25(OH) D3 alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model of BALB/c mice due to modulations in vitamin D receptor

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.

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.

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.

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)).

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.

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).

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).

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).

Hepatic fibrosis was induced in BALB/c mice by biweekly IP CCl₄ injections for 1 week (acute model) and 4 weeks (chronic model) and was compared with naive vehicle-treated mice (WT) (n = 10 mice in each group). Histological staining and αSMA expressions were performed for fibrosis assessments. Figure 1a shows staining of Trichrome and Sirius red for the three major animal groups with and without IP administration of vitamin D as described in materials and methods. Acute and chronic model of CCl₄ showed gradual extent of collagenous connective tissue fibers of red fibrosis septae with both Trichrome and Sirius red stains in consistent with CCl₄ exposure time.

Fibrotic mice of WT and acute model of CCl₄ treated with vitamin D showed attenuation in their fibrosis histology that lack red fibrosis septae. Vitamin D treatments in the chronic CCl₄ model, in the contrary, had propagated liver fibrosis as seen in liver histological samples (Fig. 1a).

To evaluate effects of vitamin D on liver injury, systemic inflammatory marker for serum ALT levels and liver samples for quantitation of fibrosis were performed. Figure 1b shows serum ALT levels highly significantly (P = 0.05) as it elevated from 33.1 ± 7.5 IU/L in naïve WT mice to 401.5 ± 45 IU/L and 178 ± 21 IU/L in acute and chronic model of CCl_4, respectively. Vitamin D did not alter phenotype changes in WT mice, it inhibited systemic inflammation through decreased ALT levels in acute model of CCl₄ to 380 ± 29.4 IU/L (p = 0.04) while propagate inflammation through increased ALT levels in the chronic model of CCl₄ mice up to 288.5 ± 30.5 IU/L (P < 0.05). Gene expressions of liver αSMA fibrotic marker by RT PCR (Fig. 1c), αSMA liver portion expressions by western blot analysis (Fig. 1d) and liver collagen quantitation (Fig. 1e) showed similar patterns of ALT. The above results indicated dual anti- and pro- fibrotic effects of vitamin D that could be achieved depending on the severity of fibrosis.

The vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], binds with high affinity to the nuclear vitamin D receptor (VDR), and is considered as primary regulator of calcium (Ca^+ 2). We aimed to evaluate Ca^+ 2, vitamin D and VDR changes in mice model treated with or without vitamin D. Prior to vitamin D treatments, similar Ca^+ 2 serum levels were obtained in both the acute and the chronic CCl₄- mice model (Fig. 2a; P = ns). Moreover, vitamin D treatments did not affect Ca^+ 2 serum levels in both the WT and the acute model of CCl₄, however, it significantly caused an elevation in Ca^+ 2 levels as presented in the chronic model of CCl₄.in Fig. 2a. We further display changes in mice phenotype and found vitamin D serum levels to be significantly elevated in the acute model of CCl₄, an effect that were inhibited in the chronic model to levels similar to WT mice (Fig. 2b). Vitamin D treatments while did not alter vitamin D serum levels in the WT and chronic model of CCl₄, it significantly dropped levels of serum vitamin D to 4.6 fold (P = 0.004) in the acute model.

Therefore, our next aim was to relate changes in serum vitamin with modulations in the VDR. For this purpose, primary hepatic stellate cells (pHSCs) obtained from livers from all mice groups were quantitated for VDR by the western blot analysis. Figure 2c shows upregulation in the expressions of VDR in acute model of CCl₄ as compared to the WT, while total loss of VDR expressions were seen in the chronic model of CCl₄ and indicate that VDR expressions were modulated along the course of the liver injury. Vitamin D treatments significantly caused increased in VDR expressions in acute model of CCl₄ (P = 0.03) while showed to have no effects in VDR expressions in the WT and the chronic model of CCl₄.

The above results suggest increased Ca^+ 2 serum levels in chronic model of CCl₄ treated with vitamin D could be a result of liver injury accompanied with vitamin D degradation. Consequently, these effects were associated with inhibited VDR and therefore, unlike the acute model, chronic mice model of CCl₄ were unresponsive to vitamin D treatments (Fig. 2b and c). Moreover, due to the upregulation of VDR in the acute model of CCl₄, vitamin D treatments were most probably up taken/ consumed and could explain the low vitamin D serum levels while calcium serum levels remained the same.

We next sought to determine effects of vitamin D on immune alterations in liver fibrosis. We previously showed that NK cells exert anti-fibrotic effects through killing of activated HSCs [13]. We examined liver resident NK cells (NK1.1/CD49+) cellular infiltrates as well as their expressions of VDR and their activity (CD107a) by using the flow cytometry. Figure 3a shows a linear correlation between NK cells infiltrates with progression of liver fibrosis. Vitamin D caused an additional increase in the NK1.1 cells infiltrates (p = 0.01) only in the acute model of CCl₄ indicating increased anti-fibrotic effects of NK1.1 cells and therefore inhibiting fibrosis in this model, a phenomena emphasized in our previous data summarized in Fig. 1 and Fig. 2. VDR expressions on liver NK1.1 increased in the acute while inhibited in the chronic model of CCl₄ to levels similar to WT. The VDR were upregulated following treatments with vitamin D in the WT and acute model of CCl₄. No changes in the VDR were noticed in the NK1.1 cell from chronic model of CCl₄ following the vitamin D treatments (Fig. 3b). These data suggest inverse relation between NK1.1 cell proliferation (Fig. 3a) and their VDR expressions (Fig. 3b) prior and following vitamin D treatments.

To further correlate whether changes in the VDR on NK1.1 cells are associated with their phenotype alterations; CD107a (activity marker) was evaluated by the flow cytometry as described in materials and methods. CD107a NK1.1 percentages were high in both CCl₄ models to similar levels of the WT. Vitamin D treatments further elevated NK1.1 cells activity in the WT and the acute model of CCl₄ (P < 0.05) while did not alter NK1.1 activities in the chronic model most probably due to low VDR in the chronic model of CCl₄. In order to determine whether changes in CD107a and VDR on NK1.1 cells could modulate liver fibrosis; we co-cultured NK1.1 cells from mice livers from all groups with WT pHSCs through an in vitro setting assay to assess cytotoxicity/killing potential. Figure 3d shows NK1.1 cells obtained from the acute model of CCl₄ reduced αSMA percentages of pHSCs as compared to their WT counterparts. NK1.1 cells from the chronic model of CCl₄ did not change αSMA of the pHSCs indicating inability of these cells to reduce fibrosis. To cite, HSCs monocultures staining revealed 90% αSMA (data not shown). Vitamin D treatments further exacerbate NK1.1 killing potentials in both the WT and the acute model and inhibited fibrosis while did not cause any changes in αSMA of the pHSCs in the chronic model of CCl₄.