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doi:10.1111/jgh.12653
H E PAT O L O G Y
Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: Regulation of Treg/Th17 cells Lanman Xu,* Yuewen Gong,† Benfu Wang,‡ Keqing Shi,* Yijun Hou,* Liping Wang,* Zuo Lin,* Yixiang Han,§ Lu Lu,¶ Dazhi Chen,** Xiuli Lin,* Qiqiang Zeng,†† Wenke Feng,*,‡‡ and Yongping Chen* Departments of *Infection and Liver Diseases of the First Affiliated Hospital and Liver Research Center and ‡Anesthesiology of the Second Affiliated Hospital, Wenzhou Medical University, §Laboratory of Internal Medicine, Departments of ¶Pathology and ††Hepatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, and **Renji College of Wenzhou Medical University, Wenzhou, Zhejiang, China; † Faculty of Pharmacy and Section of Hepatology of Department of Internal Medicine, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and ‡‡Department of Medicine, University of Louisville, Louisville, Kentucky, USA
Key words bone marrow mesenchymal stem cells, hepatitis B virus, liver cirrhosis, stem cell transplantation, Treg/Th17 cells. Accepted for publication 17 March 2014. Correspondence Dr Yongping Chen, Department of Infection and Liver Diseases of the First Affiliated Hospital and Liver Research Center, Wenzhou Medical University, 2 Fuxue Lane, Wenzhou, Zhejiang Province 325000, China. Email: [email protected] Conflicts of interest: The authors disclose no conflicts.
Abstract Background and Aim: Liver cirrhosis is one of the major consequences of hepatitis B virus (HBV) infection, and transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) is one of promising therapies for patients with HBV-related liver cirrhosis (HBV-LC). However, the mechanism is unclear. The aim of the current study was to explore the role of Treg/Th17 cells in ABMSCs transplantation in patients with HBV-LC. Methods: In this prospective study, 56 patients were enrolled and randomly assigned to transplantation group and control group. After 24-week follow-up, 39 patients completed the study (20 cases in transplantation group and 19 cases in control group). The Model for End-Stage Liver Disease scores, liver function, changes of Treg/Th17 cells, as well as related transcription factors and serum cytokines, were determined. Results: Although patients in both groups showed significant improvement after Entecavir treatment, ABMSC transplantation further improved patients’ liver function. Moreover, there was a significant increase in Treg cells and a marked decrease in Th17 cells in the transplantation group compared with control, leading to an increased Treg/ Th17 ratio. Furthermore, mRNA levels of Treg-related transcription factor (Foxp3) and Th17-related transcription factor (RORγt) were increased and decreased, respectively. In addition, serum transforming growth factor-β levels were significantly higher at early weeks of transplantation, while serum levels of interleukin-17, tumor necrosis factor-α, and interleukin-6 were significantly lower in patients in the transplantation group compared with control. Conclusion: ABMSCs transplantation was effective in improving liver function in patients with HBV-LC, which was mediated, at least in part, through the regulation of Treg/Th17 cell balance.
Introduction Liver cirrhosis caused by the infection of hepatitis B virus (HBV) is a worldwide health problem. Many patients with HBV-related liver cirrhosis (HBV-LC) died from its serious complications, such as variceal hemorrhage and hepatic encephalopathy. Although current therapies, including antivirus medications and liver protection, are used for the treatment of HBV-LC, there are no effective therapies to reverse the cirrhosis except liver transplantation. However, most patients are either unable to obtain transplantation due to shortage of donor organs, or suffer from liver rejection and serious complications posttransplantation. Recently, several 1620
studies suggested that transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) appears to be safe and effective for patients with HBV-LC.1–4 Compared with liver transplantation, the stem cell therapy has several advantages, such as availability and fewer complications. Mesenchymal stem cells (MSCs) are an important part of bone marrow stem cells and have immunomodulatory effects.5–7 They provide a hopeful approach for treatment of graft-versus-host disease and other autoimmune diseases.8–10 Recent studies indicated that immunomodulatory activities of MSCs might be associated with Foxp3+ regulatory T cells (Treg) and IL-17+ T helper cells (Th17).11–13 Treg cells are a subset of CD4+ T cells and
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primarily characterized by CD25 and forkhead family transcription factor 3 (Foxp3). They mainly produce immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β).14 Th17 cells are another newly identified subset of CD4+ T cells and closely related to Treg cells. They mediate inflammation by producing pro-inflammatory cytokines, such as IL-17, IL-6 and tumor necrosis factor-α (TNF-α). Retinoid orphan nuclear receptor γt (RORγt) is a specific transcription factor expressed in Th17 cells.15 Studies indicated that the balance between Treg/Th17 cells was disturbed in several inflammatory and autoimmune diseases, such as primary biliary cirrhosis (PBC), systemic lupus erythematosus, and rheumatoid arthritis.16–18 Although a few studies have suggested that the imbalance of Treg/Th17 cells may also play an important role in pathogenesis of HBV-LC and fibrosis,19–21 whether ABMSC transplantation corrects this imbalance in patients with HBV-LC has yet to be confirmed. Therefore, the current study was to investigate the Treg/ Th17-mediated mechanism underlying the beneficial effects of ABMSCs transplantation in patients with HBV-LC.
Methods Study participants. From March 2012 to December 2012, 81 patients with HBV-LC were recruited in the First Affiliated Hospital of Wenzhou Medical University. The inclusion criteria were as follows: (i) 18–60 years of age; (ii) the hepatitis B surface antigen of all HBV-LC patients were seropositive for more than 6 months; (iii) HBV-DNA levels were > 2 × 103 IU/mL (range, 1.63 × 104–7.80 × 107 IU/mL); and (iv) cirrhosis was diagnosed
Stem cell transplantation for cirrhosis
by evidence of a small nodular liver, or enlarged caudate lobe, widening of liver fissures, and enlargement of the spleen shown by ultrasound, computerized tomography, and magnetic resonance, respectively. The exclusion criteria were as follows: pregnant or nursing females, co-infections with HIV or hepatitis C virus or hepatitis D virus, serious bacterial infection, and/or evidence of other factors causing liver cirrhosis (e.g. a history of alcohol abuse, PBC, druginduced liver disease, inherited metabolic liver disease, etc.), antiviral or immunomodulatory therapy within 6 months before the study, concurrence of liver cancer or other malignancies, other vital organ dysfunctions (e.g. heart failure or kidney dysfunction), and patients with severe complications of liver cirrhosis (e.g. hepatic encephalopathy, variceal bleeding, etc.). High serum creatinine (Cr) level (serum creatinine > 150 μmol/L) and severe coagulopathy (platelets < 30 × 109/L and/or prothrombin time (PT) activity < 20%) were also considered exclusion criteria. Study design. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, China. It was also registered at http:// www.clinicaltrials.gov/ct2/results?term=NCT01560845&Search= Search (NCT01560845) and conformed to the ethical guidelines of the Declaration of Helsinki. All patients were hospitalized for the assessment of eligibility. According to CONSORT guidelines, the disposition of patients was shown in Figure 1. Fifty-six cases who met the above inclusion criteria were divided into transplantation group and control group by random digital table. Informed written consents were obtained from all patients before the treatment.
Figure 1 Disposition of study patients. ABMSC, autologous bone marrow mesenchymal stem cell.
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After 1 week in hospital, patients in transplantation group received ABMSC transfusion and antiviral therapy (Entecavir [ETV] 0.5 mg/day), while patients in control group received only ETV treatment. The primary end–points of this study were absolute changes in the Model for End-Stage Liver Disease (MELD) score and improvement of liver function during the 24 weeks follow-up period. Secondary end–points included the serum levels of HBV DNA and Cr, side-effects and complications, changes in Treg/ Th17 ratio, as well as in related transcription factors and serum cytokines.
Preparation of BMSCs. A total volume of 130–150 mL of bone marrow was aspirated from both sides of auto-posterior superior iliac spines and anticoagulated with heparin. BMSCs were obtained by density gradient centrifugation.1,3,22 Purification procedure of BMSCs was performed in laminar air flow room. Then, purified BMSCs were preserved in 20 mL of normal saline before transplantation. Small fractions of the cell suspension were used for cell counting and cultured to exclude fungal and/or bacterial contamination.
Identification of MSCs. For confirmation of primary isolated MSCs, typical markers were measured by flow cytometry using Human MSC Analysis Kit (BD Biosciences, CA, USA). For identification of MSCs in culture, 0.5 mL of the primary isolated cell suspension was cultured. After three passages, cells were incubated with fluorescein-conjugated antibodies, such as fluorescein isothiocyanate (FITC)-CD44, (FITC)-CD13, (FITC)-CD29, allophycocyanin (APC)-105, (APC)-HLA-DR, (APC)-CD34, and/or phycoerythrin (PE)-CD73, (PE)-CD49d, (PE)-CD31 (eBioscience, San Diego, CA, USA) for 30 min before flow cytometry analysis. Mouse isotype immunoglobulin G1 (IgG1) (eBioscience) was used as the control. We also evaluated the cells’ multipotency of MSC in vitro for osteogenic, adipogenic, and chondrogenic differentiations using different culture media (OriCellTM Human Mesenchymal Stem Cell Osteogenic Differentiation Medium, Adipogenic Differentiation Medium, and Chondrogenic Differentiation Medium from Cyagen Biosciences, respectively).
ABMSCs transplantation and follow-up. All patients from transplantation group received interventional procedures in an operating room with Seldinger technique.23 The 5F RH Catheter (Cook, Bloomington, IN, USA) was inserted into the patient’s hepatic artery via the left or right femoral artery under the monitor of digital subtraction angiography. Stem cell suspension was slowly infused (1 mL/min) into the liver via the hepatic artery. After BMSCs transplantation, patients received antiviral therapy and then followed up at weeks 1, 2, 4, 8, 12, and 24. Blood samples were collected at each visit and analyzed for alanine aminotransferase (ALT), total bilirubin (TBil), albumin (ALB), PT, international normalized ratio, HBV DNA, and Cr. Additionally, 10 mL blood sample was collected for isolation of peripheral blood mononuclear cells (PBMCs) and 5 mL blood sample was used to prepare serum for analysis of cytokines by ELISA. 1622
Flow cytometry analysis of Th17 cells and Treg cells. Isolated PBMCs were cultured at a density of 2 × 106 cells/mL in RPMI 1640 with phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA), ionomycin (SigmaAldrich), and brefeldin A (Sigma-Aldrich) at 37°C in 5% CO2 environment for 5 h. After 30 min incubation with (FITC)-CD4 (BD Biosciences), cells were fixed and permeabilized by IntraPrep Permeabilization Reagent (Beckman Coulter Inc., Fullerton, CA, USA) and then stained with intracellular antibody (PE)-IL-17A (BD Biosciences). For analysis of Treg cells, PBMCs were incubated with (FITC)-CD4 and (PE)-CD25 (BD Biosciences), and then with fix/perm reagent (eBioscience). After incubation, cells were stained with intracellular antibody (Alexa fluor 647)-FoxP3 (BD Biosciences). All labeled cells were analyzed with the FACSCalibur using the CellQuest software (Becton–Dickinson, Franklin Lakes, NJ, USA). Mouse isotype IgG1 (BD Biosciences) was used as control. Foxp3 and RORγt mRNA expression determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from PBMC by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was then converted to cDNA using Revert Aid TM first strand cDNA synthesis Kit (Life Science, Pittsburgh, PA, USA). The first strand cDNA (1 μL) was mixed with SYBR Green PCR master mix (Applied Biosystems) in a final volume of 20 μL. PCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Primers for Foxp3 (forward: 5′-ATTCCCAGA GTTCCTCCACAAC-3′, reverse: 5′-ATTGAGTGTCCGCTGC TTCTC-3′), RORγt (forward: 5′-CAATGGAAGTGGTGCTG GTTAG-3′, reverse: 5′-TTAGGGAGTGGGAGAAGTCAAAG3′), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward: 5′- ATGAGCCCCAGCCTTCTCCAT-3′, reverse: 5′GGTCGGAGTCAACGGATTTG-3′) were synthesized by SBS Genetech Co. Ltd. (Beijing, China). All samples were normalized to GAPDH. ELISA of serum TNF-α, TGF-β, IL-6, and IL-17. Serum levels of TNF-α, TGF-β, IL-6 (R&D Systems, Minneapolis, MN, USA), and IL-17 (eBioscience) were measured by their respective ELISA kits according to the manufacturer’s instructions. All samples were assayed in duplicate and repeated for three times. Intra-assay and inter-assay coefficients of variation for all ELISA were < 5% and < 10%, respectively. Statistical analysis. To achieve a statistical power of 80% and two-sided 5% type I error, with a difference in MELD score improvement by at least two points, a sample size of 22 patients in each group was calculated.24 The age, sex, and negative rates of serum HBV-DNA between transplantation group and control group were analyzed using the chi-square. The levels of ALT, TBiL, ALB, HBV-DNA, and MELD scores at baseline were analyzed using Mann–Whitney U-tests. The anova repeated test was used to assess MELD scores, ALT, TBiL, ALB, and the results from flow cytometry analyses, RT-qPCR, and ELISA in each treatment group during the follow-up time. A
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Table 1
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Clinical characteristics of the patients
Age, years Sex (male) (%) ALT (U/L) TBIL (μmol/L) ALB (g/L) MELD scores HBV-DNA (log10IU/mL)
Control group
Transplantation group
P
45 ± 10 11(58) 62.84 ± 35.61 49.21 ± 44.06 30.13 ± 6.50 13.94 ± 2.70 5.98 ± 0.88
44 ± 12 13(65) 60.05 ± 38.95 55.65 ± 48.70 30.70 ± 5.2 14.30 ± 3.53 5.42 ± 0.99
0.74 0.75 0.65 0.61 0.46 0.88 0.058
Data are shown as mean ± SEM. ALB, albumin; ALT, alanine aminotransferase; HBV-DNA, hepatitis B virus DNA; MELD scores, Model for End-Stage Liver Disease scores; TBIL, total bilirubin.
Newman–Keuls multiple comparison with an alpha level of 0.05 was used for post-hoc comparisons between groups. The analyses were conducted using Statistical Package of SPSS 13.0 (SPSS Inc., Chicago, IL, USA).
Results Patients. Of these 56 patients, 27 received ABMSC transplantation combined with ETV and 29 received ETV alone. Seven patients in the transplantation group and 10 patients in control group were lost during follow-up. Finally, 39 cases were completed and analyzed (20 cases in transplantation group and 19 cases in control group, Fig. 1). Clinical characteristics of two groups are listed in Table 1. The average age, sex ratio, levels of ALT, TBiL, and ALB, and MELD scores at baseline were similar between the two groups (P > 0.05). Characterization of BMSCs from the patients with HBV-LC. The average number of the purified bone marrow stem cells was 8.45 ± 3.28 × 108 per patient with a viability over 94%, in which approximately 0.75 ± 0.50 × 106 were MSCs. As shown in Fig. 2a, about 0.3% of isolated cells are positive in CD73 and negative in hMSC isotype control negative cocktail region, from which 56.2% cells are CD90+ and CD105+. These CD73+/CD90+/ CD105+ cells are considered as hMSC. After culture, we found that BMSCs were fusiform, uniform, and adherent to plastic. Moreover, these MSCs were also characterized by positive staining of CD44, CD13, CD29, CD105, and CD73, and negative for HLADR, CD34, CD49d, and CD31 (Fig. 2b). Furthermore, MSCs could differentiate into osteogenic, adipogenic, and chondrogenic lineages (Fig. 2c). Safety and efficacy of ABMSCs transplantation in patients with HBV-LC. For patients in transplantation group, ABMSCs were successfully infused into the liver. No serious adverse events were observed except for low-grade fever (< 38.5°C) in four patients 1 day posttransplantation, and then became afebrile without treatment. Liver function evaluated by ALT, TBil, and ALB measurements (Fig. 3) was significantly improved after ETV antiviral treatment in both transplantation and
control groups as expected. Compared with the control group, ALT, TBil, and ALB levels tended to improve after ABMSC transplantation. At week 24 posttransplantation, the alterations in serum ALB and TBil levels became statistically significant (Fig. 3b,c). Importantly, MELD scores was significantly improved in the transplantation group compared with control group at 4, 8, and 12 weeks (Fig. 3d, P < 0.05). In addition, there was no significant difference in the negative rate of HBV-DNA between the transplantation group and the control groups (Fig. 3e). Balance of Treg and Th17 cells in PBMCs from healthy and treated patients with HBV-LC. As shown in Figure 4a, Treg and Th17 cells were well balanced in number in PBMCs of healthy individuals, while there was a significant elevation of Th17 cells (approximately onefold) and a significant reduction of Treg (approximately 10%) in patients with HBV-LC. ABMSC transplantation markedly increased Treg cells population at weeks 2, 4, and 12 (Fig. 4b, P < 0.05). However, a gradual decrease in Treg cells was observed from week 12 to week 24 after transplantation. On the other hand, Th17 cells were gradually decreased over the time of treatment in both groups (Fig. 4c). At weeks 4 and 12, ABMSC transplantation significantly decreased Th17 cell population (Fig. 4c). Importantly, the ratio of Treg/Th17 was robustly increased in the transplantation group at all measuring time points except week 24, at which no change was observed (Fig. 4d). Representative flow cytometric analyses of Treg and Th17 cells were shown in Figure S1. Expression of FoxP3 and RORγt mRNA in PBMCs from treated patients with HBV-LC. The expression of RORγt decreased significantly in both groups over time during follow-up (Fig. 5a, P < 0.05). However, this reduction was more pronounced in ABMSCs transplantation group compared with that of control group at all measuring time points (Fig. 5a, P < 0.05). FoxP3 expression was not altered over time in control group, while it significantly increased at weeks 1, 2, and 4 with the peak at week 2 after ABMSC transplantation (Fig. 5b). No significant changes between control and transplantation groups were determined at weeks 8, 12, and 24 (Fig. 5b). Serum levels of TGF-β, IL-17, IL-6, and TNF-α in treated patients with HBV-LC. As shown in Figure 6, serum levels of TGF-β, IL-6, IL-17, and TNF-α decreased in control group over time. The reduction in IL-6, IL17, and TNF-α levels was more pronounced in the transplantation group, indicating an anti-inflammatory effect of ABMSC transplantation. However, TGF-β levels were significantly increased at weeks 1, 2, and 4 after transplant and were significantly higher than that in control group (P < 0.05).
Discussion Previous studies indicated that the imbalance of Treg/Th17 cells may be associated with liver diseases, such as PBC,16 autoimmune hepatitis,25 chronic hepatitis B,26 and alcoholic liver disease.27 Treg and Th17 cells are important in the progression of HBV-related diseases because liver injury caused by HBV is associated with
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Figure 2 Characterization of mesenchymal stem cells (MSCs) from the patients with hepatitis B virus-related liver cirrhosis. As shown in panel (a), the surface expression of bone marrow MSCs’ typical marker proteins was analyzed using fluorescence-assisted cell sorting flow cytometry. There were about 0.30% cells that were negative for hMSC negative cocktail PE (PE CD45, PE CD34, PE CD11b, PE CD19, and PE HLA-DR) and positive for APC CD73. Among them, about 56.2% cells were positive for fluorescein isothiocyanate CD90 and PercP-Cy5.5 CD105. After culture, the bone marrow mesenchymal stem cells were positive for CD44, CD13, CD29, CD105, and CD73, and negative for HLA-DR, CD34, CD49d, and CD31 by flow cytometry (b). As shown in panel (c), the hMSCs were induced to differentiate into osteoblasts (200×, 12 days), adipocytes (200×, 14 days), and chondrocytes (400×, 21 days).
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Figure 3 Effects of autologous bone marrow mesenchymal stem cells transplantation combined with Entecavir on several clinical results of patients with hepatitis B virus-related liver cirrhosis (HBV-LC). The levels of alanine aminotransferase (ALT) (a), albumin (ALB) (b), and total bilirubin (TBil) (c) were detected. The Model for End-Stage Liver Disease (MELD) score (d) and the negative rate of HBV DNA (e) in patients with HBV-LC were calculated. Mean ± SEM. * indicates significant difference compared with baseline at P < 0.05, # indicates significant difference between transplantation and control groups at the same time points at P < 0.05. ( ) Control group, ( ) transplantation group; ( ) control group, ( ) transplantation group.
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Figure 4 Changes of Treg/Th17 cells of patients with hepatitis B virus-related liver cirrhosis (HBV-LC) in two groups. Each vertical tick mark indicates ratio of Treg or Th17 cells to CD4+ T cells of patients by flow cytometry. Treg and Th17 cells of patients with HBV-LC and health people were detected (a). Treg cells (b) and Th17 cells (c) of patients with HBV-LC in two groups were detected. The ratio of Treg/Th17 cells was calculated (d). Mean ± SEM. * indicates significant difference compared with baseline at , The ratio of Th17 cells to CD4+ P < 0.05. T cells (a); control group (b,c,d). , the ratio of Treg cells to CD4+ Tcells (a); transplantation group (b,c,d).
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Figure 5 Expression of FoxP3 and RORγt mRNA in peripheral blood mononuclear cells (PBMCs) from patients with hepatitis B virus-related liver cirrhosis in two groups. Expression of FoxP3 mRNA (a) and RORγt mRNA (b) in PBMCs were detected. Mean ± SEM. * indicates significant difference , Control group; , transplantation group. compared with baseline at P < 0.05.
Figure 6 Changes of related cytokines in peripheral blood mononuclear cells (PBMCs) from patients with hepatitis B virus-related liver cirrhosis in two groups. Serum transforming growth factor-β (TGF-β) (a), interleukin-17 (IL-17) (b), IL-6 (c), and tumor necrosis factor-α (TNF-α) (d) were detected during the follow-up by enzyme-linked immunosorbent assay. Mean ± SEM. * indicates significant difference compared with baseline at P < 0.05, # indicates significant difference between transplantation and control groups at the same time points at P < 0.05. , Control group; , transplantation group.
impaired immune responses and imbalance of Treg/Th17 cell population.19,20 Lower Treg/Th17 ratio might be associated with high degree of liver injury and fibrosis.19 In agreement with these studies, we showed that Treg cells were significantly lower while Th17 cells were significantly higher in patients with HBV-LC as compared with healthy controls. Our findings further support the notion that imbalance between Treg and Th17 cells is a hallmark in patients with HBV-LC. 1626
ETV is considered one of the first-line therapies for the treatment of patients with HBV-LC due to its antiviral activity. Our results showed that single dose of ETV treatment decreased both Treg and Th17 cell populations in HBV-LC patients, but the ratio of Treg/Th17 cells was still lower than healthy individuals. These results might explain why single antiviral treatment could not completely retard the progression of HBV-LC. Interestingly, in comparison with single ETV treatment control, a markedly
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increased Treg cells and a significantly lowered Th17 cells in ABMSCs transplantation group were observed. These results indicated that the combination therapy of ABMSCs transplantation with ETV provides a significant anti-inflammatory effect in the HBV-LC patients through the immunoregulation of Treg/Th17 cells. Previous studies have demonstrated that transplantation of ABMSCs has the potential to improve liver regeneration, and inhibit inflammation and fibrosis.1,28,29 In the present study, we showed that patients who received ABMSCs had a significant improved liver function as shown by improved MELD scores, and serum ALB and bilirubin levels. A variety of factors could contribute to this improvement. As we demonstrated in this study, one of the major factors is the correction of Treg/Th17 ratio, which is critical for the inhibition of inflammatory responses in HBV-LC patients. In addition, ABMSCs could differentiate into hepatocytes in the liver to replace the damaged liver tissue and restore the liver function.30 ABMSCs can also have an indirect impact on the improvement of liver function through paracrine effect via secreting multiple cytokines and growth factors.31 We further examined the molecular mechanisms underlying the immunoregulatory effect of BMSC transplantation. Since BMSCs have been used to treat autoimmune and systemic inflammatory diseases,6,32 and the immune modulatory properties of BMSC may be involved in differentiation of Treg and Th17 cells,33 we examined the transcription factors FoxP3 and RORγt, which are specific to respective Treg and Th17 cells. Significant increase in FoxP3 expression after ABMSCs transplantation in patients with HBV-LC suggests an important role of FoxP3-regulated genes. Meanwhile, the inhibition of RORγt mRNA was more pronounced after transplantation. Therefore, further investigation of FoxP3and RORγt-regulated genes may reveal more precise molecular mechanisms of ABMSC transplantation. Previous studies also suggested that both increase in Th17 cells and decrease in Treg cells may trigger inflammatory disorders.34 BMSCs exhibit potent immunosuppressive and anti-inflammatory effects through regulation of serum levels of inflammatory cytokines.33,35 Our study confirmed that transplantation of BMSCs significantly decreased serum inflammatory cytokines, such as IL-17, IL-6, and TNF-α, in patients with HBV-LC. Importantly, we found that transplanted BMSCs markedly elevated serum TGF-β level, which is required for both Treg and Th17 cell proliferation,36 and increased Treg cells and reduced Th17 cell proliferation. Previous studies have demonstrated that IL-6, together with low concentrations of TGF-β, induces Th17 cells differentiation or inhibits Treg differentiation,37–39 while high concentrations of TGF-β favor FoxP3+ Treg cells, and TGF-β-induced FoxP3 could inhibit Th17 cell differentiation by antagonizing RORγt function.40 Also, it was reported that an increased ratio of Treg/ Th17 cells controls the pathogenesis and progression of collageninduced arthritis.12 Consistent with these findings, current study suggests a beneficial effect using ABMSC transplantation in the treatment of HBV-related cirrhosis. Although ABMSCs transplantation has been demonstrated effective for patients with HBV-LC, challenges still remain. Isolating and purifying sufficient numbers of BMSCs is one of the major concerns in BMSC transplantation. It is unknown whether BMSCs from health persons could effectively elevate Treg/Th17 ratio, and thus be utilized to treat HBV-LC patients. These challenges warrant further investigation.
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In conclusion, we found that transplantation of ABMSCs could improve liver function and correct the Treg/Th17 cell imbalance in patients with HBV-LC. ABMSC transplantation could be utilized as a potential therapeutic approach for patients with HBV-related cirrhosis.
Acknowledgments This work was, in part, supported by grants from Scientific Research Foundation of Wenzhou, Zhejiang Province, China (Y20100180 and Y20140073), Medicine Health Project of Zhejiang Province, China (2014KYB155), and the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012ZX10002004-101 and 2013ZX10005002-001-006).
References 1 Kharaziha P, Hellstrom PM, Noorinayer B et al. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial. Eur. J. Gastroenterol. Hepatol. 2009; 21: 1199–205. 2 Lyra AC, Soares MB, da Silva LF et al. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease. World J. Gastroenterol. 2007; 13: 1067–73. 3 Peng L, Xie DY, Lin BL et al. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes. Hepatology 2011; 54: 820–8. 4 Terai S, Ishikawa T, Omori K et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24: 2292–8. 5 Le Blanc K, Ringden O. Immunomodulation by mesenchymal stem cells and clinical experience. J. Intern. Med. 2007; 262: 509–25. 6 Ozaki K, Sato K, Oh I et al. Mechanisms of immunomodulation by mesenchymal stem cells. Int. J. Hematol. 2007; 86: 5–7. 7 Rasmusson I. Immune modulation by mesenchymal stem cells. Exp. Cell Res. 2006; 312: 2169–79. 8 Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363: 1439–41. 9 Wu Y, Ren M, Yang R et al. Reduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced CCR4+CCR6+ Th/Treg cell subset imbalance in ankylosing spondylitis. Arthritis Res. Ther. 2011; 13: R29. 10 Wang L, Li J, Liu H et al. A pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis. J. Gastroenterol. Hepatol. 2013; 28 (Suppl. 1): 85–92. 11 Madec AM, Mallone R, Afonso G et al. Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells. Diabetologia 2009; 52: 1391–9. 12 Park MJ, Park HS, Cho ML et al. Transforming growth factor beta-transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis. Arthritis Rheum. 2011; 63: 1668–80. 13 Wang Y, Zhang A, Ye Z, Xie H, Zheng S. Bone marrow-derived mesenchymal stem cells inhibit acute rejection of rat liver allografts in association with regulatory T-cell expansion. Transplant. Proc. 2009; 41: 4352–6. 14 Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008; 133: 775–87.
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15 Zhu J, Paul WE. Heterogeneity and plasticity of T helper cells. Cell Res. 2010; 20: 4–12. 16 Rong G, Zhou Y, Xiong Y et al. Imbalance between T helper type 17 and T regulatory cells in patients with primary biliary cirrhosis: the serum cytokine profile and peripheral cell population. Clin. Exp. Immunol. 2009; 156: 217–25. 17 Wang W, Shao S, Jiao Z, Guo M, Xu H, Wang S. The Th17/Treg imbalance and cytokine environment in peripheral blood of patients with rheumatoid arthritis. Rheumatol. Int. 2012; 32: 887–93. 18 Yang J, Yang X, Zou H, Chu Y, Li M. Recovery of the immune balance between Th17 and regulatory T cells as a treatment for systemic lupus erythematosus. Rheumatology 2011; 50: 1366–72. 19 Li J, Qiu SJ, She WM et al. Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis. PLoS ONE 2012; 7: e39307. 20 Niu Y, Liu H, Yin D et al. The balance between intrahepatic IL-17(+) T cells and Foxp3(+) regulatory T cells plays an important role in HBV-related end-stage liver disease. BMC Immunol. 2011; 12: 47. 21 Zhao L, Qiu K, Ma X. Th17 cells: the emerging reciprocal partner of regulatory T cells in the liver. J. Dig. Dis. 2010; 11: 126–33. 22 Li J, Tao R, Wu W et al. 3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes. Stem Cells Dev. 2010; 19: 1427–36. 23 Higgs ZC, Macafee DA, Braithwaite BD, Maxwell-Armstrong CA. The Seldinger technique: 50 years on. Lancet 2005; 366: 1407–9. 24 Salama H, Zekri AR, Zern M et al. Autologous hematopoietic stem cell transplantation in 48 patients with end-stage chronic liver diseases. Cell Transplant. 2010; 19: 1475–86. 25 Longhi MS, Meda F, Wang P et al. Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis. Hepatology 2008; 47: 581–91. 26 Zhang JY, Zhang Z, Lin F et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology 2010; 51: 81–91. 27 Lemmers A, Moreno C, Gustot T et al. The interleukin-17 pathway is involved in human alcoholic liver disease. Hepatology 2009; 49: 646–57. 28 Almeida-Porada G, Zanjani ED, Porada CD. Bone marrow stem cells and liver regeneration. Exp. Hematol. 2010; 38: 574–80. 29 Gilchrist ES, Plevris JN. Bone marrow-derived stem cells in liver repair: 10 years down the line. Liver Transpl. 2010; 16: 118–29. 30 Terai S, Sakaida I, Yamamoto N et al. An in vivo model for
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monitoring trans-differentiation of bone marrow cells into functional hepatocytes. J. Biochem. 2003; 134: 551–8. Chen Z, Qi LZ, Zeng R, Li HY, Dai LJ. Stem cells and hepatic cirrhosis. Panminerva Med. 2010; 52: 149–65. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood 2007; 110: 3499–506. Tatara R, Ozaki K, Kikuchi Y et al. Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation. Cytotherapy 2011; 13: 686–94. Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17 balance. Eur. J. Immunol. 2010; 40: 1830–5. Augello A, Tasso R, Negrini SM, Cancedda R, Pennesi G. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum. 2007; 56: 1175–86. Ye C, Li WY, Zheng MH, Chen YP. T-helper 17 cell: a distinctive cell in liver diseases. Hepatol. Res. 2011; 41: 22–9. Bettelli E, Carrier Y, Gao W et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006; 441: 235–8. Mangan PR, Harrington LE, O’Quinn DB et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441: 231–4. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006; 24: 179–89. Zhou L, Lopes JE, Chong MM et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 2008; 453: 236–40.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Representative flow cytometric analyses of Treg and Th17 cells in to two groups. Representative dot plots of Treg cells at weeks 0, 4, and 24 in control group (Panel A) and transplantation group (Panel B). Representative dot plots of Th17 cells at weeks 0, 4, and 24 in control group (Panel C) and transplantation group (Panel D).
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doi:10.1111/jgh.12653
H E PAT O L O G Y
Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: Regulation of Treg/Th17 cells Lanman Xu,* Yuewen Gong,† Benfu Wang,‡ Keqing Shi,* Yijun Hou,* Liping Wang,* Zuo Lin,* Yixiang Han,§ Lu Lu,¶ Dazhi Chen,** Xiuli Lin,* Qiqiang Zeng,†† Wenke Feng,*,‡‡ and Yongping Chen* Departments of *Infection and Liver Diseases of the First Affiliated Hospital and Liver Research Center and ‡Anesthesiology of the Second Affiliated Hospital, Wenzhou Medical University, §Laboratory of Internal Medicine, Departments of ¶Pathology and ††Hepatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, and **Renji College of Wenzhou Medical University, Wenzhou, Zhejiang, China; † Faculty of Pharmacy and Section of Hepatology of Department of Internal Medicine, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and ‡‡Department of Medicine, University of Louisville, Louisville, Kentucky, USA
Key words bone marrow mesenchymal stem cells, hepatitis B virus, liver cirrhosis, stem cell transplantation, Treg/Th17 cells. Accepted for publication 17 March 2014. Correspondence Dr Yongping Chen, Department of Infection and Liver Diseases of the First Affiliated Hospital and Liver Research Center, Wenzhou Medical University, 2 Fuxue Lane, Wenzhou, Zhejiang Province 325000, China. Email: [email protected] Conflicts of interest: The authors disclose no conflicts.
Abstract Background and Aim: Liver cirrhosis is one of the major consequences of hepatitis B virus (HBV) infection, and transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) is one of promising therapies for patients with HBV-related liver cirrhosis (HBV-LC). However, the mechanism is unclear. The aim of the current study was to explore the role of Treg/Th17 cells in ABMSCs transplantation in patients with HBV-LC. Methods: In this prospective study, 56 patients were enrolled and randomly assigned to transplantation group and control group. After 24-week follow-up, 39 patients completed the study (20 cases in transplantation group and 19 cases in control group). The Model for End-Stage Liver Disease scores, liver function, changes of Treg/Th17 cells, as well as related transcription factors and serum cytokines, were determined. Results: Although patients in both groups showed significant improvement after Entecavir treatment, ABMSC transplantation further improved patients’ liver function. Moreover, there was a significant increase in Treg cells and a marked decrease in Th17 cells in the transplantation group compared with control, leading to an increased Treg/ Th17 ratio. Furthermore, mRNA levels of Treg-related transcription factor (Foxp3) and Th17-related transcription factor (RORγt) were increased and decreased, respectively. In addition, serum transforming growth factor-β levels were significantly higher at early weeks of transplantation, while serum levels of interleukin-17, tumor necrosis factor-α, and interleukin-6 were significantly lower in patients in the transplantation group compared with control. Conclusion: ABMSCs transplantation was effective in improving liver function in patients with HBV-LC, which was mediated, at least in part, through the regulation of Treg/Th17 cell balance.
Introduction Liver cirrhosis caused by the infection of hepatitis B virus (HBV) is a worldwide health problem. Many patients with HBV-related liver cirrhosis (HBV-LC) died from its serious complications, such as variceal hemorrhage and hepatic encephalopathy. Although current therapies, including antivirus medications and liver protection, are used for the treatment of HBV-LC, there are no effective therapies to reverse the cirrhosis except liver transplantation. However, most patients are either unable to obtain transplantation due to shortage of donor organs, or suffer from liver rejection and serious complications posttransplantation. Recently, several 1620
studies suggested that transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) appears to be safe and effective for patients with HBV-LC.1–4 Compared with liver transplantation, the stem cell therapy has several advantages, such as availability and fewer complications. Mesenchymal stem cells (MSCs) are an important part of bone marrow stem cells and have immunomodulatory effects.5–7 They provide a hopeful approach for treatment of graft-versus-host disease and other autoimmune diseases.8–10 Recent studies indicated that immunomodulatory activities of MSCs might be associated with Foxp3+ regulatory T cells (Treg) and IL-17+ T helper cells (Th17).11–13 Treg cells are a subset of CD4+ T cells and
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primarily characterized by CD25 and forkhead family transcription factor 3 (Foxp3). They mainly produce immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β).14 Th17 cells are another newly identified subset of CD4+ T cells and closely related to Treg cells. They mediate inflammation by producing pro-inflammatory cytokines, such as IL-17, IL-6 and tumor necrosis factor-α (TNF-α). Retinoid orphan nuclear receptor γt (RORγt) is a specific transcription factor expressed in Th17 cells.15 Studies indicated that the balance between Treg/Th17 cells was disturbed in several inflammatory and autoimmune diseases, such as primary biliary cirrhosis (PBC), systemic lupus erythematosus, and rheumatoid arthritis.16–18 Although a few studies have suggested that the imbalance of Treg/Th17 cells may also play an important role in pathogenesis of HBV-LC and fibrosis,19–21 whether ABMSC transplantation corrects this imbalance in patients with HBV-LC has yet to be confirmed. Therefore, the current study was to investigate the Treg/ Th17-mediated mechanism underlying the beneficial effects of ABMSCs transplantation in patients with HBV-LC.
Methods Study participants. From March 2012 to December 2012, 81 patients with HBV-LC were recruited in the First Affiliated Hospital of Wenzhou Medical University. The inclusion criteria were as follows: (i) 18–60 years of age; (ii) the hepatitis B surface antigen of all HBV-LC patients were seropositive for more than 6 months; (iii) HBV-DNA levels were > 2 × 103 IU/mL (range, 1.63 × 104–7.80 × 107 IU/mL); and (iv) cirrhosis was diagnosed
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by evidence of a small nodular liver, or enlarged caudate lobe, widening of liver fissures, and enlargement of the spleen shown by ultrasound, computerized tomography, and magnetic resonance, respectively. The exclusion criteria were as follows: pregnant or nursing females, co-infections with HIV or hepatitis C virus or hepatitis D virus, serious bacterial infection, and/or evidence of other factors causing liver cirrhosis (e.g. a history of alcohol abuse, PBC, druginduced liver disease, inherited metabolic liver disease, etc.), antiviral or immunomodulatory therapy within 6 months before the study, concurrence of liver cancer or other malignancies, other vital organ dysfunctions (e.g. heart failure or kidney dysfunction), and patients with severe complications of liver cirrhosis (e.g. hepatic encephalopathy, variceal bleeding, etc.). High serum creatinine (Cr) level (serum creatinine > 150 μmol/L) and severe coagulopathy (platelets < 30 × 109/L and/or prothrombin time (PT) activity < 20%) were also considered exclusion criteria. Study design. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, China. It was also registered at http:// www.clinicaltrials.gov/ct2/results?term=NCT01560845&Search= Search (NCT01560845) and conformed to the ethical guidelines of the Declaration of Helsinki. All patients were hospitalized for the assessment of eligibility. According to CONSORT guidelines, the disposition of patients was shown in Figure 1. Fifty-six cases who met the above inclusion criteria were divided into transplantation group and control group by random digital table. Informed written consents were obtained from all patients before the treatment.
Figure 1 Disposition of study patients. ABMSC, autologous bone marrow mesenchymal stem cell.
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After 1 week in hospital, patients in transplantation group received ABMSC transfusion and antiviral therapy (Entecavir [ETV] 0.5 mg/day), while patients in control group received only ETV treatment. The primary end–points of this study were absolute changes in the Model for End-Stage Liver Disease (MELD) score and improvement of liver function during the 24 weeks follow-up period. Secondary end–points included the serum levels of HBV DNA and Cr, side-effects and complications, changes in Treg/ Th17 ratio, as well as in related transcription factors and serum cytokines.
Preparation of BMSCs. A total volume of 130–150 mL of bone marrow was aspirated from both sides of auto-posterior superior iliac spines and anticoagulated with heparin. BMSCs were obtained by density gradient centrifugation.1,3,22 Purification procedure of BMSCs was performed in laminar air flow room. Then, purified BMSCs were preserved in 20 mL of normal saline before transplantation. Small fractions of the cell suspension were used for cell counting and cultured to exclude fungal and/or bacterial contamination.
Identification of MSCs. For confirmation of primary isolated MSCs, typical markers were measured by flow cytometry using Human MSC Analysis Kit (BD Biosciences, CA, USA). For identification of MSCs in culture, 0.5 mL of the primary isolated cell suspension was cultured. After three passages, cells were incubated with fluorescein-conjugated antibodies, such as fluorescein isothiocyanate (FITC)-CD44, (FITC)-CD13, (FITC)-CD29, allophycocyanin (APC)-105, (APC)-HLA-DR, (APC)-CD34, and/or phycoerythrin (PE)-CD73, (PE)-CD49d, (PE)-CD31 (eBioscience, San Diego, CA, USA) for 30 min before flow cytometry analysis. Mouse isotype immunoglobulin G1 (IgG1) (eBioscience) was used as the control. We also evaluated the cells’ multipotency of MSC in vitro for osteogenic, adipogenic, and chondrogenic differentiations using different culture media (OriCellTM Human Mesenchymal Stem Cell Osteogenic Differentiation Medium, Adipogenic Differentiation Medium, and Chondrogenic Differentiation Medium from Cyagen Biosciences, respectively).
ABMSCs transplantation and follow-up. All patients from transplantation group received interventional procedures in an operating room with Seldinger technique.23 The 5F RH Catheter (Cook, Bloomington, IN, USA) was inserted into the patient’s hepatic artery via the left or right femoral artery under the monitor of digital subtraction angiography. Stem cell suspension was slowly infused (1 mL/min) into the liver via the hepatic artery. After BMSCs transplantation, patients received antiviral therapy and then followed up at weeks 1, 2, 4, 8, 12, and 24. Blood samples were collected at each visit and analyzed for alanine aminotransferase (ALT), total bilirubin (TBil), albumin (ALB), PT, international normalized ratio, HBV DNA, and Cr. Additionally, 10 mL blood sample was collected for isolation of peripheral blood mononuclear cells (PBMCs) and 5 mL blood sample was used to prepare serum for analysis of cytokines by ELISA. 1622
Flow cytometry analysis of Th17 cells and Treg cells. Isolated PBMCs were cultured at a density of 2 × 106 cells/mL in RPMI 1640 with phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA), ionomycin (SigmaAldrich), and brefeldin A (Sigma-Aldrich) at 37°C in 5% CO2 environment for 5 h. After 30 min incubation with (FITC)-CD4 (BD Biosciences), cells were fixed and permeabilized by IntraPrep Permeabilization Reagent (Beckman Coulter Inc., Fullerton, CA, USA) and then stained with intracellular antibody (PE)-IL-17A (BD Biosciences). For analysis of Treg cells, PBMCs were incubated with (FITC)-CD4 and (PE)-CD25 (BD Biosciences), and then with fix/perm reagent (eBioscience). After incubation, cells were stained with intracellular antibody (Alexa fluor 647)-FoxP3 (BD Biosciences). All labeled cells were analyzed with the FACSCalibur using the CellQuest software (Becton–Dickinson, Franklin Lakes, NJ, USA). Mouse isotype IgG1 (BD Biosciences) was used as control. Foxp3 and RORγt mRNA expression determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from PBMC by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was then converted to cDNA using Revert Aid TM first strand cDNA synthesis Kit (Life Science, Pittsburgh, PA, USA). The first strand cDNA (1 μL) was mixed with SYBR Green PCR master mix (Applied Biosystems) in a final volume of 20 μL. PCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Primers for Foxp3 (forward: 5′-ATTCCCAGA GTTCCTCCACAAC-3′, reverse: 5′-ATTGAGTGTCCGCTGC TTCTC-3′), RORγt (forward: 5′-CAATGGAAGTGGTGCTG GTTAG-3′, reverse: 5′-TTAGGGAGTGGGAGAAGTCAAAG3′), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward: 5′- ATGAGCCCCAGCCTTCTCCAT-3′, reverse: 5′GGTCGGAGTCAACGGATTTG-3′) were synthesized by SBS Genetech Co. Ltd. (Beijing, China). All samples were normalized to GAPDH. ELISA of serum TNF-α, TGF-β, IL-6, and IL-17. Serum levels of TNF-α, TGF-β, IL-6 (R&D Systems, Minneapolis, MN, USA), and IL-17 (eBioscience) were measured by their respective ELISA kits according to the manufacturer’s instructions. All samples were assayed in duplicate and repeated for three times. Intra-assay and inter-assay coefficients of variation for all ELISA were < 5% and < 10%, respectively. Statistical analysis. To achieve a statistical power of 80% and two-sided 5% type I error, with a difference in MELD score improvement by at least two points, a sample size of 22 patients in each group was calculated.24 The age, sex, and negative rates of serum HBV-DNA between transplantation group and control group were analyzed using the chi-square. The levels of ALT, TBiL, ALB, HBV-DNA, and MELD scores at baseline were analyzed using Mann–Whitney U-tests. The anova repeated test was used to assess MELD scores, ALT, TBiL, ALB, and the results from flow cytometry analyses, RT-qPCR, and ELISA in each treatment group during the follow-up time. A
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Table 1
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Clinical characteristics of the patients
Age, years Sex (male) (%) ALT (U/L) TBIL (μmol/L) ALB (g/L) MELD scores HBV-DNA (log10IU/mL)
Control group
Transplantation group
P
45 ± 10 11(58) 62.84 ± 35.61 49.21 ± 44.06 30.13 ± 6.50 13.94 ± 2.70 5.98 ± 0.88
44 ± 12 13(65) 60.05 ± 38.95 55.65 ± 48.70 30.70 ± 5.2 14.30 ± 3.53 5.42 ± 0.99
0.74 0.75 0.65 0.61 0.46 0.88 0.058
Data are shown as mean ± SEM. ALB, albumin; ALT, alanine aminotransferase; HBV-DNA, hepatitis B virus DNA; MELD scores, Model for End-Stage Liver Disease scores; TBIL, total bilirubin.
Newman–Keuls multiple comparison with an alpha level of 0.05 was used for post-hoc comparisons between groups. The analyses were conducted using Statistical Package of SPSS 13.0 (SPSS Inc., Chicago, IL, USA).
Results Patients. Of these 56 patients, 27 received ABMSC transplantation combined with ETV and 29 received ETV alone. Seven patients in the transplantation group and 10 patients in control group were lost during follow-up. Finally, 39 cases were completed and analyzed (20 cases in transplantation group and 19 cases in control group, Fig. 1). Clinical characteristics of two groups are listed in Table 1. The average age, sex ratio, levels of ALT, TBiL, and ALB, and MELD scores at baseline were similar between the two groups (P > 0.05). Characterization of BMSCs from the patients with HBV-LC. The average number of the purified bone marrow stem cells was 8.45 ± 3.28 × 108 per patient with a viability over 94%, in which approximately 0.75 ± 0.50 × 106 were MSCs. As shown in Fig. 2a, about 0.3% of isolated cells are positive in CD73 and negative in hMSC isotype control negative cocktail region, from which 56.2% cells are CD90+ and CD105+. These CD73+/CD90+/ CD105+ cells are considered as hMSC. After culture, we found that BMSCs were fusiform, uniform, and adherent to plastic. Moreover, these MSCs were also characterized by positive staining of CD44, CD13, CD29, CD105, and CD73, and negative for HLADR, CD34, CD49d, and CD31 (Fig. 2b). Furthermore, MSCs could differentiate into osteogenic, adipogenic, and chondrogenic lineages (Fig. 2c). Safety and efficacy of ABMSCs transplantation in patients with HBV-LC. For patients in transplantation group, ABMSCs were successfully infused into the liver. No serious adverse events were observed except for low-grade fever (< 38.5°C) in four patients 1 day posttransplantation, and then became afebrile without treatment. Liver function evaluated by ALT, TBil, and ALB measurements (Fig. 3) was significantly improved after ETV antiviral treatment in both transplantation and
control groups as expected. Compared with the control group, ALT, TBil, and ALB levels tended to improve after ABMSC transplantation. At week 24 posttransplantation, the alterations in serum ALB and TBil levels became statistically significant (Fig. 3b,c). Importantly, MELD scores was significantly improved in the transplantation group compared with control group at 4, 8, and 12 weeks (Fig. 3d, P < 0.05). In addition, there was no significant difference in the negative rate of HBV-DNA between the transplantation group and the control groups (Fig. 3e). Balance of Treg and Th17 cells in PBMCs from healthy and treated patients with HBV-LC. As shown in Figure 4a, Treg and Th17 cells were well balanced in number in PBMCs of healthy individuals, while there was a significant elevation of Th17 cells (approximately onefold) and a significant reduction of Treg (approximately 10%) in patients with HBV-LC. ABMSC transplantation markedly increased Treg cells population at weeks 2, 4, and 12 (Fig. 4b, P < 0.05). However, a gradual decrease in Treg cells was observed from week 12 to week 24 after transplantation. On the other hand, Th17 cells were gradually decreased over the time of treatment in both groups (Fig. 4c). At weeks 4 and 12, ABMSC transplantation significantly decreased Th17 cell population (Fig. 4c). Importantly, the ratio of Treg/Th17 was robustly increased in the transplantation group at all measuring time points except week 24, at which no change was observed (Fig. 4d). Representative flow cytometric analyses of Treg and Th17 cells were shown in Figure S1. Expression of FoxP3 and RORγt mRNA in PBMCs from treated patients with HBV-LC. The expression of RORγt decreased significantly in both groups over time during follow-up (Fig. 5a, P < 0.05). However, this reduction was more pronounced in ABMSCs transplantation group compared with that of control group at all measuring time points (Fig. 5a, P < 0.05). FoxP3 expression was not altered over time in control group, while it significantly increased at weeks 1, 2, and 4 with the peak at week 2 after ABMSC transplantation (Fig. 5b). No significant changes between control and transplantation groups were determined at weeks 8, 12, and 24 (Fig. 5b). Serum levels of TGF-β, IL-17, IL-6, and TNF-α in treated patients with HBV-LC. As shown in Figure 6, serum levels of TGF-β, IL-6, IL-17, and TNF-α decreased in control group over time. The reduction in IL-6, IL17, and TNF-α levels was more pronounced in the transplantation group, indicating an anti-inflammatory effect of ABMSC transplantation. However, TGF-β levels were significantly increased at weeks 1, 2, and 4 after transplant and were significantly higher than that in control group (P < 0.05).
Discussion Previous studies indicated that the imbalance of Treg/Th17 cells may be associated with liver diseases, such as PBC,16 autoimmune hepatitis,25 chronic hepatitis B,26 and alcoholic liver disease.27 Treg and Th17 cells are important in the progression of HBV-related diseases because liver injury caused by HBV is associated with
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Figure 2 Characterization of mesenchymal stem cells (MSCs) from the patients with hepatitis B virus-related liver cirrhosis. As shown in panel (a), the surface expression of bone marrow MSCs’ typical marker proteins was analyzed using fluorescence-assisted cell sorting flow cytometry. There were about 0.30% cells that were negative for hMSC negative cocktail PE (PE CD45, PE CD34, PE CD11b, PE CD19, and PE HLA-DR) and positive for APC CD73. Among them, about 56.2% cells were positive for fluorescein isothiocyanate CD90 and PercP-Cy5.5 CD105. After culture, the bone marrow mesenchymal stem cells were positive for CD44, CD13, CD29, CD105, and CD73, and negative for HLA-DR, CD34, CD49d, and CD31 by flow cytometry (b). As shown in panel (c), the hMSCs were induced to differentiate into osteoblasts (200×, 12 days), adipocytes (200×, 14 days), and chondrocytes (400×, 21 days).
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(a)
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Figure 3 Effects of autologous bone marrow mesenchymal stem cells transplantation combined with Entecavir on several clinical results of patients with hepatitis B virus-related liver cirrhosis (HBV-LC). The levels of alanine aminotransferase (ALT) (a), albumin (ALB) (b), and total bilirubin (TBil) (c) were detected. The Model for End-Stage Liver Disease (MELD) score (d) and the negative rate of HBV DNA (e) in patients with HBV-LC were calculated. Mean ± SEM. * indicates significant difference compared with baseline at P < 0.05, # indicates significant difference between transplantation and control groups at the same time points at P < 0.05. ( ) Control group, ( ) transplantation group; ( ) control group, ( ) transplantation group.
(b) CD25+ FoxP3+ /CD4+ T cells %
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Figure 4 Changes of Treg/Th17 cells of patients with hepatitis B virus-related liver cirrhosis (HBV-LC) in two groups. Each vertical tick mark indicates ratio of Treg or Th17 cells to CD4+ T cells of patients by flow cytometry. Treg and Th17 cells of patients with HBV-LC and health people were detected (a). Treg cells (b) and Th17 cells (c) of patients with HBV-LC in two groups were detected. The ratio of Treg/Th17 cells was calculated (d). Mean ± SEM. * indicates significant difference compared with baseline at , The ratio of Th17 cells to CD4+ P < 0.05. T cells (a); control group (b,c,d). , the ratio of Treg cells to CD4+ Tcells (a); transplantation group (b,c,d).
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Figure 5 Expression of FoxP3 and RORγt mRNA in peripheral blood mononuclear cells (PBMCs) from patients with hepatitis B virus-related liver cirrhosis in two groups. Expression of FoxP3 mRNA (a) and RORγt mRNA (b) in PBMCs were detected. Mean ± SEM. * indicates significant difference , Control group; , transplantation group. compared with baseline at P < 0.05.
Figure 6 Changes of related cytokines in peripheral blood mononuclear cells (PBMCs) from patients with hepatitis B virus-related liver cirrhosis in two groups. Serum transforming growth factor-β (TGF-β) (a), interleukin-17 (IL-17) (b), IL-6 (c), and tumor necrosis factor-α (TNF-α) (d) were detected during the follow-up by enzyme-linked immunosorbent assay. Mean ± SEM. * indicates significant difference compared with baseline at P < 0.05, # indicates significant difference between transplantation and control groups at the same time points at P < 0.05. , Control group; , transplantation group.
impaired immune responses and imbalance of Treg/Th17 cell population.19,20 Lower Treg/Th17 ratio might be associated with high degree of liver injury and fibrosis.19 In agreement with these studies, we showed that Treg cells were significantly lower while Th17 cells were significantly higher in patients with HBV-LC as compared with healthy controls. Our findings further support the notion that imbalance between Treg and Th17 cells is a hallmark in patients with HBV-LC. 1626
ETV is considered one of the first-line therapies for the treatment of patients with HBV-LC due to its antiviral activity. Our results showed that single dose of ETV treatment decreased both Treg and Th17 cell populations in HBV-LC patients, but the ratio of Treg/Th17 cells was still lower than healthy individuals. These results might explain why single antiviral treatment could not completely retard the progression of HBV-LC. Interestingly, in comparison with single ETV treatment control, a markedly
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increased Treg cells and a significantly lowered Th17 cells in ABMSCs transplantation group were observed. These results indicated that the combination therapy of ABMSCs transplantation with ETV provides a significant anti-inflammatory effect in the HBV-LC patients through the immunoregulation of Treg/Th17 cells. Previous studies have demonstrated that transplantation of ABMSCs has the potential to improve liver regeneration, and inhibit inflammation and fibrosis.1,28,29 In the present study, we showed that patients who received ABMSCs had a significant improved liver function as shown by improved MELD scores, and serum ALB and bilirubin levels. A variety of factors could contribute to this improvement. As we demonstrated in this study, one of the major factors is the correction of Treg/Th17 ratio, which is critical for the inhibition of inflammatory responses in HBV-LC patients. In addition, ABMSCs could differentiate into hepatocytes in the liver to replace the damaged liver tissue and restore the liver function.30 ABMSCs can also have an indirect impact on the improvement of liver function through paracrine effect via secreting multiple cytokines and growth factors.31 We further examined the molecular mechanisms underlying the immunoregulatory effect of BMSC transplantation. Since BMSCs have been used to treat autoimmune and systemic inflammatory diseases,6,32 and the immune modulatory properties of BMSC may be involved in differentiation of Treg and Th17 cells,33 we examined the transcription factors FoxP3 and RORγt, which are specific to respective Treg and Th17 cells. Significant increase in FoxP3 expression after ABMSCs transplantation in patients with HBV-LC suggests an important role of FoxP3-regulated genes. Meanwhile, the inhibition of RORγt mRNA was more pronounced after transplantation. Therefore, further investigation of FoxP3and RORγt-regulated genes may reveal more precise molecular mechanisms of ABMSC transplantation. Previous studies also suggested that both increase in Th17 cells and decrease in Treg cells may trigger inflammatory disorders.34 BMSCs exhibit potent immunosuppressive and anti-inflammatory effects through regulation of serum levels of inflammatory cytokines.33,35 Our study confirmed that transplantation of BMSCs significantly decreased serum inflammatory cytokines, such as IL-17, IL-6, and TNF-α, in patients with HBV-LC. Importantly, we found that transplanted BMSCs markedly elevated serum TGF-β level, which is required for both Treg and Th17 cell proliferation,36 and increased Treg cells and reduced Th17 cell proliferation. Previous studies have demonstrated that IL-6, together with low concentrations of TGF-β, induces Th17 cells differentiation or inhibits Treg differentiation,37–39 while high concentrations of TGF-β favor FoxP3+ Treg cells, and TGF-β-induced FoxP3 could inhibit Th17 cell differentiation by antagonizing RORγt function.40 Also, it was reported that an increased ratio of Treg/ Th17 cells controls the pathogenesis and progression of collageninduced arthritis.12 Consistent with these findings, current study suggests a beneficial effect using ABMSC transplantation in the treatment of HBV-related cirrhosis. Although ABMSCs transplantation has been demonstrated effective for patients with HBV-LC, challenges still remain. Isolating and purifying sufficient numbers of BMSCs is one of the major concerns in BMSC transplantation. It is unknown whether BMSCs from health persons could effectively elevate Treg/Th17 ratio, and thus be utilized to treat HBV-LC patients. These challenges warrant further investigation.
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In conclusion, we found that transplantation of ABMSCs could improve liver function and correct the Treg/Th17 cell imbalance in patients with HBV-LC. ABMSC transplantation could be utilized as a potential therapeutic approach for patients with HBV-related cirrhosis.
Acknowledgments This work was, in part, supported by grants from Scientific Research Foundation of Wenzhou, Zhejiang Province, China (Y20100180 and Y20140073), Medicine Health Project of Zhejiang Province, China (2014KYB155), and the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012ZX10002004-101 and 2013ZX10005002-001-006).
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Representative flow cytometric analyses of Treg and Th17 cells in to two groups. Representative dot plots of Treg cells at weeks 0, 4, and 24 in control group (Panel A) and transplantation group (Panel B). Representative dot plots of Th17 cells at weeks 0, 4, and 24 in control group (Panel C) and transplantation group (Panel D).
Journal of Gastroenterology and Hepatology 29 (2014) 1620–1628 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
