Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury
© Wang et al.; licensee BioMed Central. 2015
Received: 11 December 2014
Accepted: 24 February 2015
Published: 11 March 2015
Tumor necrosis factor-inducible gene 6 protein (TSG-6), one of the cytokines released by human mesenchymal stem/stromal cells (hMSC), has an anti-inflammatory effect and alleviates several pathological conditions; however, the hepatoprotective potential of TSG-6 remains unclear. We investigated whether TSG-6 promoted liver regeneration in acute liver failure.
The immortalized hMSC (B10) constitutively over-expressing TSG-6 or empty plasmid (NC: Negative Control) were established, and either TSG-6 or NC-conditioned medium (CM) was intraperitoneally injected into mice with acute liver damage caused by CCl4. Mice were sacrificed at 3 days post-CM treatment.
Higher expression and the immunosuppressive activity of TSG-6 were observed in CM from TSG-6-hMSC. The obvious histomorphological liver injury and increased level of liver enzymes were shown in CCl4-treated mice with or without NC-CM, whereas those observations were markedly ameliorated in TSG-6-CM-treated mice with CCl4. Ki67-positive hepatocytic cells were accumulated in the liver of the CCl4 + TSG-6 group. RNA analysis showed the decrease in both of inflammation markers, tnfα, il-1β, cxcl1 and cxcl2, and fibrotic markers, tgf-β1, α-sma and collagen α1, in the CCl4 + TSG-6 group, compared to the CCl4 or the CCl4 + NC group. Protein analysis confirmed the lower expression of TGF-β1 and α-SMA in the CCl4 + TSG-6 than the CCl4 or the CCl4 + NC group. Immunostaining for α-SMA also revealed the accumulation of the activated hepatic stellate cells in the livers of mice in the CCl4 and CCl4 + NC groups, but not in the livers of mice from the CCl4 + TSG-6 group. The cultured LX2 cells, human hepatic stellate cell line, in TSG-6-CM showed the reduced expression of fibrotic markers, tgf-β1, vimentin and collagen α1, whereas the addition of the TSG-6 antibody neutralized the inhibitory effect of TSG-6 on the activation of LX2 cells. In addition, cytoplasmic lipid drops, the marker of inactivated hepatic stellate cell, were detected in TSG-6-CM-cultured LX2 cells, only. The suppressed TSG-6 activity by TSG-6 antibody attenuated the restoration process in livers of TSG-6-CM-treated mice with CCl4.
These results demonstrated that TSG-6 contributed to the liver regeneration by suppressing the activation of hepatic stellate cells in CCl4-treated mice, suggesting the therapeutic potential of TSG-6 for acute liver failure.
Acute liver failure and chronic liver disease are life-threatening diseases for which liver transplantation is the only permanent remedy. However, the number of available organs from donors is vastly insufficient for the number of patients requiring such procedures. Even if transplant patients receive a whole liver transplantation, several post-transplant complications may arise, such as immune rejection response and death of the donor or recipient in worst-case scenarios . Therefore, extensive studies are being conducted to develop new treatments for liver diseases, and stem cell based therapy has been suggested as an alternative treatment strategy for patients who suffer from various hepatic diseases .
Mesenchymal stem cells (MSCs) found in most adult and postnatal organs are capable of self-renewing and differentiating into several lineages of cells, including hepatocytes [3,4]. This differentiation potential of MSCs into hepatocytes provides new and promising therapeutics for patients with liver disease. These therapeutic effects of MSCs in the treatment of liver disease have been reported both in animal and clinical studies . In those studies, MSCs were shown to contribute to liver regeneration by secreting tropic and immunomodulatory molecules [6,7]. However, there are still a number of technical limitations or possible undesirable side effects associated with the therapeutic application of MSCs to patients with end-stage liver diseases . In particular, engrafted MSCs can differentiate into not only hepatocytes but also myofibroblasts, a main source of collagen fiber in a fibrotic liver, depending on the timeframe of differentiation and route of MSC injection . Hence, further characterization of MSCs may be critical for ensuring the safety of MSC-based cell therapy. The beneficial effect of MSC transplantation is based on autologous transplantation. However, it is difficult to try MSC transplantation with patients with end-stage liver disease . Although allogeneic stem cell transplantation might be more effective for these patients, it also brings several obstacles, such as immune rejection or engraftment of virus-carrying MSCs . The paracrine effect, which results from biologically active soluble factors secreted from human MSCs (hMSCs), such as angiopoietin-1, interleukin-10, keratinocyte growth factor, and so on, has been shown to be therapeutically valid in both animal and clinical studies [10,11]. Since many kinds of tropic and immunomodulatory factors secreted from MSCs are also known to create a favorable micro-environment for liver regeneration , it is necessary to identify and characterize such biologically active soluble factors.
Tumor necrosis factor-inducible gene 6 protein (TSG-6), a 35 kDa glycoprotein , was identified as an inflammatory factor as its expression increased in response to inflammatory mediators . However, upregulated TSG-6 during the inflammatory process has been shown to contribute to modulate the inflammatory response in adverse . Recent studies demonstrate that TSG-6 is identified as an important immune modulator secreted from hMSCs and shown to be responsible for hMSCs’ therapeutic effects, such as improvement of cardiac function, peritonitis and wound healing . However, these associations of TSG-6 with response in the liver are unknown.
Liver inflammation occurs in response to damage . Liver injuries stimulate the repair response, such as proliferation of hepatocytes and inflammation, and a successful repair response reconstitutes a functional liver . However, continued damage perpetuates injury and promotes progressive fibrogenesis . Liver inflammation also accelerates fibrosis . Several inflammatory factors, such as tumor necrosis factor (TNF-α) and interleukin (Il) -1β, and Il-6, secreted by inflammatory cells are involved in the recruitment of circulating macrophages into the liver and the transition of hepatic stellate cells into myofibroblasts . This progressive fibrogenesis ends with death from cirrhosis and/or liver cancer . Thus, the modulation of liver inflammation in this setting is a key target for reducing fibrosis. For this reason, we hypothesized that TSG-6 could influence liver regeneration by reducing inflammation and fibrosis because TSG-6 exerts an anti-inflammatory effect. To prove our hypothesis, we made immortalized hMSCs stably expressing TSG-6. Due to constant secretion of TSG-6 from hMSCs in the conditioned medium (CM), TSG-6-rich CM could be readily applied to mice with acute liver injury caused by CCl4. Our results showed that TSG-6 promoted liver regeneration by decreasing fibrosis.
Generation of hMSCs stably expressing TSG-6
We sub-cloned P/I-TSG-6 (kindly provided by Dr. Tae-Hee Lee in Yonsei Univ.) into a CSII-EF-MCS vector. hMSCs stably expressing TSG-6, were generated as previously reported . In detail, each viral plasmid (3 μg) (CSII-EF-MCS and CSII-EF-TSG-6) was transfected into 293FT cells with pLP1, pLP2, pLP/VSVG, using lipofectamine 2000 (cat. #11668-027, Life Technologies, Inc. Grand Island, NY, USA). After 48 hours, the culture media containing the lenti-viruses were collected from the transfected 293FT cells. Virus medium was filtered (0.45 μm filter, EMD Millipore, Billerica, MA, USA) and then treated to hMSCs for an additional 24 hours in the presence of 4 μg/ml of polybrene (Sigma–Aldrich, St. Louis, MO, USA). B10-hMSCs were maintained as described previously .
Preparation of conditioned media
For preparation of CM, cells were seeded at more than 90% confluence. The next day, cells were washed with PBS twice, changed to 0.2% fetal bovine serum (FBS)-containing media and incubated for another three days. CM were further concentrated with YM-10 (Millipore, Cat. No. 4205).
Immune suppression assay
Splenocytes, 1.5 × 106 per well (24-well) (freshly isolated from C57BL6 female mice as described previously ), were cultured with or without CM supplement for three days. To stimulate T cells, 1 μg/ml of anti-CD3ε (2C11) (BD Pharmingen, San Jose, CA, USA) was added. Cells were fluorescence labeled with carboxyfluorescein succinimidyl ester (CFSE) and then proliferation after activation was determined by flow cytometry (BD, FACS Calibur™).
Animal care and surgical procedures were approved by the Pusan National University Institutional Animal Care and Use Committee and carried out in accordance with the provisions of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Measurement of AST/ALT
Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using Chemi Lab GOT/GPT (IVD Lab Co., Korea) according to the manufacturer’s instructions.
Liver histology and immunohistochemistry
Liver specimens were fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 4 μm sections. Specimens were dewaxed, hydrated, and stained in the usual manner with standard hematoxylin and eosin (H & E) to examine morphology. For immunohistochemistry (IHC), sections were incubated for 10 minutes in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) or incubating with pepsin for 10 minutes. After washing with TBS, sections were treated with Dako protein block (X9090; Dako Envision, Carpinteria, CA) for 30 minutes and incubated with primary antibody Ki67 (NCL-Ki67, Novocastra, Leica Microsystems, Newcastle, Upon Tyne, UK), pancytokeratin (PanCK) (Z0622; Dako), Sox9 (AB5535; Millipore), or α-Sma (ab5694; Abcam, Cambridge, Massachusetts, USA), at 4°C overnight. Other sections were also incubated at 4°C overnight in non-immune sera to demonstrate staining specificity. Polymer horseradish peroxidase (HRP) anti-rabbit (K 4003; Dako) or anti-mouse (K 4001; Dako) was used as secondary antinody. 3,3′-Diaminobenzidine (DAB) was employed in the detection procedure.
To quantify the number of Ki67-positive cells, 10 central vein (CV) areas were randomly selected per section at × 40 magnification for each mouse. CV chosen for analysis ranged from 90 to 150 μm. The Ki67-positive cells were quantified by counting the total number of Ki67-positive cells per field.
Quantitative real-time PCR
Sequences of primers used for QRT-PCR
Western blot assay
Total protein was extracted from freeze-clamped liver tissue samples that had been stored at -80°C. Whole tissues were homogenized in RIPA (78510; Thermo, Rockford, IL) supplemented with protease inhibitors (Complete Mini 11 836 153 001; Roche, Indianapolis, IN). Equal amounts of total protein (120 μg) were fractionated by polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. Primary antibodies against TGF-β (3711S; Cell Signaling) and α-Sma (A5228-200UL; Sigma-Aldrich) were used in this experiment. Membranes were developed by chemiluminescence (ATTO Corporation, Tokyo, Japan). The blots that were obtained from three independent experiments were scanned and a region of interest (ROI) around the band of interest was defined. Band intensities were calculated by using the CS analyzer 2.0 program (ATTO Corporation).
The human hepatic stellate cell line LX2, a well-characterized cell line derived from human hepatic stellate cells , B10-NC and B10-TSG-6 were cultured at a density of 2 × 106 in Minimum Essential Medium alpha (MEM α, Gibco, Life Technologies) supplemented with 10% FBS (Gibco, Life Technologies, Grand Island, NY) and 1X antibiotics at 37°C in a humidified atmosphere containing 5% CO2. LX2 was a generous gift from Won-il Jung (Korea Advanced Institute of Science and Technology). When B10-NC or B10-TSG-6 cells were 70% to 80% confluent, CM of these cells were collected to treat LX2. For biochemical analysis of gene expression changes, LX2 at 70% to 80% confluence were starved in medium containing no FBS for six hours. Activation of LX2 was verified by examining the expression of pro-fibrotic signaling genes at 24 and 48 hours after addition of FBS. Based on the gene expression data, we considered LX2 as fully activated cells at 48 hours (data not shown). Fully activated LX2 was cultured in LX2 cell medium, B10-NC-CM or TSG-6-CM. In addition, those cells cultured in TSG-6-CM were treated with TSG-6 antibody (10 μg/ml) or control IgG (10 μg/ml). These experiments were repeated three times .
Results are expressed as the mean ± standard deviation (SD). Statistical differences were determined by Student’s t-test or one-way analysis of variance (ANOVA) using the SPSS statistics 20, followed by Scheffe’ post hoc test. P-values <0.05 were considered to be statistically significant.
Generation of TSG-6-overrexpressing MSCs
TSG-6 attenuates liver injury
Decreased hepatic fibrosis in TSG-6 treated mice
TSG-6 induced inactivation of hepatic stellate cells
Acute liver failure shows a higher inflammatory response to liver damage, which impairs the parenchymal function that extends into systemic organ failure, eventually leading to death [16,31]. Thus, researchers must develop new treatments for acute liver disease. The potential for MSCs to differentiate into hepatocytes and their immunomodulatory capabilities make MSCs an attractive choice in the therapy of acute or chronic liver diseases . Several clinical trials are currently being performed to investigate the therapeutic potential of MSCs in the treatment of chronic liver diseases, such as Hepatitis B or C virus-infected liver or alcoholic liver with cirrhosis [32-34]. Previous studies have shown that transplanted MSCs replace the damaged cells and repopulate in the injured tissues or organs . However, the number of long-term substituted MSCs is open to dispute because those numbers differed depending on the injury models, the transplant route and time, and so on . Nevertheless, it seems to be uncontroversial that MSCs and the CM from MSCs contribute to improving damage from liver injuries [9,36]. The transplanted MSCs help or allow the survival or proliferation of endogenous cells by direct contact or modulating inflammation . MSCs are known to alleviate acute and chronic liver injury by changing the disease environment; for example, they decreased inflammation and cell death, but they increased cell proliferation . In the present study, TSG-6 released by MSCs suppressed the proinflammatory signal and reduced the collagen accumulation which was likely due to the inactivation of hepatic stellate cells by TSG-6 in the acute liver injury of mice.
Although the anti-inflammatory action of TSG-6 is shown to promote improvement in the damaged tissue [14,37], this effect of TSG-6 in the liver remains unknown. Thus, we investigated whether TSG-6 might be involved in liver regeneration, and how it contributed to this process. Our results demonstrated that TSG-containing CM led to liver regeneration. This protective effect of TSG-6 was also observed in another experimental model; specifically, chorionic plate-derived MSCs (CP-MSCs) that shared common characteristics and differentiation potentials with bone marrow-MSCs promoted liver regeneration in rat livers that had been chronically damaged by CCl4 [38,39]. In this model, the CP-MSCs contained a greater expression of TSG-6, and the regenerating liver transplanted with CP-MSC showed a higher expression of TSG-6, compared to a non-transplanted liver (Additional file 1: Figure S1). This evidence strongly supported the regenerative effect of TSG-6 in the liver.
In this experimental model, acutely injured livers showed distorted histomorphology, changes in LW/BW, and increased levels of the liver enzymes, ALT and AST, but TSG-6-CM treatment attenuated those abnormalities and even contributed to restoring the liver function which was evidenced by g6pc expression. G6pc hydrolyzes glucose-6-phosphate, which is the central metabolite of glucose metabolism . The data showing almost normal glucose metabolic homeostasis in TSG-6-CM treated livers strongly support the restoration of liver function through the action of TSG-6. In addition, the downregulated proinflammatory factors in the TSG-6-CM group demonstrated that TSG-6 exerted an anti-inflammatory effect on the CCl4-treated mouse livers.
Liver injury induces activation of hepatic stellate cells and proliferation of progenitors [16,26]. Activated stellate cells have been shown to be associated with the proliferation of hepatic progenitors [27,41]. In Ki67 staining, more Ki67-positive hepatocytic cells and fewer Ki67–positive hepatic stellate cells were observed in livers of the TSG6 group. Compared with liver of CCl4 + TSG-6-treated mice, livers of CCl4 or CCl4 + NC-treated mice contained more PanCK-expressing cells and more SoX9-expressing cells (Figure 5). We examined if these changes in the activation and /or proliferation of hepatic stellate cells and the reduced liver damage influenced the proliferation of progenitors. As we expected, decreased expansion of progenitors was observed in livers of the TSG-6 group, compared to the CCl4 and NC groups. Therefore, these results indicated that micro-environmental changes by TSG-6 might contribute to the proliferation of hepatocytes by protecting hepatocytes from injury. Also, it could be possible that TSG-6 might promote differentiation of progenitors into hepatocytes. However, neither the direct effects of TSG-6 on progenitors nor the progenitor proliferation at the early time point after CM injection was investigated in the present studies. Hence, further studies are required to provide the evidence for this possibility.
The degree of inflammatory response parallels the fibrotic severity in the liver . The activated hepatic stellate cells play a key role in hepatic fibrogenesis [43,44]. Both RNA and the protein level of fibrotic markers were downregulated in the livers of TSG-6-CM-treated mice. Activated LX2 cultured in TSG-6-CM showed a decreased expression of fibrotic markers and an increased number of lipid drops. Conversely, the addition of the TSG-6 antibody neutralized these inhibitory effects of TSG-6 on the activation of LX2, although this addition was less effective than NC-CM. It is possible that the TSG-6 antibody does not block all TSG-6, and the amount of TSG-6 that escaped blocking by the antibody is higher than it is in NC-CM. One explanation is that the TSG-6-overexpressing MSCs and the CM of those cells show a greater expression of TSG-6, but TSG-6 is rarely detected in NC and NC-CM. Hence, these results proved that TSG-6 influenced the activation of hepatic stellate cells, suggesting that TSG-6 contributed to the reduced fibrosis in damaged livers of mice.
CM obtained from empty plasmid-transfected MSC was less protective in the damaged liver. In line with our finding, Herrera et al. , demonstrated that both human liver stem cells (HLSCs) and HLSC-CM improved liver injury and had a protective effect for liver, but MSC-CM was totally ineffective in their experimental model . In addition, they employed the concentrated CM in their experimental model of liver injury, like other researchers did. However, the CM used in the present studies was not concentrated. MSC-CM includes many kinds of cytokines, growth factors, and even microparticles [37,45]. Because we used 0.5 ml CM per mouse from a total 12 ml CM which was collected at 70% to 80% confluent cells, this small volume of CM seemed to contain a significantly lower amount of those factors, compared to the concentrated CM. Hence, the effects of other factors were likely negligible in this model. Also, we examined the regulation of hepatic stellate cells by TSG-6, not the apoptosis of hepatocytes, and demonstrated that hepatic stellate cells were inactivated in TSG-6-CM, but not in NC-CM or TSG-6-CM with the TSG-6 antibody. Furthermore, CCl4-treated mice injected with TSG-6-CM incubated with TSG-6 antibody induced the liver damage which was ameliorated in CCl4-treated mice with TSG-6-CM. These results suggest that the reduced inflammation and fibrosis caused by TSG-6 might provide a beneficial microenvironment which contributes to the proliferation or survival of hepatocytes, leading to liver regeneration.
Our results first demonstrate that TSG-6 influences liver regeneration and place TSG-6 into the central position in the regulation of liver regeneration. The protective and/or improving effects of TSG-6 on the damaged liver through attenuating inflammation and fibrosis help to develop novel therapeutic approaches to treat acute liver failure. However, further studies are required to examine the underlying mechanism for the regenerative actions of TSG-6 and those effects in other liver diseases, including chronic diseases.
analysis of variance
carboxyfluorescein succinimidyl ester
fetal bovine serum
- H & E:
hematoxylin and eosin
human mesenchymal stem/stromal cells
transforming growth factor-β
tumor necrosis factor-α
tumor necrosis factor-inducible gene 6 protein
α-smooth muscle actin
The authors thank Dr. Gi Jin Kim (Cha University, Korea) for providing the CP-MSC and rat liver tissue. This work was supported by the National Research Foundation (NRF) of Korea funded by the Korean government (MEST) (2013R1A2A2A01068268) to YJ.
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