Immunology Letters 162 (2014) 222–228

Contents lists available at ScienceDirect

Immunology Letters journal homepage: www.elsevier.com/locate/immlet

Mesenchymal stem cells ameliorate experimental autoimmune hepatitis by activation of the programmed death 1 pathway Yi Chen a,1 , Si Chen a,1 , Li-Yuan Liu a , Zhuo-Lin Zou a , Yi-Jing Cai a , Jin-Guo Wang b , Bi Chen c , Lan-Man Xu a , Zhuo Lin a , Xiao-Dong Wang a , Yong-Ping Chen a,∗ a Hepatology Institute of Wenzhou Medical University, Department of Infectious Disease, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China b Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China c Department of Spine Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

a r t i c l e

i n f o

Article history: Available online 28 October 2014 Keywords: Mesenchymal stem cells Autoimmune hepatitis Programmed death ligand 1 Interleukin-17 Interleukin-23

a b s t r a c t Previous studies have shown beneficial effects of mesenchymal stem cells (MSCs) transplantation in many autoimmune diseases. However, few studies have focused on the effects of MSCs on autoimmune hepatitis. In our study, we investigated the therapeutic effects of BMSCs (bone mesenchymal stem cells) transplantation in mouse experimental autoimmune hepatitis (EAH) and explored the potential mechanism. BMSCs were injected intravenously into EAH mice. Then, serum levels of ALT and AST, and pathologic alteration of liver tissue were measured to evaluate the liver function and inflammation degree. The expressions of programmed death ligand 1, IL-17 and IL-23 were detected by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. Upon serum biochemical levels and pathological examination, the BMSCs-treated mice especially with multiple dosing administration showed significantly reduction of liver damage. Moreover, the expression of IL-17 was down-regulated by BMSCs intervention as compared to the model group, whereas the PD-L1 and IL-23 were up-regulated following the administration of MSCs. In conclusion, the results of this study suggest that BMSCs transplantation, especially on multiple dosing, may exert immunosuppression effect to ameliorate EAH through the inhibition of IL-17 and up-regulation of PD-L1. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver with unclear etiology. It is characterized serologically by elevated aminotransferase levels, hypergammaglobulinemia with high levels of IgG and production of characteristic autoantibodies [1,2], primarily antinuclear antibodies (ANA), antismooth muscle antigen (SMA), antiliver kidney microsomial antibody (LKM) and anti-soluble liver antigen/liver pancreas (SLA/LP). The histological hallmark of AIH is periportal or periseptal interface hepatitis infiltrated with immune cell consisting of lymphocytes, macrophages and plasma cells [3,4]. A working model for its pathogenesis postulates that environmental triggers,

∗ Corresponding author at: The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China. Tel.: +86 13505777281. E-mail address: [email protected] (Y.-P. Chen). 1 These authors equally contributed to this work. http://dx.doi.org/10.1016/j.imlet.2014.10.021 0165-2478/© 2014 Elsevier B.V. All rights reserved.

a failure of immune tolerance mechanisms, and a genetic predisposition collaborate to induce a T cell-mediated immune attack upon liver antigens, leading to a progressive necroinflammatory and fibrotic process in the liver. The therapy of corticosteroid or associated with azathioprine has been the time-honored treatment for autoimmune hepatitis. Nevertheless, not all patients respond to the conventional treatment and those who do respond may be accompanied by strong side effects related to the cure or relapse after drug withdrawal [5]. Thus, the emergence of new immunosuppressive agents or novel alternative treatment is urgent. Mesenchymal stem cells (MSCs) represent a heterogeneous progenitor cell population which possess multipotent differentiation and regenerative potential, immune modulation and migratory capacity. Other than their capacity to differentiate into various cell lineages such as adipocytes, osteoblasts or chondrocytes, MSCs display potent T-cell suppressive properties both in vitro and in vivo [6–8]. In various experimental models of Th17-derived autoimmune diseases, administration of MSCs has been shown to suppress inflammation and autoimmunity [9,10]. In vitro, Ghannam et al.

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

have demonstrated that MSCs inhibit Th17 cells differentiation and induce regulatory T cell characteristics in an inflammatory environment [11]. In addition, MSCs have recently been explored in trials of various autoimmune diseases, including inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA) [12,13]. However, there are few reports regarding the effects of MSCs on the autoimmune hepatitis. In this study, we discussed the effects of MSCs on autoimmune hepatitis in EAH mice which mimicked the human autoimmune hepatitis. The activation of naïve T cells not only need the first signal conducted by the T-cell antigen receptor but also the second signal of co-stimulatory molecules, without which will lead to immune tolerance or immune excessive activation [14]. There have been many reports indicating that the pathogenesis of autoimmune diseases is related to the co-stimulatory molecules and their ligands such as CD28/cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) and CD80/CD86 [15]. Programmed death-1 (PD-1), found in various tissues, acts as an inhibitory co-stimulatory molecule during immune responses [15]. PD-L1, the ligand of PD-1 is expressed on B cells, monocytes, dendritic cell (DCs), mesenchymal stem cells and cultured bone marrow-derived mast cells [14]. Engagement of PD-1 with PD-L1 during TCR signaling can block T-cell proliferation, cytokine production and impair T-cell survival [16,17]. Consistent with its negative regulatory function, PD-1 deficiency in mice results in the development of systemic and organ-specific autoimmune disorders [15]. Moreover, there is mounting evidence that PD-1 is linked to human autoimmunity, such as autoimmune liver disease [18], rheumatoid arthritis, Type I diabetes, multiple sclerosis [19]. A recent study has showed that knockdown of PD-L1 in MSCs abolish their immunosuppression function [20]. However, the expression of PD-L1 in the EAH mice with MSCs therapy has not been determined. Proinflammatory cytokine IL-17 was initially identified from a subset of CD4+ T cells termed as Th17 cells [21]. IL-17 and Th17 cells play a fundamental role in the pathogenesis and development of various autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease [22,23]. In Lafdil and Nagata’s study [24,25], IL-17-deficient mice was reported to develop reduced liver injury compared with wild type mice. IL-23 is a heterodimeric molecule produced by the DCs and macrophages principally, and the major functions of IL-23 consist of stimulation of T cell differentiation into Th17 cells and DC antigen presentation [26]. The studies that IL-23–deficient (IL-23p19) mice were resistant to collagen-induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE) [27,28] and IBD [29] highlighted a pivotal role of this cytokine in autoimmune pathogenesis. A link between PD-L1 costimulatory pathway and Th17 has been discovered in the fetomaternal tolerance that PD-L1 blockade inhibit antigen specific alloreactive T cell apoptosis and induce Tregs apoptosis and a switch toward higher frequency of Th17 cells [30]. Moreover, Luz-Crawford et al. have demonstrated that MSCs repress the function and proliferation of Th17 cell which is mediated by PD-L1 up-regulation on primed MSCs [31]. The aim of this study was to determine the influence of immune therapy of MSCs against autoimmune hepatitis in mouse model and to explore the possible underlying mechanisms. In particular, we focused on the alteration of PD-L1, IL-17 and IL-23.

2. Materials and methods 2.1. Mesenchymal stem cell prepared MSCs were obtained from Cyagen Biosciences Inc. (Cyagen Biosciences, Guangzhou, China) from the femurs and tibias of

223

C57BL/6 mice. The MSCs phenotypic properties were performed by flow cytometry analysis. Cyagen Biosciences Inc provided identification results: CD29 99.97%, CD44 99.32%, CD34 98.48%, Sca-1 96.85% and CD117 1.77%. Confirmation of differentiation of the cells to adipocytes, osteocytes and chondrocytes were performed using standard protocol. MSCs were cultured in the complete C57BL/6 mouse mesenchymal stem cell medium (Cyagen Biosciences, Guangzhou, China) for further study. At all from our study, we used the same BMSC cell line. A frozen vial of murine bone marrow MSCs was thawed and expanded according to the supplier’s instructions. MSCs used in all experiments were passages 6. 2.2. Murine hepatitis induced by hepatic S100 injection Sixty male SPF C57BL/6 mice, 10–12 weeks old, obtained from the Shanghai Laboratory Animal Center (Shanghai, China), were maintained under specific pathogen-free conditions and had free access to standard laboratory water and chow. All the animal experiments were approved by the institutional animal committee of Wenzhou Medical University. Among sixty mice, six were chosen randomly as control group and ten were used for offering the hepatic cytosolic S100 fraction as described [32]. Briefly, after the mouse was intraperitoneally injected with 0.004 ml/g 10% chloral hydrate solution, the liver was exposed and continually perfused with cold phosphate-buffered saline (PBS) through the portal vein with an angiocatheter. Then, livers were minced and homogenized with cold PBS on ice under ultrasonic dispersion for 30 min. The homogenate was centrifuged down at 150 g for 10 min to take out nuclei and the supernatants were further centrifuged by the Beckman ultracentrifuge for an hour at 100,000 × g. The remaining supernatants were used for immunization (called S100). Subsequently, the freshly prepared S100 was separated by running through a 90-cm CL-6b Sepharose column (Pharmacia, Freiburg) [33]. The column was extensively washed with physiological saline overnight and then the separation was also performed in physiological saline. Of the three resulting protein peaks, the first peak was only used to avoid toxicity. The eluate was reached to 0.5–2 g/l protein concentration using an Amicon Ultra-15 filter. The remaining 44 mice were injected intraperitoneally of this protein 0.5 ml emulsified in an equal volume of complete Freund’s adjuvant (CFA, sigma, USA) per animal on day 0 and a repeat injection on day 7. Eight mice died in the progress of modeling EAH. With six animals sacrifice, histology and blood biochemistry assay were performed to evaluate the disease severity on day 21. 2.3. Treatment with MSCs in experimental autoimmune hepatitis (EAH) The rest 30 mice were randomly divided into five groups: the model group (n = 6), the drug-treated group (n = 6), MSCs-treated one group (n = 6), MSCs-treated two group (n = 6) and MSCs-treated three group (n = 6). Also, there was the control group (n = 6) mentioned above. The mice in the control group were only injected intraperitoneally with 1.0 ml physiological saline on day 0 and day 7. After the first administration of S100, the animals in the drug-treated group were given prednisolone and azathioprine respectively in a dose of 5 mg per animal intraperitoneally on day 21, 28 and 35 [32]. Meanwhile, the mice in the model group were injected with 0.1 ml physiological saline through tail vein. The MSCs-treated one group was injected with 1 × 105 MSCs via the tail vein on day 21; the MSCs-treated two group was injected with 1 × 105 MSCs via the tail vein on day 21 and 28; and the MSCstreated three group was injected with 1 × 105 MSCs via the tail vein on day 21, 28 and 35. All the animals were sacrificed on day 42 and the liver tissues and blood were collected for further study.

224

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

2.4. Liver histological examination Harvested liver samples were fixed in 10% neutral formalin and embedded in paraffin. The tissue sections were then cut into 4-␮m sections, and histological alterations were observed using hematoxylin–eosin and Masson’s staining. Three sections per animal were blindly examined by an experienced liver pathologist. Fibrosis and necroinflammatory activity indices were evaluated according to the Ishak grading system [34]. 2.5. Examination of serum chemical levels The serum was isolated from blood samples of each group by 1300 rpm centrifugation for 10 min. According to the manufacturer’s protocol, the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were evaluated using an automatic biochemistry analyzer (Abbott Laboratories, USA). 2.6. Protein isolation and western blotting Total liver protein was isolated using 1 ml 1× radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail from Roche (Summerville, NJ, USA). Following heat denaturation at 95 ◦ C for 5 min, the samples (20 ␮g protein each) were subjected to 10% polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred onto a nitrocellulose membrane. The membrane was then blocked by skimmed milk for 1 h at room temperature. The primary antibodies against PD-L1 1:500 (MAB1019; R&D Systems), and GAPDH 1:1000 (SC-51907; Santa Cruz Biotechnology, Inc.) were used for incubation with the membrane overnight at 4 ◦ C, respectively. After being washed with TBS/T (1 × Tris Buffered Saline + 0.1% Tween-20) three times for 6 min each, the membrane was incubated respectively with the PD-L1 secondary antibody, goat anti-mouse IgG conjugated with HRP (1:2500; Cell Signaling Technology), and GAPDH secondary antibody, goat anti-rabbit IgG conjugated with HRP (1:8000; Cell Signaling Technology) at room temperature for 75 min. Again, the membrane was washed 3 times for 6 min each in TBS/T. Then the immunoreactive bands were visualized by chemiluminescent reagent (Millipore Corporation, Billerica, USA) and exposed to Kodak film. 2.7. Determination of cytokine IL-17 and IL-23 Concentrations of IL-17 and IL-23 in isolated liver protein in each group were determined by Enzyme-linked Immunosorbent Assay

(ELISA) kits (eBioscience, San Diego, CA, USA). Each experiment was carried out three times with three replicates, and the results were normalized relative to total protein. 2.8. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was prepared with EASYspin Plus RNA kit (Aidlab Biotechnologies Co., China), and cDNAs were reverse transcribed from 2.5 ␮g total RNA using a 2× Power Taq PCR MasterMix (BioTeke, China). The resulting cDNA was amplified with the matching primers: mouse ␤-actin: forward, 5 -AACAGTCCGCCTAGAAGCAC-3 , reverse, 5 -CGTTGACATCCGTAAAGACC-3 (product size: 281 bp); PD-L1: forward, 5 -CTCGCCTGCTGCAGATAGTTCCC-3 , reverse, 5 -TAAACGCCCGTAGCAAGTGA-3 (product size: 83 bp); forward, 5 -TACCTCAACCGTTCCACGTC-3 , reverse, IL-17:  5 -TTTCCCTCCGCATTGACACA-3 (product size: 119 bp); IL-23: forward, 5 -AAAGGATCCGCCAAGGTCTG-3 , reverse, 5 -GCAGGCTCCCTTTGAAGAT-3 (product size: 70 bp). PCR was performed for 40 cycles at 95 ◦ C for 30 s, 58.4 ◦ C for 30 s (IL-17), 61.8 ◦ C for 30 s (PD-L1 and IL-23), 61.0 ◦ C for 30 s (␤-actin) and then extension at 72 ◦ C for 5 s. 2.9. Statistical analysis SPSS19.0 software was used to analyze the statistical differences among multiple groups determined by one-way analysis of variance (ANOVA) or the least significant difference (LSD) test. All values are expressed as the means ± standard deviation (SD), and values of P < 0.05 were considered to be statistically significant. 3. Results 3.1. Liver antigen S100 induces autoimmune hepatitis in C57BL/6 mice A model of AIH was established by intraperitoneal injection of the S100/adjuvant. As shown in Fig. 1, after administration of S100/adjuvant, ALT and AST levels in the model group were increased, as compared to the control group (P < 0.05). However, MSCs was found to significantly reverse those changes induced by S100/adjuvant with distinct decreased serum markers levels. The same change was observed after the administration of prednisolone and azathioprine. Histological examination using HE and Masson’s staining were also adopted to show the degree of liver damage (shown in Fig. 2).

Fig. 1. Effect of MSCs on liver function and histological changes of EAH mice. The activities of alanine aminotransferase (ALT) (A) and aspartate aminotransferase (AST) (B) were assayed using an automated blood chemistry analyzer. (C) Three histological sections per animal were examined to evaluate the fibrosis and necroinflammatory activity indices according to the Ishak grading system (*, significant compared to control group, P < 0.05; #, significant compared to model group, P < 0.05; , significant compared between the subgroups of MSCs treated groups, P < 0.05).

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

225

Fig. 2. Representative photographs of liver sections using H&E staining (magnification: ×400, scale bar = 50 ␮m). (A) Liver sections from the control group; (B) the model group; (C) the drug-treated group; (D) the MSCs-treated one group; (E) the MSCs-treated two group; and (F) the MSCs-treated three group.

Mice received only physiological saline showed normal hepatic cells and central veins by H&E staining (Fig. 2A). In the animals treated with S100/adjuvant, the liver lesions were characterized by infiltrates of polymorphonuclear leukocytes with some lymphocytes localized mainly in the centrilobular or portal areas as well as intralobular inflammatory lesions and necrosis, which displayed the typical histologic features of autoimmune hepatitis (Fig. 2B) as previously reported [32,35]. However, after prednisolone and azathioprine treatment, hepatitis was attenuated based on preserved lobular structure without cell focal necrosis and with no lymphocytes infiltration (Fig. 2C). Likewise, MSCs significantly ameliorated tissue lesions and perivascular infiltrates. MSCs-treated three group showed better histological picture than MSCs-treated one and two groups (Fig. 2D–F). In addition, the tissue sections were assessed by Ishak grading system. A significantly higher histological hepatitis score (mean score = 6.33 ± 2.25 vs. 0.33 ± 0.51; P < 0.05; Fig. 1C) was found in the model group compared with control group. After treatment of drugs or MSCs, the score was declined predominantly. However, MSCs-treated two group, although lower than the model group, was not significantly different from MSCs-treated one group (mean score = 3.17 ± 1.70 vs. 3.50 ± 1.52; P > 0.05). The score of MSCs-treated three group was restored to almost the same level as control group. In the six groups, fibrosis, as determined by Masson’s staining for collagen deposition, was mild (figures not shown). These results suggested MSCs played a significant role in the relief of polymorphonuclear leukocytes infiltration and inflammation. 3.2. Function of MSCs on the expression of PD-L1 in autoimmune hepatitis mice To clarify the effect of MSCs on the expression of PD-L1, the change of the cytokine was examined using RT-PCR and western blotting. The mRNA level of the PD-L1 in the EAH model group was significantly higher than those in the control group (1.43 ± 0.27 vs. 0.80 ± 0.18, P < 0.05), which was reversed in the drug and MSCstreated one group. Moreover, the expression of PD-L1 in the MSCstreated three groups was increased significantly compared to the MSCs-treated one group (Fig. 4). Similarly, the increased protein expression of PD-L1 in the model group was reversed in the drug

and MSCs-treated one group. Following the administration of MSCs, PD-L1 was on a rise. It was statistically different between MSCstreated one group and MSCs-treated three group (2.09 ± 0.29 vs. 2.77 ± 0.23, P < 0.05) (Fig. 3).

3.3. Influence of MSCs on the expression of IL-17 in autoimmune hepatitis mice RT-PCR and ELISA were used to measure the expression of IL-17 in this experiment. The protein level of the IL-17 in the EAH model was elevated for more than 1-fold compared to the control group (198.16 ± 30.30 vs. 78 .45 ± 20.23, P < 0.05). However, treatment with MSCs significantly attenuated these changes. Furthermore, IL17 was gradually decreased following the increasing times of MSCs transplant, which was just the opposite of the variation tendency of PD-L1, indicating that MSCs may inhibit IL-17 production via promotion of PD-L1. In the MSCs-treated three group, IL-17 was returned to normal level, as in the control group and drug group. There was no statistically significant difference among these three groups (Fig. 5). The similar trend was observed in the gene change of IL-17 (Fig. 4).

3.4. Effect of MSCs on the expression of IL-23 in autoimmune hepatitis mice To further investigate the specific anti-inflammatory effect of MSCs, the expression pattern of interleukin-23 was evaluated by RT-PCR and ELISA. Not the same as PD-L1 and IL-17, IL-23 level was reduced in the model group. However, infusion of MSCs significantly inhibited the decline. In addition, the increasing times of MSCs injection lead to the gradually rise of IL-23 level with the similar trend of PD-L1. It was statistically different between the MSCs-treated subgroups (0.84 ± 0.12 vs. 1.07 ± 0.11 vs. 1.31 ± 0.19, P < 0.05). That variation tendency between the level of protein and mRNA was almost the same. Furthermore, the expression pattern of PD-L1 and IL-23 were both increased following the alleviation of histology image in the MSCs treated groups in autoimmune hepatitis (Figs. 4 and 5).

226

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

Fig. 3. Effect of MSCs on the expression of PD-L1 in EAH mice. The protein level of PD-L1 in the liver was determined by western blotting and was further normalized to the level of GAPDH. Data represent the mean ± SD of 6 mice, and the experiments were repeated three times (*, significant compared to control group, P < 0.05; #, significant compared to model group, P < 0.05; , significant compared between the subgroups of MSCs treated groups, P < 0.05).

Fig. 4. Effect of MSCs on gene levels of PD-L1, IL-17 and IL-23 in EAH mice. (A) Reverse transcription polymerase chain reaction was employed to investigate mRNA level of programmed death ligand 1 (PD-L1) (B), IL-17 (C) and IL-23 (D) in the liver. Data represent the mean ± SD of 6 mice, and the experiments were repeated three times (*, significant compared to control group, P < 0.05; #, significant compared to model group, P < 0.05; , significant compared between the subgroups of MSCs treated groups, P < 0.05).

Fig. 5. Effect of MSCs on protein expression of IL-17 and IL-23 in EAH mice. The concentrations of IL-17 and IL-23 in the liver were detected by ELISA assay. Data represent the mean ± SD and the experiments were repeated three times (*, significant compared to control group, P < 0.05; #, significant compared to model group, P < 0.05; , significant compared between the subgroups of MSCs treated groups, P < 0.05).

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

4. Discussion In this study, we established an experimental autoimmune hepatitis (EAH) model by intraperitoneal injection of S100, which was generally considered to mimic human AIH [33]. The mice exposed to S100 presented significant increase of serum ALT and AST, whereas this increase was attenuated by the transplantation of BMSCs, especially with multiple dosing administration. In addition to the decrease of serum makers, the histopathological alterations showed that BMSCs transplantation significantly alleviated the infiltration of lymphocytes as well as intralobar inflammatory lesions and necrosis. Besides, we could see fatty degeneration in part of the liver parenchyma and capsule of the mice injected with S100 in complete Freund’s adjuvant. In our speculation, it was probably related to the Freund’s adjuvant which was full of lipid. Taken together, we reported that experimental autoimmune hepatitis (EAH) could be alleviated by the administration of culture-expanded bone mesenchymal stromal cells (BMSCs) with a dose dependent way. Recent investigations have displayed that MSCs are recruited to sites of tissue damage and activated by local inflammatory cytokines which are generated by activated immune cells. The activation of MSCs induces the production of immunoregulatory factors and trophic factors. MSCs may either result in the repair of the destroyed tissue and attenuate the inflammatory response, or keep a consistent chronic inflammatory response, ending in fibrosis and deformation of tissue architecture [36]. In 2002, the finding that baboon MSCs could inhibit the mixed lymphocyte reaction in vitro, and block the rejection of allogeneic skin graft in vivo suggested that MSCs could regulate immune responses [7]. Later on numerous clinical and pre-clinical researches both in vivo and in vitro demonstrated that MSCs were immunosuppressive [13]. In the mouse model of GvHD, multiple administrations of MSCs were often used to maintain and extend their inhibitory effect, and also probably increased the exposure of MSCs to released inflammatory cytokines thus offering more beneficial effects [37]. Our study was in line with this result that therapeutic effect in the group given MSCs three times was better than the one given once. Furthermore, a recent study exhibited that the immunosuppressive drug mycophenolate mofetil (MMF) strengthened the effect of MSCs significantly in facilitating the survival of allogeneic heart grafts in a mouse model [38]. Nevertheless, we did not consider the combined use of MSCs with immunosuppressive drugs which would be worth studying. The BMSCs used in our study expressed CD34. Similarly, Peister et al. [39] discovered that mMSCs obtained from 4 inbred strains of mice (C57Bl/6, BALB/c, FVB/N, and DBA1) varied in their expression of CD34 as assayed by the mouse antibody that was not specific for murine hematopoietic cells. C57Bl/6 cells expressed high levels of CD34. Although the prevailing school of thought contents that mesenchymal stem cells do not express CD34, available evidence points to CD34 being expressed in tissue-resident MSCs, and its negative finding being a consequence of cell culturing [40]. In 1991, Simmons and Torok-Storb’ s study [41] performed a detailed analysis of uncultured BMSCs and provided convincing evidence that BMSCs were CD34+. After in vitro expansion, MSCs that originated from the CD34+ fraction became CD34− [42]. Thus, MSC’s negativity for CD34 is likely a cell culture-induced phenomenon, not indicative of their actual in vivo status. In addition, one of the most distinctive features of MSCs is their ability to adhere to plastic surface, as opposed to other bone marrow cells, such as hematopoietic stem cells (HSCs), which cannot. Also MSCs are defined by their ability to differentiate into mesenchymal tissues such as adipose, bone, and cartilage. In our research, we found that the mRNA and protein expression of PD-L1 in EAH mice livers were up-regulated compared with control group, which was in parallel with the discovery presented

227

in the study by Cao et al. and Kassel et al. [43,44], suggesting PD-L1 expression was enhanced in order to limit inflammation and tissue damage. The expression of IL-17 was also increased in the model group, indicating its pro-inflammatory role in EAH mice. These phenomena suggested that in EAH mice the pro-inflammatory and anti-inflammatory activities occurred in the meantime. After administration of MSCs once, the expression of PD-L1 was reduced compared to the model group; while it was increased following the increasing times of MSCs administration. In contrast with PD-L1, the rising level of IL-17 in the model group was reversed in the MSCs treated groups following the increasing times of MSCs administration. These data showed that MSCs might suppress IL-17 secretion and up-regulated PD-L1 production. Moreover, pro-inflammatory and anti-inflammatory activities in the liver achieved a better balance after MSCs transplant. In accordance with our conclusion, Luz-Crawford et al. [31] supported a MSC-mediated repression on mature Th17 cells and a major role of PD-L1 expressed highly by primed MSC in their suppressive activity on mature Th17 program. Several studies also showed that MSCs inhibited Th17 cells differentiation in an inflammatory environment [11,45] and MSCs suppressed T cell proliferation via up-regulation of PD-L1 [20,46]. Furthermore, neutralization of PD-L1 induced a skew toward higher frequency of Th17 cells in breaking fetomaternal tolerance and completely reversed the effect of IL-27-primed naïve CD4+ T cells to inhibit Th17 cell differentiation in non-primed CD4+ T cell, suggesting a link between PD-L1 costimulatory pathway and Th17 [30,47]. In the light of these discoveries, we speculate that MSCs inhibiting the immune response in our experiment is involved with the fact that MSCs may inhibit the pro-inflammatory cytokine IL-17 via inducing the inhibitory factor PD-L1. The negative co-stimulatory binding of PD-1 by PD-L1 can also block the phosphorylation of Akt by interfering on CD28-mediated PI3K activation, decreasing the ability of T cells to proliferation and survival, metabolize glucose and cytokine synthesis [48]. Current viewpoints consider IL-23 drives the development and expansion of activated CD4 + T cells which generate an array of pro-inflammatory cytokines including IL-17, IL-17F, TNF, and IL-6 upon antigen-specific stimulation [49,50]. IL-23-expanded T cells were able to induce EAE and CIA in an animal model [27,28]. IL23 appears to be essential for the expansion and stabilization of proinflammatory Th17 responses. It was noteworthy that our data showing declined expression of IL-23 in the mouse EAH model while elevated expression in the MSCs treatment groups was paradoxically at odds with some of the literatures because in their work the effect of IL-23 appeared to be proinflammatory. Nevertheless, there existed some researches in keeping with our study. IL-23p19-deficient mice had increased susceptibility to Con Ainduced hepatitis [51]. In addition, Olewicz-Gawlik et al. observed decreased levels of IL-23 and IL-17 in patients with systemic sclerosis (SSc) compared to healthy subjects [52]. In another study, compared with EAE control group, the level of IL-17 mRNA of EAE mice transplant with BMSCs was decreased significantly, while IL-23 mRNA was increased [53]. Therefore, further studies are necessary to elucidate the decided role of IL-23. In conclusion, our research demonstrated that the transplant of BMSCs was effective to EAH mice. The potential mechanism is inferred that the MSCs may repress the IL-17 production via the up-regulation of PD-L1. Though the exact mechanism of BMSCs therapy on EAH still remains unclear, the knowledge we gained will facilitate our investigations using human MSCs in the treatment in human AIH. Conflict of interest No other potential conflict of interest relevant to this article could be reported.

228

Y. Chen et al. / Immunology Letters 162 (2014) 222–228

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 Five-year science and technology major projects (2012ZX10002004-010). References [1] Zachou K, Rigopoulou E, Dalekos GN. Autoantibodies and autoantigens in autoimmune hepatitis: important tools in clinical practice and to study pathogenesis of the disease. J Autoimmun Dis 2004;1:2. [2] Vierling JM. Diagnosis and treatment of autoimmune hepatitis. Curr Gastroenterol Rep 2012;14:25–36. [3] Ichiki Y, Aoki CA, Bowlus CL, Shimoda S, Ishibashi H, Gershwin ME. T cell immunity in autoimmune hepatitis. Autoimmun Rev 2005;4:315–21. [4] Krawitt EL. Autoimmune hepatitis. N Engl J Med 2006;354:54–66. [5] Selvarajah V, Montano-Loza AJ, Czaja AJ. Systematic review: managing suboptimal treatment responses in autoimmune hepatitis with conventional and nonstandard drugs. Aliment Pharmacol Ther 2012;36:691–707. [6] Djouad F, Bouffi C, Ghannam S, Noel D, Jorgensen C. Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases. Nat Rev Rheumatol 2009;5:392–9. [7] Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42–8. [8] Djouad F, Plence P, Bony C, Tropel P, Apparailly F, Sany J, et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 2003;102:3837–44. [9] Wang J, Wang G, Sun B, Li H, Mu L, Wang Q, et al. Interleukin-27 suppresses experimental autoimmune encephalomyelitis during bone marrow stromal cell treatment. J Autoimmun 2008;30:222–9. [10] Bouffi C, Bony C, Courties G, Jorgensen C, Noel D. IL-6-dependent PGE2 secretion by mesenchymal stem cells inhibits local inflammation in experimental arthritis. PLoS ONE 2010;5:e14247. [11] Ghannam S, Pene J, Moquet-Torcy G, Jorgensen C, Yssel H. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol (Baltimore, MD: 1950) 2010;185:302–12. [12] Kim N, Cho SG. Clinical applications of mesenchymal stem cells. Korean J Intern Med 2013;28:387–402. [13] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726–36. [14] Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002;2:116–26. [15] Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 2002;20:29–53. [16] Riley JL. PD-1 signaling in primary T cells. Immunol Rev 2009;229:114–25. [17] Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000. [18] Oikawa T, Takahashi H, Ishikawa T, Hokari A, Otsuki N, Azuma M, et al. Intrahepatic expression of the co-stimulatory molecules programmed death-1, and its ligands in autoimmune liver disease. Pathol Int 2007;57:485–92. [19] Fife BT, Pauken KE. The role of the PD-1 pathway in autoimmunity and peripheral tolerance. Ann NY Acad Sci 2011;1217:45–59. [20] Sheng H, Wang Y, Jin Y, Zhang Q, Zhang Y, Wang L, et al. A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1. Cell Res 2008;18:846–57. [21] Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem 2003;278:1910–4. [22] Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 cells. Annu Rev Immunol 2009;27:485–517. [23] Haak S, Gyulveszi G, Becher B. Th17 cells in autoimmune disease: changing the verdict. Immunotherapy 2009;1:199–203. [24] Lafdil F, Wang H, Park O, Zhang W, Moritoki Y, Yin S, et al. Myeloid STAT3 inhibits T cell-mediated hepatitis by regulating T helper 1 cytokine and interleukin-17 production. Gastroenterology 2009;137:2125–35, e2121–2. [25] Nagata T, McKinley L, Peschon JJ, Alcorn JF, Aujla SJ, Kolls JK. Requirement of IL-17RA in Con A induced hepatitis and negative regulation of IL-17 production in mouse T cells. J Immunol 2008;181:7473–9. [26] Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 2000. [27] Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, et al. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 2003;198:1951–7.

[28] Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003;421:744–8. [29] Gheita TA, El Gazzar II, El-Fishawy HS, Aboul-Ezz MA, Kenawy SA. Involvement of IL-23 in enteropathic arthritis patients with inflammatory bowel disease: preliminary results. Clin Rheumatol 2014. [30] D’Addio F, Riella LV, Mfarrej BG, Chabtini L, Adams LT, Yeung M, et al. The link between the PDL1 costimulatory pathway and Th17 in fetomaternal tolerance. J Immunol (Baltimore, MD: 1950) 2011;187:4530–41. [31] Luz-Crawford P, Noel D, Fernandez X, Khoury M, Figueroa F, Carrion F, et al. Mesenchymal stem cells repress Th17 molecular program through the PD-1 pathway. PLOS ONE 2012;7:e45272. [32] Lohse AW, Dienes HP, Meyer zum Buschenfelde KH. Suppression of murine experimental autoimmune hepatitis by T-cell vaccination or immunosuppression. Hepatology 1998;27:1536–43. [33] Lohse AW, Manns M, Dienes HP, Meyer zum Buschenfelde KH, Cohen IR. Experimental autoimmune hepatitis: disease induction, time course and T-cell reactivity. Hepatology 1990;11:24–30. [34] Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22: 696–9. [35] Schramm C, Protschka M, Kohler HH, Podlech J, Reddehase MJ, Schirmacher P, et al. Impairment of TGF-beta signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice. Am J Physiol: Gastrointest Liver Physiol 2003;284:G525–35. [36] Shi Y, Su J, Roberts AI, Shou P, Rabson AB, Ren G. How mesenchymal stem cells interact with tissue immune responses. Trends Immunol 2012;33: 136–43. [37] Yanez R, Lamana ML, Garcia-Castro J, Colmenero I, Ramirez M, Bueren JA. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells 2006;24:2582–91. [38] Eggenhofer E, Renner P, Soeder Y, Popp FC, Hoogduijn MJ, Geissler EK, et al. Features of synergism between mesenchymal stem cells and immunosuppressive drugs in a murine heart transplantation model. Transpl Immunol 2011;25:141–7. [39] Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 2004;103:1662–8. [40] Lin CS, Ning H, Lin G, Lue TF. Is CD34 truly a negative marker for mesenchymal stromal cells. Cytotherapy 2012;14:1159–63. [41] Simmons PJ, Torok-Storb B. CD34 expression by stromal precursors in normal human adult bone marrow. Blood 1991;78:2848–53. [42] Kaiser S, Hackanson B, Follo M, Mehlhorn A, Geiger K, Ihorst G, et al. BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype. Cytotherapy 2007;9:439–50. [43] Cao J, Liu FX, Yu MX. Expression of programmed death 1 and its ligands in the liver of autoimmune hepatitis C57BL/6 mice. Chin Med J (Engl) 2009. [44] Kassel R, Cruise MW, Iezzoni JC, Taylor NA, Pruett TL, Hahn YS. Chronically inflamed livers up-regulate expression of inhibitory B7 family members. Hepatology 2009;50:1625–37. [45] Luz-Crawford P, Kurte M, Bravo-Alegria J, Contreras R, Nova-Lamperti E, Tejedor G, et al. Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells. Stem Cell Res Ther 2013;4:65. [46] Augello A, Tasso R, Negrini SM, Amateis A, Indiveri F, Cancedda R, et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur J Immunol 2005;35:1482–90. [47] Hirahara K, Ghoreschi K, Yang XP, Takahashi H, Laurence A, Vahedi G, et al. Interleukin-27 priming of T cells controls IL-17 production in trans via induction of the ligand PD-L1. Immunity 2012;36:1017–30. [48] Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 2010;236:219–42. [49] Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005;201:233–40. [50] Croxford AL, Mair F, Becher B. IL-23: one cytokine in control of autoimmunity. Eur J Immunol 2012;42:2263–73. [51] Xu M, Morishima N, Mizoguchi I, Chiba Y, Fujita K, Kuroda M, et al. Regulation of the development of acute hepatitis by IL-23 through IL-22 and IL-17 production. Eur J Immunol 2011;41:2828–39. [52] Olewicz-Gawlik A, Danczak-Pazdrowska A, Kuznar-Kaminska B, GornowiczPorowska J, Katulska K, Trzybulska D, et al. Interleukin-17 and interleukin-23: importance in the pathogenesis of lung impairment in patients with systemic sclerosis. Int J Rheum Dis 2014. [53] Liu P, Xuan X, Zhu J, Guan S, Du Y, Li Q. Therapeutic effect of allogenic bone marrow mesenchymal stem cell transplantation on EAE mouse. Xi bao yu fen zi mian yi xue za zhi 2013;29:798–801.