed as pg/ml. *P<0.05 vs. early NASH fibrosis.

Statistical analysis

Results are expressed as mean ± SEM. Statistical significance was determined using the Student’s t-test. Significance was accepted at the 5% level, *p<0.05.

Results

NKT-deficient mice develop less fibrosis than wild type (WT) mice after MCD diets

Compared to WT mice that were fed normal chow (n = 10), MCD diet-treated mice (n = 10) developed significant steatohepatitis and fibrosis after 8 weeks (Fig 1A, Supplemental Fig 1). The latter was demonstrated by increased Sirius red staining (Fig 1B– C), and hepatic hydroxyproline quantification (Fig 1D). Collagen deposition was accompanied by the accumulation of α–SMA-immunoreactive cells (Fig 2A–B), and induction of pro-fibrogenic genes, including α-sma, collagen 1α1, and TGFβ (Fig 2C–D, Supplemental Fig 2A).

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. 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

Jα18−/− mice which lack invariant NKT cells, developed significantly less hepatic fibrosis (Fig 1B–D, 2A–D) and injury (Fig 2E) than WT mice after MCD diet. This finding in mice that lack invariant NKT cells is consistent with our previous report that CD1d−/− mice (which lack all NKT subsets) exhibited reduced fibrogenic responses after diet induced NASH (21) (Fig 1C–D, 2B–D, Supplemental Fig 2A).

We reported that Hh pathway activation induces the transition of quiescent HSC to collagen-producing myofibroblasts (26), and promotes fibrogenic repair in NASH (29). Consistent with this, Indian Hh (a Hh ligand), and Gli2 (a Hh-regulated transcription factor) were significantly induced during diet-induced NASH (Supplemental Fig 2B–C). Conversely, NKT-deficient mice, which developed less fibrosis, exhibited attenuated Hh signaling (Supplemental Fig 2B–C).

NKT-deficient mice express less Osteopontin during MCD diet-induced NASH than WT mice

We recently showed that OPN acts in paracrine and autocrine fashions to promote HSC activation and fibrogenesis (24). After MCD diet, mice genetically deficient in OPN exhibited reduced fibrosis, while mice that over-expressed OPN developed worse fibrosis. Here, we show that livers from MCD fed WT mice upregulate OPN protein expression by nearly 5 fold (Fig 3A, C). In contrast, livers of both NKT cell deficient strains that developed less fibrosis than WT mice after diet-induced NASH (Fig 1B–D, 2A–D, Supplemental Fig 2) had significantly reduced levels of OPN (Fig 3A–C, Supplemental Fig 3). These findings raise the possibility that NKT cell associated factors modulate OPN expression and resultant hepatic fibrogenesis.

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

Previously, others reported that plasma OPN levels correlated with severity of liver fibrosis in subjects with chronic hepatitis B and C (33, 34). In this study, plasma from WT mice with fibrosing-NASH contained nearly 2 fold more OPN by ELISA than chow-fed controls (Fig 3D). Consistent with reduced hepatic OPN expression, plasma OPN levels were 2 fold lower in NKT cell deficient mice after diet-induced NASH. The aggregate data, therefore, suggest that elevated plasma OPN levels may be a biomarker of advanced NASH fibrosis.

Primary mouse liver NKT cell associated Osteopontin promotes myofibroblastic activation of primary hepatic stellate cells

In addition to HSC and cholangiocytes (24), OPN is expressed by activated immune cells (23) (Fig 3). Therefore, we examined if activated liver NKT cells generate OPN that acts in a paracrine fashion to promote HSC fibrogenesis. Primary liver mononuclear cells (LMNC) were isolated from healthy mice and incubated with αGC (which specifically activates iNKT cells) or vehicle overnight; LMNC-conditioned medium (CM) was then collected for OPN ELISA. αGC-activated LMNC-CM contained 4 fold more OPN protein than vehicle treated LMNC-CM, and similar levels of OPN as CM from cholangiocytes, which are known to be a source of OPN in liver (Fig 4A). To ascertain if activated LMNC in vivo also express higher amounts of OPN, we harvested primary LMNC from chow or MCD fed WT mice (n=4/group), and OPN expression was analyzed by QRTPCR. Primary LMNC from MCD fed mice harbored 12 fold more OPN mRNA than LMNC from chow fed mice (Fig 4B).

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

αGC-activated LMNC-CM was then added to primary cultures of mouse HSC with either OPN-aptamers or sham-aptamers for 48 hours. The addition of OPN-aptamers to neutralize OPN activity in LMNC-CM resulted in a significant repression of HSC fibrogenic genes (OPN, Collagen) (Fig 4C–D).

To ascertain if differences in the fibrogenic outcomes observed in Figure 1 could be attributed to fewer NKT cells, and hence, reduced NKT associated OPN, we isolated primary LMNC from WT, Jα18−/− and CD1d−/− mice, and incubated them with αGC or vehicle overnight (as above), and LMNC-CM was collected for OPN ELISA, or added to primary cultures of HSC. We found that WT-LMNC mice secreted 5 fold more OPN protein (Fig 4E) and induced greater collagen expression in HSC than NKT deficient-LMNC (Figure 4F). These findings, in concert, support the hypothesis that activated NKT cell associated OPN promotes HSC activation in a paracrine fashion.

NKT cell associated Hedgehog (Hh) promotes myofibroblastic activation of primary hepatic stellate cells

Previously, we had reported that progressive NASH is associated with Hh pathway activation (29), that activated hepatic NKT cells are capable of secreting Hh ligands (25), and that Hh ligands could directly induce HSC activation and fibrogenesis (26). Because OPN expression in HSC is regulated, at least in part, by Hh signaling (24), we examined if NKT derived Hh ligands could directly promote OPN expression in HSC and mediate fibrogenic responses. Primary WT LMNC were incubated with αGC or vehicle (as above); LMNC-CM was then added to primary cultures of mouse HSC, with or without Hh neutralizing antibody, 5E1, or GDC, a pharmacologic inhibitor of the Hh pathway. 48 hours later, HSC RNA was harvested for PCR analysis. We confirm by FACS analysis that primary hepatic NKT cells express the Hh ligand (Supplemental Fig 4A–B). CM from αGC-LMNC (that contained activated NKT cells) upregulated HSC expression of collagen and OPN (Fig 5A–B). The addition of 5E1 or GDC (inhibition of Hh signaling) led to a significant abrogation of these fibrogenic effects (Fig 5A–B). The aggregate data suggest that in addition to generating OPN (Fig 4A, E), activated hepatic NKT cells secrete Hh ligands to promote liver fibrogenesis.

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.

NASH progression in humans in accompanied by increased liver and plasma OPN

NAFLD progression in mice is associated with activation of the Hh pathway, induction of Hh-regulated fibrogenic factors, such as OPN (24), and the accumulation of intrahepatic NKT cells (19, 21). Herein we demonstrate that fibrogenesis in mice with diet-induced NASH depends upon hepatic NKT cells, and show the mechanism involves NKT cell-mediated production of Hh ligands and OPN (Fig 1–5). NKT cells comprise a much greater proportion of the resident hepatic immune cells in mice than humans (4, 35). Therefore, we examined livers from patients with NASH to assess the relationship between hepatic NKT cell accumulation, Hh pathway activation, OPN expression and fibrosis in humans.

Human livers with advanced NASH (stage 3–4 fibrosis) contained 3 fold more of the Hh-regulated cytokine, OPN, particularly in the fibrous septa, than those with early NASH (no fibrosis or stage 1–2 fibrosis) (Fig 6A–B). Livers from individuals with advanced NASH also contained more cells that expressed the Hh ligands, Shh and Ihh (Supplemental Figure 5A–C), and the Hh-regulated transcription factor, Gli2 (Supplemental Figure 6A). Previously, we reported that the hepatic content of cells that co-express CD57 and CD3 or CD56 and CD3 (i.e., NKT cells) is increased significantly in patients with NASH-related cirrhosis (21). Here we show that NKT cells that accumulate during fibrosing NASH express Ihh and OPN protein (Supplemental Figure 6B–E). Moreover, we found that increased hepatic OPN expression was mirrored by higher plasma OPN levels in NASH patients with advanced fibrosis than those with mild fibrosis (Fig 6C). Overall plasma OPN levels in NASH were approximately 2 fold higher than levels detected in healthy volunteers (mean OPN level, normal: 3000pg/ml) (33). The findings in humans with NASH, therefore, are similar to those in mice with NASH (Fig 3) and together suggest that OPN may be a useful biomarker of advanced NASH fibrosis.

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.

Discussion

This study shows that hepatic NKT cells are critical drivers of fibrogenesis in NASH, and demonstrates that the mechanism involves NKT cell-mediated stimulation of hepatic Hh pathway activity and resultant increases in the liver content of OPN, a Hh-regulated, pro-fibrogenic factor. Importantly, plasma levels of OPN reflect the activity of this process in liver and thus, may prove to be a useful non-invasive marker of advanced NASH.

Previously, we reported that Hh pathway activity correlates with severity of NAFLD-related liver injury (29), and showed that Hh signaling enhances hepatic accumulation of immune cells, including CD1d-restricted NKT cells (21). Here, we prove that fibrogenesis in mice with diet-induced NASH directly depends upon NKT cells. Compared with WT mice, two strains of NKT cell deficient mice (Jα18−/− and CD1d−/−) exhibited dramatically attenuated fibrosis after 8 weeks of MCD diets. NKT cell deficient mice also demonstrated blunted Hh pathway activity (less expression of Hh ligand and the Hh-regulated transcription factor, Gli2), and reduced expression of OPN, a Hh-regulated cytokine which acts in both paracrine and autocrine manners to promote HSC activation and fibrogenesis (24).

In the present study, liver mononuclear cells (LMNC) from WT mice with diet-induced NASH expressed 12 fold higher levels of OPN mRNA than chow-fed controls. Conditioned medium (CM) from WT mice-derived LMNC that were treated with αGC to activate resident NKT cells stimulated cultured hepatic stellate cells (HSC) to express collagen mRNA, whereas similarly treated LMNC from two different strains of NKT cell deficient mice did not. These results indicate that activated liver NKT cells promote the production of factors that drive fibrogenic responses in HSC, and suggest that one of these pro-fibrogenic factors might be OPN. Indeed, ELISA demonstrated that CM from αGC-activated WT LMNC contained four times more OPN than CM from similarly-treated LMNC populations from NKT cell deficient mice. Moreover, neutralizing OPN activity with OPN-specific RNA aptamers significantly abrogated the HSC fibrogenic responses to WT LMNC-derived factors, reducing HSC expression of collagen mRNA to basal levels. The aggregate findings, therefore, complement and extend earlier evidence that liver NKT cells are capable of producing OPN by showing that important fibrogenic actions of NKT cells are directly mediated by OPN (23, 25, 36).

OPN expression is induced by activating Hh signaling in Hh-responsive cells (24). We previously reported that NKT cells produce and respond to Hh ligands (25, 36). In the present study we show that adding the pan-Hh-neutralizing antibody (5E1) to LMNC-CM completely blocked its ability to up-regulate expression of collagen mRNA in HSC. CM from LMNC of NKT cell deficient mice is also completely unable to induce collagen gene expression in cultured HSC. Thus, NKT cells appear to be a major source of the Hh ligands that drive fibrogenic responses in HSC. The latter likely involves both NKT cell-derived OPN and HSC-derived OPN because treating HSC that were grown in LMNC-CM with a drug that blocks intra-cellular Hh signaling (GDC) reduced HSC expression of OPN mRNA. Thus, fibrogenesis in NASH seems to result from the combined actions of several autocrine/paracrine signaling loops that re-enforce Hh pathway activation and OPN production in different types of hepatic non-parenchymal cells, particularly NKT cells and HSC.

Our studies in human subjects support the pathophysiological relevance of excessive OPN production and Hh pathway activation during NASH-related fibrogenesis. Notably, we provide novel evidence that the upregulation of liver OPN production in advanced NASH is mirrored by increased plasma OPN levels. Recent reports suggest that high plasma OPN levels may also be predictive of cirrhosis in patients with chronic hepatitis B and C (33, 34). In addition, elevated plasma OPN levels are associated with cancer development, recurrence, and bad prognosis (37, 38). Given these observations, it is conceivable that plasma OPN levels might be used to identify NASH patients who are at high risk of bad outcomes. Further studies, in a larger cohort of individuals with NAFLD, will be necessary to confirm the clinical utility of this novel biomarker.

Summary Box.

What is known about this subject?

• Immune responses are important in dictating liver disease outcomes

• Natural killer T (NKT) cells accumulate in fibrosing nonalcoholic steatohepatitis (NASH) of mice and humans

• Hedgehog pathway activation occurs in nonalcoholic fatty liver disease progression

• Osteopontin is a proximal effector of Hedgehog and promotes hepatic stellate cell activation

What are the new findings?

• Livers from NKT deficient (CD1d−/− and Jα18−/−) mice contained significantly less Hedgehog and Osteopontin, and had dramatically less fibrosis than wild type mice after diet-induced NASH

• Human NASH livers with advanced fibrosis contain more NKT cells, and more Hedgehog and OPN protein than NASH livers with early fibrosis

• NKT cell activation is associated with increased expression of Hedgehog ligands and Osteopontin

• Plasma Osteopontin mirrors hepatic Osteopontin content

• Plasma Osteopontin may be a useful biomarker of advanced NASH fibrosis

How might it impact on clinical practice in the foreseeable future?

• Modulating NKT cell function or numbers may be a useful strategy to inhibit fibrosis progression

• Plasma Osteopontin may be used as one of the biomarkers of advanced NASH fibrosis

Abbreviations

αGalCer

alpha-Galactosylceramide

α–sma

alpha-smooth muscle actin

FACS

fluorescent activated cell sorting

FFPE

formalin-fixed paraffin embedded

Gli

glioblastoma

Hh

Hedgehog

iNKT

invariant natural killer T

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

NKT

natural killer T

MCD

methionine choline deficient

OPN

osteopontin

Shh

sonic hedgehog

Tgf β

transforming growth factor beta