Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of NKT cells, which comprise a substantial sub-population of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of pro-fibrogenic iNKT cell populations during certain types of fibrosing liver damage.
Many types of chronic liver disease are associated with hepatic accumulation of leukocytes, including various types of lymphocytes, monocytes, and macrophages. These cells are thought to contribute to liver injury by producing inflammatory mediators and exerting direct cytotoxic actions on hepatic epithelial cells. They may also modulate liver repair responses by modulating local accumulation of pro-fibrogenic cytokines 1–3
NKT cells are specialized T cells that respond to glycolipid antigens (as opposed to typical peptide immunogens) 2, 4–8. The precise identity of the endogenous glycolipid antigen(s) for NKT cells remains controversial, but it is generally acknowledged that glycolipids are presented to NKT cells by CD1d molecules on antigen-presenting cells 9, 10. Several types of resident liver cells, namely hepatocytes, bile ductular cells, and various sinusoidal lining cells, including hepatic stellate cells, are capable of presenting CD1d-associated glycolipids to NKT cells 3, 11–13, which comprise the largest sub-population of lymphocytes in murine livers 2, 14–18. NKT cells are also found in human livers, although at lower frequencies when compared with mice 19–21. Whereas the murine hepatic NKT cell population is comprised predominately of classical, invariant NKT (iNKT) cells, the human hepatic NKT cell population includes a larger proportion of non-classical CD8+ and/or γδTCR NKT cells 19, 22, 23.
In both mice and humans, NKT cells are believed to contribute to certain types of liver damage. For example, portal tract accumulation of NKT cells has been demonstrated in patients with primary biliary cirrhosis 24, 25. A role for NKT cells in disease pathogenesis/progression is supported by studies showing that mice with genetic NKT cell deficiency are protected from experimental primary biliary cirrhosis 26. NKT cell accumulation has also been associated with disease progression in patients with chronic hepatitis C 3, 27, and parallels the evolution from chronic hepatitis to fibrosis and cirrhosis. Conversely, mice with relatively stable, obesity-associated hepatic steatosis that do not advance to fibrosis/cirrhosis have a lower frequency of intrahepatic NKT cells 28–30. The association between liver disease progression and the size of hepatic NKT cell populations suggests that common factor(s) might mediate both responses.
Whereas the mechanisms that control NKT cell accumulation during liver damage remain poorly understood, fibrogenic responses to diverse types of adult liver injury are mediated, at least in part, by reactivation of the hedgehog (Hh) pathway in resident hepatic stellate cells and ductular-type hepatic epithelial progenitors 31–33. Hh ligands are pleiotropic morphogens that regulate the viability and differentiation of many types of progenitor cells, including those required for thymic and lymphoid system development 34–37. Adult NKT cells are thought to be derived from thymic precursors and may undergo terminal differentiation either prior to their release from the thymus or after redistributing to peripheral depots, such as the liver 38–41.
Whether or not NKT cells produce or respond to Hh ligands in adults has not been reported, but might be relevant to the pathogenesis of cholestatic liver damage because both Hh pathway activation 31, 33, 42 and NKT cell accumulation 13, 43, 44 characterize biliary injury. NKT cells might also generate Hh ligands, as has been shown recently for adult CD4+ T lymphocytes in peripheral blood 36. Secretion of Hh ligands by NKT cells could provide a mechanism by which NKT cell accumulation might contribute to liver fibrogenesis, because Hh ligands are known to enhance the viability and growth of myofibroblastic hepatic stellate cell populations. Conversely, if NKT cells, themselves, were proven to be Hh-responsive, this might explain why they accumulate as biliary fibrosis advances, because the latter is generally associated with dramatic expansion of resident liver cell types that produce Hh ligands 45, 46. The current study reports that adult iNKT cells produce and respond to Hh ligands, and provides novel evidence that the Hh pathway regulates iNKT cell activation and cytokine production. Hence, Hh signaling likely modulates both the size and the actions of iNKT populations during liver damage.
DN32 iNKT cells produce and respond to Hh ligands
To begin our investigation of possible Hh reactivity in NKT cells, DN32 cells, a mouse iNKT cell line 47, were evaluated for expression of sonic hedgehog (Shh, a Hh ligand), Patched (ptc, the cell surface Hh receptor), and glioblastoma-2 (Gli2, a Hh-regulated transcription factor). Expression of Shh was demonstrated by immunocytochemistry (Fig. 1A) and Western blot (Fig. 1B). The latter approach also revealed that iNKT cells produce both full-length (45 kD) Shh precursor and truncated (20 kD) Shh, which is the signaling competent form of the Hh ligand. Subsequent flow cytometry showed that>80% of the iNKT cells in this line produce Shh constitutively (Fig. 1C and D and Supporting Information Fig. 1) and also express both ptc (Fig. 1E) and Gli2 (Fig. 1F), demonstrating the potential to transduce intracellular Hh-initiated signals.
Shh treatment alters the phenotype of DN32 iNKT cells
To determine whether or not Shh influences the behavior of iNKT cells, DN32 iNKT cells were treated with recombinant Shh (0–1000 ng/mL) for 72 h. Each well was initially seeded with 1×105 cells and by the end of the treatment period, cell proliferation had occurred in all wells, including wells treated only with vehicle (Shh, 0 ng/mL), which showed a threefold increase in cell number. Addition of Shh (10–1000 ng/mL) evoked further dose-related increases in iNKT cell numbers (Fig. 2A). However, this effect was minor, and reached statistical significance only in wells that received the highest Shh dose (1000 ng/mL). The latter showed a 20% increase in cell numbers over vehicle-treated (control) wells, indicating that Shh (Hh ligand) promotes iNKT cell proliferation. Treatment with Shh (10–1000 ng/mL) resulted only in minor (non-statistically significant) reductions in caspase 3/7 activity. However, when cells were treated with 5E1 (10 μg/mL) antibody to neutralize endogenous Shh, basal caspase 3/7 activity increased by almost twofold (Fig. 2B). Flow cytometry confirmed that 5E1 antibody increased the percentage of Annexin V-positive cells (Supporting Information Fig. 2), suggesting that endogenously produced Shh (Fig. 1) functions as an autocrine factor to maintain iNKT cell viability. This autocrine loop could explain why treating DN32 iNKT cells with exogenous Shh had only a small effect on growth or survival.
iNKT cells are capable of producing both pro-inflammatory (Th1) and anti-inflammatory/pro-fibrogenic (Th2) cytokines that affect the local outcome of liver cell injury 48–50. To determine if the Hh pathway regulated iNKT cell cytokine production, DN32 iNKT cells were treated with various doses of Shh; RNA and conditioned medium were harvested and analyzed for changes in prototypical Th1 cytokines (IFN-γ), Th2 cytokines (IL-13 and IL-4) and IL-10. Shh at 100 ng/mL significantly increased IL-13 secretion, but had little effect on IL-4, IL-10 or IFN-γ(Fig. 2C).
Given that the effects of Shh on IL-13 secretion plateaued between 100 and 1000 ng/mL, doses of Shh ranging between 0 and 100 ng/mL were used to investigate whether Shh influences expression of cytokines and SOCS. Seventy-two-hour incubation with 100 ng/mL Shh elicited a twofold increase in IL-13 mRNA, but did not significantly affect IL-4, IL-10 or IFN-γFig. 2D) These results suggest that Hh pathway activation promotes a selective increase in iNKT cell secretion of IL-13, a pro-fibrogenic (Th2) cytokine.
The balance between production of pro-inflammatory (Th1) and anti-inflammatory/pro-fibrogenic (Th2) cytokines is regulated by the relative predominance of SOCS-2 and SOCS-3, with the former favoring Th1 cytokine production and the latter promoting Th2 cytokine polarization 51–53. To assess if Shh influences this balance, DN32 iNKT cells were treated with Shh (0–100 ng/mL) and mRNA levels of SOCS-2 and SOCS-3 measured by quantitative RT-PCR (qRT-PCR) after 72 h. Shh increased expression of both SOCS-2 and SOCS-3 but the magnitude of the responses differed. Expression of SOCS-2 mRNA increased ∼sixfold after Shh treatment (Fig. 2E), whereas a similar dose of Shh stimulated ∼50-fold induction of SOCS-3 mRNA (Fig. 2F).
Primary mouse hepatic iNKT cells produce Shh
To verify that DN32 iNKT cells are appropriate model cells to study intrahepatic iNKT cells, studies were repeated with primary cells that were isolated from livers of healthy adult mice. For each experiment, liver leukocytes were pooled from at least eight mice and all experiments were repeated at least three times, such that the final data reflect information gathered on three separate occasions from a total of at least 24 mice. Results showed that more than half of the intrahepatic CD1d-tetramer-reactive iNKT cells produce Shh (Fig. 3A and B and Supporting Information Fig. 3).
Shh regulates the proliferation and viability of primary mouse NKT cells
To determine if manipulating Hh activity influenced NKT cell growth and/or viability, primary mouse iNKT cells were exposed to Shh (0–1000 ng/mL) or treated with anti-Shh antibody (5E1; 10 μg/mL) to neutralize endogenous Shh activity. Treatment with exogenous Shh evoked dose-dependent increases in the proliferation of primary mouse iNKT cells (Fig. 3C). Compared with DN32 iNKT cells, primary liver iNKT cells were more sensitive to the proliferative effects of exogenous Shh; treatment with Shh 1000 ng/mL elicited a twofold increase in primary liver iNKT cells, compared with a 20% increase in DN32 cells (Fig. 2A). Exogenous Shh (10–1000 ng/mL) had no effect on apoptosis of either DN32 cells or primary iNKT cells (Fig. 3D). However, neutralization of endogenous Shh by treatment with anti-Shh antibody markedly increased NKT cell apoptosis, (Fig. 3D) resulting in reduced cell numbers (Fig. 3C). Studies were repeated using a more physiologically relevant model of iNKT cell activation in which α-galactosylceramide (αGalCer)-primed antigen-presenting cells were used to activate primary liver iNKT cells in the absence or presence of 5E1 antibody 54. Neutralization of endogenous (iNKT-cell-generated) Shh with 5E1 antibody significantly increased the numbers of iNKT cells that labeled with Annexin V (Supporting Information Fig. 4). Thus, mouse liver iNKT cells respond to Shh by increased proliferation and reduced apoptosis, suggesting that Shh functions as an autocrine viability factor for primary mouse liver iNKT cells that would promote the expansion of Hh-responsive liver iNKT cell populations.
Shh promoted IL-13 secretion in DN32 iNKT cells (Fig. 2). To determine if it has a similar effect on mouse primary hepatic iNKT cells, the latter were treated with Shh (10–1000 ng/mL) and expression of Th1 and Th2 cytokines measured. Shh stimulated mouse primary liver iNKT cells to secrete IL-13 (Fig. 3E), but did not change levels of IFN-γ, IL-10, or IL-4. Thus, Hh pathway activation reproducibly stimulates IL-13 secretion by rodent hepatic iNKT cells.
Human iNKT cells are Hh-responsive
Although qualitative and quantitative differences in hepatic NKT cell populations have been noted between mice and humans, both species are known to harbor iNKT cells 15, 47, 55. To screen human iNKT cells for Hh reactivity, iNKT cells were isolated from the peripheral blood of normal volunteers. RNA was obtained from the pooled cells and examined for the expression of ptc and Gli1, two Hh-target genes. Results were compared with expression of ptc and Gli1 in LX-2 cells, a human stellate cell line that has constitutive Hh pathway activity 56. We found that human iNKT cells express both Hh-target genes (Fig. 4A and B), demonstrating that they also possess an active Hh pathway.
iNKT cells are cytotoxic and pro-fibrogenic when cultured with Hh-producing bile ductular cells
Many types of chronic fibrosing liver disease are accompanied by the accumulation of ductular-type cells, myofibroblasts, and inflammatory cells within fibrous septa 57. Previously, we reported that ductular cells and myofibroblasts are a rich source of Hh ligands 58. These cell types also use Hh ligands to regulate each other's growth and viability in a paracrine fashion 31, 33. The present studies suggest that iNKT cells might participate in paracrine regulation of the fibroductular response. To explore this concept, we compared cytokine production and cell viability in DN32 iNKT cells and cholangiocyte (603B cells) monocultures and cholangiocyte-DN32 iNKT cell co-cultures. Compared with monocultures of either DN32 iNKT cells or cholangiocytes, co-cultures of DN32 iNKT cells and cholangiocytes produced tenfold more IL-2 (Fig. 5A), 5–6-fold more IL-13 (Fig. 5B), and 6–7-fold more IL-4 (Fig. 5C), whereas expression of IFN-γ (Fig. 5D) and IL-10 (Fig. 5E) remained relatively constant. Enhanced cytokine production was only observed when GalCer, a known CD1d-presented glycolipid antigen, was added to co-cultures, demonstrating that cytokine production required iNKT cell activation by antigen presented by cholangiocytes. Together with the earlier evidence that Hh ligands promote pro-fibrogenic cytokine secretion by iNKT cells (Fig 2 and 3), these results support the concept that hepatic iNKT cells contribute to the fibroductular response in some types of chronic liver disease.
Hepatic accumulation of iNKT cells occurs in rodents during cholestatic liver injury induced by bile duct ligation (BDL) 59, and ptc+/− mice that have an overly active Hh pathway due to haplo-insufficiency of the Hh signaling inhibitor, ptc 60, develop increased fibroductular response to BDL compared with WT ptc+/+ mice 31. To determine if ptc+/− mice also have more liver iNKT cells, we compared the size of liver iNKT cell populations in ptc+/− mice and their WT littermates. The livers of ptc+/− mice harbored twice as many iNKT cells compared with ptc+/+ littermate controls (p<0.05). Thus, findings in vivo are consistent with the in vitro evidence that Hh signaling promotes the accumulation of liver iNKT cells.
To more directly evaluate the impact of liver iNKT cell accumulation on liver injury, we co-cultured DN32 iNKT cells with cholangiocytes. Co-culture with DN32 NKT cells resulted in a relatively rapid disruption of the cholangiocyte monolayers (Fig. 6A–D). This finding was unanticipated because our earlier results demonstrated that iNKT cells produce Hh ligands (Shh) (Fig. 1 and 3), which generally enhance the growth and survival of cholangiocytes 31. An alternative explanation was that Shh treatment of iNKT cells promotes a more cytotoxic phenotype. In support of this, treatment of DN32 iNKT cells with Shh (10 ng/mL) increased Hh signaling, as evidenced by up-regulation of Gli1 mRNA expression (Fig. 7A), and resulted in significant increases in mRNA expression of several iNKT cell activation markers, including CD69 (Fig. 7B), and particularly the TNF super-family members, CD154 (Fig. 7C), and CD178 (FasL, Fig. 7D), both of which are implicated in the ability of effector cells to kill target cells including cholangiocytes 44, 61. Thus, although additional research is required to establish cause–effect relationships, the net effect of expanding hepatic populations of Hh-responsive iNKT cells might be ductular destruction.
Although there is little debate that the liver is both a normal reservoir of leukocytes, as well as a target for immune cell attack during certain types of liver disease, the mechanisms regulating interactions between immune cells and liver epithelial cells are not well understood. Hepatic accumulation of lymphocytes is a key feature of chronic hepatitis 62, 63, and chronic hepatitis is a major risk factor for progressive liver fibrosis in liver diseases of diverse etiologies 64–66. Liver lymphocyte populations in healthy livers are heterogeneous, but include sizeable sub-populations of NKT cells 2, 15. NKT cells are specialized lymphocytes that are selectively activated by glycolipid antigens presented by CD1 molecules on the surface of antigen-presenting cells 2, 4, 5. Many types of liver cells, including cholangiocytes and hepatic stellate cells, express CD1 and are capable of presenting antigen to NKT cells 3, 11–13. The fact that ductular cells and hepatic stellate cells can interact with and activate NKT cells might have important pathogenic implications because both of the former cell types progressively accumulate as liver fibrosis progresses.
We reported that immature ductular cells and myofibroblastic hepatic stellate cells produce and respond to Hh ligands 31–33, and showed that Hh pathway activation promotes ductular cell proliferation and liver fibrosis during liver injury 42. In damaged livers, lymphocytes typically accumulate in and around fibrous septa that are comprised of proliferating ductules and myofibroblasts 57. In the present study, therefore, we investigated the possibility that NKT cells, which comprise a substantial fraction of liver lymphocyte populations 2, 15, might be Hh-responsive. Our results provide novel information about this issue. Although the Hh pathway plays a critical role in thymic development 34 and regulates lymphopoeisis 35–37, to our knowledge, no information has been published about Hh signaling in adult iNKT cells. Our work demonstrates, for the first time, that iNKT cells are capable of producing Shh. This discovery has important implications for adult liver repair because hepatic accumulation of iNKT cells would, therefore, be predicted to support the outgrowth of Shh-responsive myofibroblasts, thereby enhancing fibrotic responses to liver injury. In addition, we showed that iNKT cells are, themselves, Hh-responsive, relying on Shh for their own growth and survival. Thus, the Shh-rich microenvironment that develops during many types of fibrotic liver disease would be predicted to promote expansion of hepatic iNKT cell populations. This might help explain earlier observations that numbers of hepatic iNKT cells increase with progression of fibrosis in primary biliary cirrhosis and chronic hepatitis C 3, 24–26.
Interestingly, we also discovered that Shh stimulates iNKT cells to acquire an activated, more cytotoxic phenotype, while increasing their production of IL-13 in vitro. The integrity of the ductal epithelial barrier becomes compromised in many types of chronic liver injury and this is thought to permit “regurgitation” of toxic bile acids into the parenchyma 67, 68. Hepatic accumulation of Shh-responsive iNKT cells may contribute to this process by promoting duct disruption. This concept is supported by recent publications, which reported that genetic or acquired depletion of hepatic NKT cells protects mice from cholestatic liver damage 22, 59. Our finding of increased iNKT cells in the livers of ptc+/− mice, which have an overly active Hh pathway 60 and develop an exaggerated fibroductular response to BDL 31, provides further evidence that hepatic iNKT cells influence the outcomes of biliary injury. The finding that Shh induced increased expression of CD154 and Fas-L on iNKT cells provides a mechanism to explain the enhanced killing of cholangiocytes because these TNF family members act in a cooperative way to increase apoptotic death of cholangiocytes in response to effector cells 61. Data showing that Shh stimulates iNKT cells to produce IL-13 may also be pertinent to this issue because IL-13 is a major fibrogenic cytokine and plays a pivotal role in hepatic fibrosis 65, 69. Thus, accumulation of Hh-sensitive immune cells that generate IL-13 may also be an important mechanism for increasing local production of this potent fibrogenic factor.
In summary, our results identify a novel mechanism that regulates immune responses to adult liver injury, namely Hh pathway activation. Our findings also suggest that both chronic hepatitis and progressive liver fibrosis might be outcomes of increased Hh signaling. Although further research will be necessary to prove (or disprove) this hypothesis, the existing data support a model for disease progression in which activation of Hh signaling in various types of resident liver cells in injured livers (e.g. hepatic stellate cells, certain ductular cells, and some immune cells) triggers a variety of self-re-enforcing/feed-forward mechanisms that perpetuate accumulation of immune cells and epithelial damage (i.e. chronic hepatitis), as well as expansion of myofibroblast populations and matrix deposition (i.e. fibrosis). If validated by future research, this model suggests novel diagnostic and therapeutic targets, and may also prove to be helpful in predicting the outcomes of certain types of liver injury.
Materials and methods
Murine cholangiocyte 603B line 70 was kindly provided by Yoshiyuki Ueno (Tohoku University, Sendai, Japan) and G. Gores (Mayo Clinic, Rochester, MN). The murine iNKT hybridoma cells (DN32) was provided by Dr Albert Bendelac (University of Chicago, Chicago, IL) and human hepatic stellate cell line (LX2) was obtained from Dr. S.L. Friedman (Mount Sinai School of Medicine, NY) 71.
C57BL/6 (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME). B6.129 Sv/J ptc+/− and ptc+/+ littermates were obtained from Dr. R.J. Wechsler-Reya (Duke University Medical Center, NC). ptc+/− mice have only one copy of ptc, a Hh pathway repressor. Therefore, they are unable to silence Hh signaling and exhibit excessive Hh pathway activity 60. Mice are maintained in a temperature- and light-controlled facility, and permitted ad libitum consumption of water and standard pellet chow. Animal care and procedures were approved by the Duke University Medical Center 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.
Primary mouse hepatic NKT cell isolation and culture
Primary hepatic leukocytes were isolated from C57BL/6 mice following a series of enzymatic digestion (collagenase 0.05%, DNAse I 0.002%), mechanical digestion (Seward Stomacher 80; Biomarker Lab systems) and 30% Percoll density gradient. For each experiment, leukocytes were isolated from eight healthy adult mice and then pooled. All experiments were replicated at least one time. Hence, over 100 mice were used for to assess expression of Hh signaling components and analyze effects of Hh pathway activation on primary hepatic iNKT cells (see below).
Immediately after isolation, hepatic leukocytes were washed, re-suspended in flow cytometry buffer (eBiosciences) (1×106 cells/mL) and incubated with anti-mouse CD16/32 (Mouse Fc Block, 1 μg/106 cells; BD) for 30 min. Leukocytes were then stained with FITC-conjugated CD3 (Santa Cruz; sc18843) and PE-conjugated PBS57loaded-CD1d-tetramers (provided by NIH, Atlanta, GA) and sorted using FACS. FACS was performed at the flow cytometry core facility at the Human Vaccine Institute, Duke University Medical Center, using the FACS VantageSE (Beckton Dickinson). Cells were kept at 4°C throughout the purification protocol. Primary iNKT cells were sorted as CD3+ CD1d-tetramer+ double-positive cells. Purity of sorted NKT fractions was checked by FACS re-analysis (>90% purity).
Primary hepatic iNKT cells were cultured in complete NKT media (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL streptomycin and 100 units/mL penicillin, 10 mM HEPES, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate and 5.5 uM 2-mercaptoethanol) 72. For growth and viability assays, cells were cultured in CD3-coated, sterile, 96-well, black, tissue culture plates (Costar 3603, Corning, NY), at a concentration of 1×105 cells per well, with recombinant mouse IL-2 (10 ng/mL; Biolegend), recombinant mouse IL-12, (1 ng/mL; R&D) and anti-CD28 (1μg/mL; eBiosciences).
In additional experiments, primary hepatic leukocytes were incubated in RPMI with the NKT cell ligand, αGalCer (100 ng/mL), in the absence of anti-CD3, anti-CD28, IL-2 and IL-12, for 24 h. 100 ng/mL of αGalCer was used in experiments, as this dose has been shown to elicit the maximum iNKT responses 54, 73. The co-expression of CD3 and CD1d-tetramers was used to identify iNKT cells within each culture.
In experiments where recombinant Shh protein (0–1000 ng/mL) (StemCell Tech, Canada), 5E1-neutralizing antibody (10 μg/mL) (Iowa Hybridoma bank, University of Iowa) or isotype control antibodies were utilized, these were added at the initiation of cell-culture. As the range of Shh concentration in disease states is currently unknown, we have utilized a spectrum of Shh dosing (0–1000 ng/mL) as previously described 36, 74. In all experiments, cultures were harvested for analysis 24 or 72 h later, as specified. In each experiment, all assesses were performed in triplicate. Every experiment was replicated at least one time.
DN32 hybridoma cells were cyto-spun onto VWR superfrost® plus micro slides (VWR) using the Shandon Cytospin 4 (Thermo Scientific, UK) at 300 rpm for 3 min. Slides were air-dried and then fixed with cold (−20°C) methanol for 5 min. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide and non-specific binding of antibodies blocked using Dakocytomation serum-free protein block (Dako). Slides were then incubated with primary antibodies over night in 4°C. After washing with TBS-Tween20 0.1%, HRP-conjugated secondary antibodies were added for 30 min. Antigens were detected by the addition of liquid diaminobenzidine substrate (Dako), with haematoxylin (Sigma, MHS16) counterstain. Isotype-matched antibodies were used as negative controls. Primary antibodies used were as follows: Shh H-160 (200 μg/mL, 1:100 dilution; Santa Cruz), ptc G19 (200 μg/mL, 1:50 dilution; Santa Cruz) and Gli2 (1 mg/mL, 1:50 dilution; abCam). Secondary antibodies used were as follows: ECL™ donkey anti-rabbit IgG HRP-linked whole antibody (1:1000 dilution, Amersham, GE Healthcare, UK) and donkey anti-goat IgG HRP-linked antibody (200 μg/mL, 1:1000 dilution; Santa Cruz).
DN32 hybridoma cells were homogenized using standard RIPA buffer (TBS, 1% NP-40, 0.1% SDS) containing Protease Inhibitor Cocktail Tablets from Roche (Indianapolis, IN). Protein concentration was measured using BCA Protein Assay Kit from Pierce Biotechnology (Rockford, IL). Approximately 15–20 μg of protein was loaded per lane on Tris-Glycine 4–20% gels (Invitrogen, Carlsbad, CA). Separated proteins were then transferred to nitrocellulose membranes (0.45 μm, Invitrogen). After blocking with 5% non-fat milk (Carnation, Swampscott, MA) in TBS (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCL) containing 0.1% Tween-20 (TBS-T), nitrocellulose membranes were incubated with primary antibodies (Shh: 1:100 dilution) overnight at 4°C. ECL™ donkey anti-rabbit IgG HRP-conjugated secondary antibody (Amersham, UK) was added after washing, at a dilution of 1:2000 in 5% non-fat milk for 1 h. SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) was used to detect specific antibody-HRP complexes.
Intrahepatic leukocytes were labeled with anti-mouse CD3-FITC (Santa Cruz), PBS-loaded CD1d-tetramers-allophycocyanin or PE (NIH tetramer core facility, Atlanta), Shh-PE (R&D systems), CD4-Pacific blue (BioLegend), CD8-allophycocyanin (AbCam) or matched isotype controls and analyzed using the FACS VantageSE (Beckton Dickinson).
iNKT cell viability assays
iNKT cells (primary murine liver iNKT cells or DN32 iNKT cells) were cultured in 96-well tissue culture plates, as above. 1×105 cells were seeded in each well and either vehicle (no Shh) or a various doses of Shh (StemCell Tech, Canada, 10–1000 ng/mL) were added. Cultures were harvested 72 h later and iNKT cell numbers were determined by the commercially available Cell Counting Kit-8 (CCK-8, Dojindo, Maryland). Briefly, 10 μL of the CCK8 substrate was added to cell cultures 72 h after plating (end of incubation period) and absorbance in each well measured. A calibration curve was prepared using wells containing a fixed numbers of viable cells. A FLUOstar OPTIMA micro-plate reader (BMG Labtech, Durham, NC) was used for absorbance measurements.
iNKT cell apoptosis assays
Apoptotic activity was assayed using the Apo-ONE Homogeneous Caspase 3/7 Apoptosis Assay (Promega, Madison, WI), according to the manufacturer's instructions. Results were expressed as relative fluorescent units (RFU). A FLUOstar OPTIMA micro-plate reader (BMG Labtech, Durham, NC) was used for fluorescence measurements. Apoptotic activity of primary hepatic leukocyte in culture was assessed by FACS analysis of Annexin-V-FITC (BioVision, CA) staining. Identification of iNKT cell fraction was determined by CD3-CD1d-tetramer double positive staining.
mRNA quantification by real-time PCR
Total RNA was extracted from DN32 hybridoma cells using Trizol (Invitrogen). 1.5 μg of RNA was reverse-transcribed using random primers and Superscript RNase H-reverse transcriptase (Invitrogen). Samples were incubated at 25°C for 15 min, 42°C for 55 min; reverse transcriptase was inactivated by heating at 70°C for 15 min followed by cooling at 4°C for 10 min. mRNA were quantified by real-time reverse-transcriptase-PCR per the manufacturer's specifications (Eppendorf, Mastercycler Real-Time PCR). Amplification was performed using SYBR Green PCR Master Mix (Applied Biosystems). Five microliters of diluted cDNA samples (1:5 dilution) were used for quantitative two-step PCR (a 10-min step at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 65°C) in the presence of 400 nM specific forward and reverse primers, 5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer (pH 8.3), 200 μM dATP, dCTP, dGTP, and 400 μM dUTP and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin Elmer Applied Biosystems). Each sample was analyzed in triplicate. S9 (mouse) or 18S (human) rRNA was used as housekeeping control. Threshold cycles (Ct) were automatically calculated by the iCycler iQ Real-Time Detection System. Ct values were normalized to the housekeeping control to give a relative mRNA level. Sequences of primers used were as follows: MOUSE: 9S: sense: GGGAGCTGTTGACGCTAGAC, anti-sense: CGGGCATGGTGAATAGATTT; shh: sense: CTGGCCAGATGTTTTCTGGT, anti-sense: TAAAGGGGTCAGCTTTTTGG; Gli1: sense: AACTCCACAGGCACACAGG, anti-sense: GCTCAGGCTTCTCCTCTCTC; ptc: sense: ATGCTCCTTTCCTCCTGAAACC, anti-sense: TGAACTGGGCAGCTATGAAGTC; IL-4: sense: TCCTGCTCTTCTTTCTCG, anti-sense: CTTCTCCTGTGACCTCGTT; IL-10: sense: TGTGAAAATAAGAGCAAGGCAGTG, anti-sense: CATTCATGGCCTTGTAGACACC; IL-13: sense: CAGCATGGTATGGAGTGTGG, anti-sense: TGGGCTACTTCGATTTTGGT; IFN-γ: sense: CATCAGCAACAACATAAGCGTCA, anti-sense: CTCCTTTTCCGCTTCCTGA; TNF-α: sense: TCGTAGCAAACCACCAAGTG, anti-sense: AGATAGCAAATCGGCTGACG; CD69: sense: GTACAATTGCCCAGGCTTGT, anti-sense: TCCAATGTTCCAGTTCACCA; CD154: sense: CAGTGGGCCAAGAAAGGATA, anti-sense: GGTATTTGCCGCCTTGAGTA; CD178: sense: CATCACAACCACTCCCACTG, anti-sense: GTTCTGCCAGTTCCTTCTGC; SOCS2: sense: TCAGCTGGACCGACTAACCT, anti-sense: TGTCCGTTTATCCTTGCACA; SOCS3: sense: AGCTCCAAAAGCGAGTACCA, anti-sense: TGACGCTCAACGTGAAGAAG; HUMAN: Gli1: sense: GTGCAAGTCAAGCCAGAACA, anti-sense: ATAGGGGCCTGACTGGAGAT; ptc: sense: ACAAACTCCTGGTGCAAACC, anti-sense: CTTTGTCGTGGACCCATTCT; 18S: sense: TGCATGTCTAAGTACGCACG, anti-sense: TTGATAGGGCAGACGTTCGA.
Cytokine analysis by ELISA
CD3+ CD1d-tetramer+ (double-positive) mouse primary hepatic iNKT and DN32 cell-culture supernatants were collected and assayed using the Eli-pair ELISA kit (IL-10: ab47600; IFN-γ: ab47619; Abcam), BD OptEIA ELISA set (IL-4: Cat. No. 55232; BD Pharmingen) and eBiosciences (IL-13: Cat 887137), following the manufacturers' protocols.
Luminex assay (Bio-Plex assay)
Supernatants of DN32 cells were harvested and analyzed by Bio-Plex Cytokine Assay (BIO-RAD, Bio-Plex Reagent Kit: Cat.171304000; Mouse Grp I Cytokine 6-Plex Panel: Cat.X60000ZGYK), according to manufacturer's recommendations.
Human peripheral blood iNKT cells
NKT cells were isolated from healthy donor peripheral blood and iNKT expanded with αGalCer (Axxora, San Diego). Briefly, PBMC were isolated from Buffy Coats by Ficoll-Hypaque density centrifugation. NKT cells were selected by Mo-Flo cell sorting CD3+CD56+ cells. For expansion of iNKT, cells were first cultured for a 2-wk period in RPMI-1640 containing L-glutamine and 10% human serum (HD Supplies) in the presence of αGalCer at 100 ng/mL, supplemented with 100 U/mL IL-2 (Peprotec). iNKT cells were then selected by Mo-Flo cell sorting CD3+ cells expressing the Vα24/Jα18 iNKT TCR (6B11; BD Biosciences). Blood samples were obtained with informed consent of donors and in accordance with local ethical approval 04/Q2708/41 and REC 2003/242 from the South Birmingham Research Ethics Committee, UK.
Co-culture experiments with DN32 iNKT and 603B cholangiocytes
603B cells were cultured until 90% confluent in standard culture media as previously described 75. Cells were then loaded overnight with vehicle or 100 ng/mL αGalCer. DN32 hybridoma iNKT cells (1×105/well) were added to individual wells for 6–24 h. Culture supernatants were collected for cytokine analyses. 603B cell monolayer was then washed with PBS and the proportion of 603B cells remaining intact on the culture plate determined. For each experiment, a minimum of ten high power fields of view (20×) were examined using a phase-contrast microscope. All experiments were performed twice.
Results are expressed as mean±SEM. For analyses of individual columns, significance was established using the Student's t-test. ANOVA was used for multiple group comparisons. Differences were considered significant when p<0.05.
The authors thank Dr. Y. Ueno (Tohoku University, Sendai, Japan) and Dr. G. Gores (Mayo Clinic, Rochester, MN) for providing the murine cholangiocyte cell line (603B); Dr. Albert Bendelac (University of Chicago, Chicago, IL) for providing the murine invariant NKT hybridoma cells (DN32); and Dr. S.L. Friedman (Mount Sinai School of Medicine, NY, USA) for providing the human hepatic stellate cell line (LX2). The authors also thank Dr. Jiawen Huang for his assistance with animal care, Mr. W.C. Stone for his administrative support and Ms. Roxana M. Teisanu for technical assistance. The 5E1 antibody was obtained from the Developmental Studies Hybridoma Bank, developed under Department of Biological Sciences, Iowa City, IA 52242, USA. Funding: This work was supported by the National Institute of Health grants RO1 DK077794 and RO1 DK053792 to A.M.D.
Conflict of interest: The authors declare no financial or commercial conflict of interest.