Background
Vitamin D is an important prohormone with known effect on calcium homeostasis [
1], but recently there is increasing recognition that vitamin D also is involved in cell proliferation and differentiation, it has immunomodulatory and anti-inflammatory properties [
2]. The effects of vitamin D are mediated through the vitamin D receptor (VDR) [
3]. VDR is a member of the nuclear receptor super-family of ligand-inducible transcription factors, which are involved in many physiological processes, including cell growth and differentiation, embryonic development and metabolic homeostasis [
4]. The transcriptional activity of this receptor is modulated by several ligands, such as steroids, retinoids and other lipid soluble compounds, and by nuclear proteins acting as co-activators and co-repressors [
5]. The liganded VDR heterodimerizes with the retinoid X receptor and binds to vitamin D response elements in the promoter of target genes, thereby affecting their transcription. The genomic organization of the
VDR at locus 12q13.1 shows that the
VDR gene itself is quite large (over 100 kb) and has an extensive promoter region capable of generating multiple tissue-specific transcripts [
6].
Clinical observations have recently demonstrated that 25-OH D serum levels were significantly lower in patients with chronic hepatitis C than in controls and that low 25-OH D serum levels were associated with more severe fibrosis and lower responsiveness to interferon-based therapy in those patients [
7,
8]. Other studies have shown that low 25-OH D serum levels are associated with poor liver function and more advanced stages of liver fibrosis in hepatitis C virus patients [
9]. Moreover, a possible role for vitamin D in liver fibrosis has gained further support from the finding that VDR is expressed in human as well as in rat liver non-parenchymal cells, such as Hepatic stellate cells (HSCs) [
10]. It has recently been suggested that VDR polymorphism is associated with primary biliary cirrhosis [
11]. Vitamin D deficiency is a common phenomenon in chronic liver disease, particularly in advanced fibrosis and cirrhosis [
12]. Whether this association reflects the cause of accelerated fibrosis progression or the consequence of impaired liver function in advanced disease is still unclear. In our current study, we aimed to evaluate changes in liver NK cells cytotoxicity due to modulations in VDR in CCl
4 fibrosis model following 25(OH) D3 injections.
Methods
Ethics statement and animals
Male mice on the BALB/c background, 12 weeks of age, weighing 22 ± 0.5 g, received care according to NIH guidelines. Mice were purchased commercially from Harlan Laboratories, Jerusalem-Israel. All animal protocols were approved by the institutional animal care ethical committee of the Hebrew University and housed in a barrier facility under the ethic number: MD-18-154,943.
CCl4 model of liver injury and fibrosis
BALB/c mice were IP injected with 0.5 ml/kg body weight CCl4 (1:10 v/v in corn oil from Sigma) or vehicle (corn oil) twice a week for 1 week (acute model) and 4 week (chronic model). 25(OH) D3 (Biogems, Cat# 3220632–10 mg) at the dose of 0.5 microgram/100 g body was IP injected twice a week, commencing one day after the first dose of CCl4. The animals were terminated 72 h. after the final CCl4 injection through intramuscularly anesthetize with 0.1 ml of ketamine: xylazine: acepromazine (4:1:1) per 30 g of body-weight prior to cervical dislocation. The whole livers and serum were collected for histological, cytological and biochemical analyses.
Alanine aminotransferase (ALT)
Blood samples were collected from BALB/c male hearts at the volume of 1 ml blood; the samples were centrifuged for 5 min at 4000 rpm. Serum samples were drawn to an Eppendorf tubes, after blood centrifuge. Serum samples of 32 μl were dropped into the strip of the ALT (Reflotron Company) and analyzed by Reflovet® plus Roche.
Liver NK cells isolation
Under deep ether anesthesia, mice were euthanized by isoflurane, USP 100% (INH), then the liver was removed and a part of the liver was transferred to Petri dish that contains 5 ml DMEM medium (Biological industries; Cat# 01–055-1A). The liver tissue was thoroughly dissected by stainless steel mesh, the cells were harvested with the medium and added to 50 ml tubes containing 10 ml DMEM, and then carefully cells were transferred to new tubes that contain Ficoll (Abcam; Cat# AB18115269). Tubes were centrifuged for 20 min, at 1600 rpm at 20 °C. The supernatant in each tube was transferred to a new tube, for another centrifuge for 10 min, at 1600 rpm at 4 °C. After the second centrifuge, the pellet in each tube was suspended in 1 ml of DMEM for NK isolation kit (StemCells; Cat# 19665)).
Primary HSCs isolation
HSCs were isolated from 12-week-old male BALB/c mice by in situ pronase, collagenase perfusion, and single-step Histogenz gradient as previously reported (Hendriks et al., 1985; Knook et al., 1982). Isolated HSCs were cultured in Dulbecco’s modified Eagle’s medium (Mediatech) containing 20% fetal bovine serum (Hyclone) on six-well plates for 40 h. prior to end-point assays.
Flow cytometry
Harvested mice liver NK cells were adjusted to 106/ml in buffer saline containing 1% bovine albumin (Biological Industries; Cat# 02–023-5A), and were stained for the following antibodies: Anti-mice NK1.1 (murine NK cell marker) (Biogems; Cat# 83712–70), Anti CD49a (MACS; Lot# 5150716246), and anti-mice Lysosomal-associated membrane protein-1 (CD107a; NK1.1 cells cytotoxicity marker) (eBioscience; Cat# 48–1071). All antibodies were incubated for 45 min at 4 °C. The cells were washed with 0.5 ml of staining buffer and fixed with 20 ml of 2% paraformaldehyde. All stained cells analyzed with a flow cytometer (The BD LSR Fortessa™, Becton Dickinson, Immunofluorometry systems, Mountain View, CA).
Real time PCR
Tissue RNA isolation
Total cellular RNA was isolated from liver tissue, using 2 ml TRI Reagent (Bio LAB; Cat# 90102331) for each cm3 of tissue. The samples were homogenized for 5 min at room temperature of 25 °C. Chloroform at volume of 0.2 ml (Bio LAB; Cat# 03080521) were added to each sample, incubated for 15 min at room temperature and centrifuged (1400 rpm) for 15 min at 4 °C. RNA precipitation: The supernatant in each sample was transferred to new Eppendorf, 0.5 ml of isopropanol (Bio LAB; Cat# 16260521) left for 10 min at 25 °C and centrifuged (12,000 rpm) for 10 min at 4 °C. The supernatants were removed and one ml of ethanol 75% were added to the pellet and centrifuged (7500 rpm) for 5 min. The pellets were dried in air at room temperature for 15 min, 50 μl of DEPC were added, and the samples were heated for 10 min at 55 °C.
cDNA preparation
Liver RNA was extracted as described above. Preparing of c-DNA was performed using High Capacity cDNA Isolation Kit (R&D; Cat # 1406197).
Real time PCR
Real-time PCR is performed for the quantification of the expression of the gene that encoded alpha Smooth Muscle Actin (αSMA) and Vitamin D Receptor (VDR), compared to GAPDH as a housekeeping gene by using Taqman Fast Advanced Master Mix (Applied Biosystem; Cat # 4371130).
Western blot analysis
Immunoblot analysis of αSMA in liver extracts performed. Following isolation, whole protein extracts were prepared in liver homogenization RIPA buffer (Sigma; R0278-50ML) with protease phosphatase inhibitor cocktail (Roche; 1,183,617,011). Next, proteins (30 μg per lane) were resolved on a 10% (wt/vol) SDS-polyacrylamide gel (Novex, Groningen, The Netherlands) under reducing conditions. For immunoblotting, proteins transferred to a PVDF membrane. Blots incubated for 1 h. at 4 °C in a blocking buffer containing 5% skim milk. Later on, the mixture was incubated with either mouse anti- human\mice α-SMA (Novusbio; NB600–531), rabbit anti-collagen I (ab34710), rabbit anti-VDR (ab109234) and mouse anti-human\mice β-Actin (R&D System; 937,215), diluted 1/1000, overnight in 4 °C, and subsequently, with peroxidase-conjugated goat anti-mouse and Rabbit IgG (PARIS, Compiegne, France) diluted 1/5000, for 1.5 h at room temperature. Immuno-reactivity revealed by enhanced chemiluminescence using an ECL Kit (Abcam; ab133406).
Histological assessments of liver injury
The posterior one third of the liver was fixed in 10% formalin for 24 h and then paraffin-embedded in an automated tissue processor. Seven-millimeter liver sections were cut from each animal. Sections (15 mm) were then stained in 0.1% Sirius red F3B in saturated picric acid as well as Masson trichrome stain for connective tissue stain (both from Sigma). Sirius red staining assessed using the modified Histological Activity Index (HAI) criteria, incorporating semi quantitative assessment of periportal/periseptal interface hepatitis (0–4), confluent necrosis (0–6), focal lytic necrosis/apoptosis and focal inflammation (0–4), portal inflammation (0–4), and architectural changes/fibrosis and cirrhosis (0–6).
Statistical analysis
The results were evaluated using the Student’s t-test, with statistical significance set at P < 0.05. Comparison between the mean values of different experiments was carried out. All data were presented as mean ± SE.
Discussion
The cell-specific expression pattern of VDR suggests that the liver could be responsive to vitamin D during liver fibrosis through its non-parenchymal cells, in particular, HSCs. However, this was true only in the acute model of CCl4 for the following reasons: (1) VDR on HSCs was upregulated in the inflammatory model in contrary to the chronic (fibrotic) model. (2) Liver histology structure of collagen and αSMA is less pronounced in the inflammatory model and therefore vitamin D is less likely to undergo degradation. (3) Serum calcium levels were within the normal physiological range and is not expected to cause dramatic decrease in VDR as resulted in the fibrotic model.
Abramovitch and colleagues showed, in 2011, that ligation of VDR in HSCs inhibited their proliferation, and activation and reduced thioacetamide (TAA)-induced liver fibrosis in rats [
14]. Further, Ding and colleagues revealed that VDR ligation in activated hepatic stellate cells has anti-fibrotic effects, which are mediated through a VDR/SMAD3/TGF-β signaling loop [
15]. The authors also carried out genetic studies in mice which resulted in spontaneous liver fibrosis when one or both
Vdr alleles were knocked out, with more severe fibrosis occurred in
Vdr−/− animals [
15]. In our chronic fibrotic model, serum vitamin D levels was unchanged as compared to WT mice and progressions of liver fibrosis were due to inhibitions of VDR in HSCs. Also, vitamin D serum levels were unchanged following vitamin D treatments (Fig.
2b), in contrary to other studies that suggested that not only may vitamin D deficiency contribute to liver fibrosis [
9].
Furthermore, the acute inflammatory model of CCl4 showed low serum vitamin D levels following vitamin D treatments a result that indicate consumption of vitamin D because of the high expressions of VDR. Therefore, low vitamin D serum levels in this case may not reflect liver injury while in the contrary showed alleviation in the fibrotic and the inflammatory profile. Moreover, our results showed modulation of VDR in liver resident NK cells of the chronic CCl4 model. NK cells of the fibrotic model showed less expressions of VDR and was correlated with their functional alterations. VDR inhibitions in NK cells from the chronic model of CCl4 attenuated their potentials to kill HSCs in our in vitro co-culture assay.
These findings, taken together with the correlation between liver VDR expressions and the severity of liver fibrosis, suggest that not only activated HSCs through their secretion of αSMA and collagen may contribute to liver fibrosis, it is also suggested that NK cells also lose their anti-fibrotic potential to kill HSCs, which exacerbate fibrosis especially when treated with vitamin D.
Conclusion
Vitamin D showed to have dual effects on liver injury. In inflammatory acute model of CCl4, vitamin D reduced inflammatory and fibrotic markers while in the fibrotic chronic model of CCl4, vitamin D worsen fibrosis. These findings were associated with changes in liver NK cells phenotype that were seen to lose their activity and inability to kill activated HSCs in an in vitro settings. These results suggest that VDR changes is crucial for NK cell stimulations and consequently modulate liver fibrosis progressions. More studies are needed to clarify the role of VDR in attenuation of liver fibrosis.
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