International Immunopharmacology 42 (2017) 67–73

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International Immunopharmacology

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Compact bone-derived mesenchymal stem cells attenuate nonalcoholicsteatohepatitis in a mouse model by modulation of CD4cells differentiation

Huafeng Wang a,⁎,1, Dong Wang b,1, Luhong Yang a,1, Yanxia Wang a, Junli Jia a, Dongchen Na a, Huize Chen a,Yongping Luo a, Chengfang Liu c,d,⁎⁎a Modern College of Arts and Science, or School of Life Science, Shanxi Normal University, Linfen, Chinab Central Blood Station of Tianjin, Tianjin, Chinac Department of Human anatomy, Shanxi Medical University, Taiyuan, Chinad Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

⁎ Corresponding author.⁎⁎ Correspondence to: C. Liu, Department of HumUniversity, Taiyuan, China.

E-mail addresses: shilvshe@163.com (H. Wang), ljjlcf@1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.intimp.2016.11.0121567-5769/© 2016 The Authors. Published by Elsevier

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 August 2016Received in revised form 3 November 2016Accepted 14 November 2016Available online 24 November 2016

Increasing evidence has accrued which indicates that mesenchymal stem cells (MSCs) have a potential clinicalvalue in the treatment of certain diseases. Globally, nonalcoholic steatohepatitis (NASH) is awidespreaddisorder.In the present study, MSCs were isolated successfully from compact bone and a mouse model of NASH wasestablished as achieved with use of a methionine-choline deficient (MCD) diet. Compact bone-derived MSCstransplantation reduced MCD diet-induced weight loss, hepatic lipid peroxidation, steatosis, ballooning, lobularinflammation and fibrogenesis. It was shown that MSCs treatment hampered MCD diet-induced proliferation ofCD4+ IFN-γ+ and CD4+IL-6+ T spleen cells. In addition, CD4+IL-17+ lymphocytes that associated with anti-in-flammation show little change inMCD aswell as inMCD+MSCs splenocytes.We conclude that MSCsmay havea potential clinical value upon NASH, through their capacity to suppress activation of CD4+ IFN-γ+ and CD4+IL-6+ lymphocytes.

© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Keywords:Compact boneMSCsMCD dietNASHCD4+ lymphocytes

1. Introduction

Nonalcoholic steatohepatitis (NASH), progressive form of non-alco-holic fatty liver disease (NAFLD), is a significant predisposing factor forthe development of cryptogenic cirrhosis and hepatic failure [1,2]. Theprevalence of NAFLD correlates with the rising incidence of obesityandmetabolic syndromes in thewesternworld [2]. In the United States,the National Health and Nutrition Examination Surveys from 2009 to2010 reported obesity rates of 35.5% among men and 35.8% amongwomen [3]. In Asia, prevalence of NAFLD has been found to be in therange of 15–30% in the general population and exceeds 50% in patientswith diabetes andmetabolic syndromes [4]. In the general population ofthe United States, the prevalence of NASH is approximately 3% but in-creases to N25% in obese individuals [5]. Since the mid-1990s, ultra-sound surveys for assessing fatty liver (any cause) were firstpublished in mainland China [6,7]. From these surveys, the median

an Anatomy, Shanxi Medical

126.com (C. Liu).

B.V. This is an open access article u

prevalence of ultrasonographic steatosis in the Chinese populationswas found to be 10%, but ranged from 1% to N30% [6,8].

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stro-mal stem cells that can be isolated from many adult tissues [9]. Withinthe past few years,many studies have focused on the therapeutic effectsof different types of stem cells, which can improve liver function via dis-tinct mechanisms [10]. Bone marrow-derived MSCs transplantationwas shown to be effective in rescuing experimental liver failure inducedby carbon tetrachloride gavage [11]. It was also reported that MSCs canindirectly protect against hepatocyte death and increase survival in ful-minant hepatic failure. Such effects resulted from the secretion of largefractions of chemotactic, cytokines or chemokines by MSCs [12]. Othershave reported the hepatic engraftment of transplanted MSCs as ob-served in sublethal animal models of liver injury, further signifying theclinical potential for MSCs in the treatment of liver diseases [13–15].However, the exact effects of MSCs upon NAFLD remain poorlyunderstood.

For a long time we have been interested in liver diseases. We havedemonstrated that adiponectin-derived active peptide ADP355protected the mice from hepatic inflammation and fibrosis [16]. Wehave also been interested in the isolation and characterization of mes-enchymal stem cells (MSCs) and in the potential application of thesecells to liver diseases especially [17,18]. We have used compact bone-

nder the CC BY license (http://creativecommons.org/licenses/by/4.0/).

68 H. Wang et al. / International Immunopharmacology 42 (2017) 67–73

derivedmethod to isolate and culturemouseMSCs (mMSCs) [19]. MSCsand their secreted molecules exhibited therapeutic potency in themodels of carbon tetrachloride-induced liver fibrosis andthioacetamide-induced fulminant hepatic failure (FHF) by function asimmuno-suppressor [19].

In the present study,we examined the effects ofMSCswithin NAFLD.To accomplish this goal, a mouse model of NASH was induced by treat-ment with a methionine-choline deficient (MCD) diet, whose histolog-ical changes occur rapidly and are morphologically similar to thoseobserved in human NASH [1]. MSCs were isolated from compact boneaccording to procedures described previously [20]. We found thatNASH induced by a MCD diet was attenuated when treated withMSCs, as indicated by reductions in weight loss, hepatic steatosis, hepa-tocyte ballooning, liver inflammation and fibrosis. The probable mecha-nism of this MSC-mediated immunomodulation likely results from aninhibition in the activation of CD4+ IFN-γ+ and CD4+IL-6+

lymphocytes.

2. Materials and methods

2.1. Animals and diet (Fig. 1)

Six- to eight-week-old male C57BL/6 mice were purchased from theAcademy of Military Medical Science (Beijing, China) and were main-tained on a lipogenic diet deficient in methionine and choline (MCD)as obtained from TROPHIC Animal Feed High-tech Co., Ltd. (Nantong,Jiangsu, China). The diet has made into solid by manufacturer and ad-ministrated randomly. At first, the mice were randomly divided into 2groups: (i) Normal - controls fed with a regular diet, n = 6; (ii) MCD -mice fed with the MCD diet, n = 12. Six weeks later, MCD - mice wererandomized into 2 groups again: (ii) MCD - mice as before, n = 6; (iii)MCD+MSC -mice fedwith the MCD diet supplemented with an intra-venously injection of 1 × 106MSCs administered at 6 and 7weeks of the10-week experiment, n= 6. At 10weeks, all micewere euthanized andtissue was harvested.

This experiment was approved by the Animal Ethics Committee ofTianjin Medical University.

2.2. Isolation and culture of bone-derived MSCs

The way to isolate and culture mMSCs from murine compact bonewas modified from the previously described one [19]. In brief, we firstpulled down the skin and cut many of the muscles that attached tothe hindlimbs and humeri by surgical scissors, and then disconnectedthem from the trunk and at axillaries. Use two forceps to remove rem-nants of the muscles and tendons attached to the bone. After bone

Fig. 1. Diagram of the experimental protocol. Themicewere randomly divided into 3 groups: (i(iii) MCD+MSC -mice fedwith theMCD diet supplementedwith an intravenously injection omice were euthanized and tissue was harvested.

marrow beingflushed and discardedwith PBS by a syringe, the compactboneswere excised into chips of about 1mm,washedwith PBS until thereleased cells were removed. Two weeks of being incubated in α-MEMcontaining 10% FBS later, mMSCs sprouted from chips and permeated inthe dishes. Harvest the cells by a cell scraper. The primary obtained cellswere then passaged by 0.25% trypsin-EDTA (GIBCO) digestion. After 5–8passages, cells were used for the following experiments.

2.3. Examination of immunophenotypic features of MSCs

MSCs prepared as described above were stained with the followingfluorescein-linked antibodies: CD29, CD44, CD11b, CD45 or CD135 (allfrom eBioscience, CA, USA). Cells were analyzed using a FACScalibur(BD Biosciences, USA). Flow cytometry data were analyzed using FlowJo 7.6 software (BD Biosciences, USA).

2.4. Histology of livers

Livers were perfused with PBS, removed, weighed and sliced intosmall sections (0.5 × 0.5 cm). Liver sections were embedded in paraffinafter being fixed in 4% (weight/volume) paraformaldehyde and werecut into 6-μm-thick sections. Paraffin sections were stained with hema-toxylin–eosin (HE) for assessing inflammation and steatosis andPicrosirius red for assessing fibrosis.

Evaluation of the extent of resultant NASH [2] was performed usingthe following scaling scores:

Steatosis. Hepatocytes containing fat vacuoles were subjectively vi-sualized and graded according to the following scale: 0 - normal,none of hepatocytes were affected, 1 - tiny, b5% of hepatocyteswere affected, 2 - mild, 5%–33% of hepatocytes were affected, 3 -moderate, 34% - 66% of hepatocytes were affected and 4 - severe,N66% of hepatocytes were affected.

Ballooning. 0 - none, 1 - rare or few, 2 - moderate and 3 - many.Lobular inflammation. 0 - None, 1 - 1-2 foci/20 × field, 2 - 2-4 foci/20 × field and 3 - N4 foci/20 × field.Stages of NASH. 0 - none, 1 - extensive zone 3 perisinusoidal fibrosis,2 - zone 3 perisinusoidal andportal or periportalfibrosis, 3 - bridgingfibrosis and 4 - cirrhosis.

2.5. Hepatic lipid peroxidation

Malondialdehyde (MDA) is one of major aldehydic metabolites oflipid peroxidation and has been used to reflect lipid peroxidation [21].Lipid Peroxidation MDA Assay Kit (Beyotime, China) was used to mea-sure MDA in the liver.

) Normal - controls fedwith a regular diet, (ii) MCD -mice fedwith theMCD diet only andf 1 × 106MSCs administered at 6 and 7 weeks of the 10-week experiment. At 10weeks, all

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2.6. Analysis of spleen leukocytes

Single-cell suspensions derived from splenic tissues (splenocytes)were prepared by mechanical disruption, filtered through a 40-μm cellstrainer (BD, USA), followed by lysis of red blood cells. For intracellularcytokine staining, splenocytes were resuspended in complete RPMI1640 medium (Gibco, Auckland, NZ) supplemented with 1 mmol/lMEM sodium pyruvate, 0.1 mmol/l MEM nonessential amino acids,2 mM L-glutamine, 100 IU penicillin, 100 mg/ml streptomycin and4.5 × 10−5 M 2-mercaptoethanol. The cells were stimulated with50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin(Enzo life sciences, Farmingdale, USA) and 3 μg/ml brefeldin A(eBioscience, CA, USA) for 5 h. Next, cells were stained for surfacemarkers using rat anti-mouse CD4-APC (eBioscience, CA, USA). For in-tracellular staining, the cells were fixed and permeabilized withCytofix/Cytoperm buffers (BD Biosciences, USA) for 20 min at 4 °C andthen washed with permeabilization wash buffer (BD Biosciences,USA). The Fc receptors were blocked with 2% rat serum and 10% bovineserum albumin prior to intracellular cytokine staining followed bystaining with rat anti-mouse IFN-γ-FITC, IL-17A-PE or IL-6-FITC(eBioscience, CA, USA). The cells were then analyzed using aFACSCalibur and the data generated were analyzed using FlowJo 7.6.1software (Tree Star, Inc., USA).

Fig. 2.Morphology and immunophenotypic features of MSCs isolated frommouse compact bonof compact bone chips. (B) Immunophenotypic features of MSCs. As shown in the upper panelwhile negative surface markers of MSCs, such as CD11b, CD45 and CD135 were not expressed

2.7. Statistical analysis

Data were expressed as means ± SDs (standard deviations). GraphPad PRISM (version 5.0) software were used for statistical analyses.

3. Results

3.1. Cells isolated from compact bone are characterized as MSCs

As described above, MSCs were obtained from 2 to 3-week-old fe-male C57BL/6 mice euthanized by cervical dislocation. Hematopoieticcells were depleted and released cells discarded by flushing with PBSor α-MEM [20]. After 1–2 weeks in culture, cells were observed togrow out from proximity of compact bone chips and appeared to be fi-broblast-like (MSCs), but not polygon shaped (osteocytes) (Fig. 2A). Tofurther identify adherent cells, immunophenotypic features were ana-lyzed. As shown in the upper panel of Fig. 2B, the cells expressed surfacemarkers characteristic of MSCs, such as CD29 and CD44; while negativesurface markers of MSCs, such as CD11b, CD45 and CD135 were notexpressed (Fig. 2B, lower panel). Taken together, these findings indicatethat these cells were MSCs.

e. (A) Photograph ofMSCsmorphology. Fibroblast-likeMSCs grew out from the proximityof B, the cells expressed surface markers characteristic of MSCs, such as CD29 and CD44;(B, lower panel). Representative images are shown for all panels.

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3.2. MCD diet-induced weight losses and oxidative stress were relieved byMSCs treatment

MCD diet-induced NASH has been reported to be accompanied byweight loss [1]. Here, as compared with Normals, we also observedweight loss in MCDmice when recorded at weeks 6 and 7 of the exper-iment, prior to treatment with MSCs. Following MSC treatment, theMCD+MSCmice began to showweight gains (Fig. 3A). As the aldehy-dic metabolites of lipid peroxidation, MDA is often used to reflect lipidperoxidation [21]. It was demonstrated that MCD diet induced-hepaticMDA accumulation was hampered with MSCs treatment (Fig. 3B).

3.3. MCD diet-induced steatosis, ballooning and lobular inflammation wereattenuated by MSCs treatment

Hepatocytes containing fat vacuoles can be easily visualized as clearbubbles following HE staining [2]. When compared with Normals, MCDdiet induced lipid deposition and inflammation within the liver, effectsthat were reduced by MSCs treatment (Fig. 4A). As indicated by scoresobtained from the scales described above, increases in steatosis (Fig.4B), ballooning (Fig. 4C) and lobular inflammation (Fig. 4D) as inducedby MCD were all decreased in response to MSC treatment.

3.4. MSCs treatment ameliorated MCD diet-induced liver fibrosis

Progression of NASH is often accompanied with fibrosis [2]. There-fore, we also assessed the stages of fibrosis in these cells as achievedwith picrosirius red staining. MCD fed mice showed a progression intoextensive fibrogenesis within their livers whereas MSCs treatment re-duced hepatic fibrogenesis as observed in MCD + MSC mice (Fig. 5A).Differences in fibrosis stage scores and fibrosis area between MCD andMCD+ MSCs mice were significantly different (Fig. 5B, C).

3.5. MSCs treatment reduced percent of CD4+ IFN-γ+ and CD4+IL-6+ Tspleen cells

MSCs can interact with both innate and adaptive immune systemcells, leading to the modulation of several different effector functions[9]. Analysis of spleen leukocytes was performed to explore the effectof MSCs on immunological responses. We found that as comparedwith Normals, percent of CD4+ IFN-γ+ and CD4+IL-6+ lymphocytesthat were associated with pro-inflammation were decreased withMSCs (Fig. 6 A, B). However, percent of CD4+IL-17+ lymphocytes that

Fig. 3. MCD diet-induced weight loss and hepatic lipid oxidation stress was hampered by MSrepeated measures ANOVA. (B) MSCs treatment with an intravenously injection of 1 × 106 Mexamined by repeated measures ANOVA. (C) Hepatic lipid oxidation stress was determined by

associated with anti-inflammation show little change in MCD as wellas in MCD+ MSCs splenocytes (Fig. 6 C).

4. Discussion

MSCs, also known as multipotent mesenchymal stromal cells, wereoriginally isolated by Friedenstein and his colleagues frombonemarrowin 1976 [22], and have subsequently been found to exist in other organsand tissues [20]. However, the method established by Friedenstein asbased on plastic adherence, has proved unsuccessful for mouse MSCs(mMSCs) due to the low frequency of MSCs and the contamination ofhematopoietic cells in the culture [23]. A recent adaption of this proto-col, which has offered an easy and reproducible method to harvestmouse MSCs, has been provided by Heng Zhu [20]. In the presentstudy, we utilized this method and successfully isolated MSCs thatwere fibroblast-like and expressed putative surface markers of MSCssuch as CD29 and CD44, but not CD11b, CD45 and CD135, which wasconsistent with a previous report [20].

Increasing evidence has been accruedwhich indicate thatMSCshavea potential clinical value in the treatment of certain diseases [9]. Inter-estingly, there is limited data available that show any promising or de-sirable therapeutic effects of MSCs in ameliorating fulminant hepaticfailure, chronic liver fibrosis and cirrhosis. Findings from a previous re-port have demonstrated that fulminant hepatic failure can be inducedin Sprague-Dawley rats with an intraperitoneally administration oftwo injections of 1.2 g/kg hepatotoxin, D-galactosamine. When theserats were treated 24 h later with an intravenous injection of MSCs ly-sates, cellular lysates showed an increased survival trend [12]. In thecarbon tetrachloride-induced hepatic fibrosis model, Flk1 mouse MSCswere systemically infused immediately or at 1 week after mice werechallengedwith carbon tetrachloride. In thesemice,mMSCs administra-tion immediately and significantly reduced carbon tetrachloride-in-duced liver damage and collagen deposition [14]. In a previous study,we indicated that compact bone-derived mMSCs ameliorated grossand microscopic liver histopathology and reduced death of mice inthioacetamide-induced FHF and also suppressed carbon tetrachloride-induced chronic liver fibrosis [19]. In addition, we also demonstratedthat MSC-CM tendentiously improves chronic liver fibrosis [19].

Recently, it was reported by Boeykens that MSC-therapy could havea limited effects on NASH, which was observed that MCD diet modelboth serum CHE and TG levels of non-treated and cell-treated ratswith PHx after MCD remained significantly lower as compared tothose of control rats [24]. Despite this, Boeykens also found that en-hanced the recovery to normal body weight, liver weight and NAS

Cs treatment. (A) Effects of MCD diet on body weight growth. P value was examined bySCs administered at 6 and 7 weeks reversed MCD diet-induced weight loss. P value wasLipid Peroxidation MDA Assay Kit. *P b 0.05 with t-TEST.

Fig. 4. MCD diet-induced steatosis, ballooning, and lobular inflammation were ameliorated by MSCs treatment. (A) HE staining of mice liver sections. Fat vacuoles (black arrows) andinflammation foci (green arrows) were reduced by MSCs treatment in MCD + MSCs mice. Scale scores of steatosis (B), ballooning (C) and lobular inflammation (D) were allsignificantly decreased in MCD+MSCs versus MCD mice. Representative images are shown for all panels. The data are presented as the means ± SDs. *P b 0.05; **P b 0.01 with t-TEST.

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score [24]. In the present study, we demonstrated that MSCs treatmentsignificantly reduced MCD diet-induced weight loss, steatosis, balloon-ing and lobular inflammation, which consistent with the report de-scribed above. Altogether, it could be yet proposed that MSCs maypossess a potential clinical value upon NASH.

A primary basis for disease protection afforded by MSCs likely in-volves their potent anti-inflammatory and immunomodulatory activi-ties [25]. Mice injected with MSCs (5 × 106 MSCs/kg body weight)showed that host T-cell responses to donor alloantigenswere decreasedin the majority of recipients [26]. There are data which indicate thatMSCs did not preferentially target any T-cell subset, and their capacityfor inhibition extended to B cells, with the result being that MSC-medi-ated inhibition induces an unresponsive T-cell profile. Such findings areconsistent with that observed in division arrest energy [27]. In our pre-vious study, we also demonstrated the immunosuppressive effects ofMSC treatment, converting the body into an anti-inflammatory stateby up-regulating anti-inflammatory Treg cells and reducing pro-inflam-matory Th1 and Th7 cells in thioacetamide-induced FHF and carbon tet-rachloride -induced chronic liver fibrosis [19]. In the present study,among potential spleen leukocytes, we selected CD4+T cells to explorethe immunomodulatory effects of MSCs on NASH. We found that MSCstreatment suppressed multiplication of CD4+ IFN-γ+ and CD4+IL-6+ T

Fig. 5. Improvement of MCD diet-induced liver fibrogenesis byMSCs. (A) Picrosirius red staininRepresentative images are shown for all panels. The data are presented as the means ± SDs. *P

spleen cells, which was consistent with previous reports [25]. The mo-lecular mechanism may be due to the molecules secreted by MSCs[19]. Accordingly, we conclude that MSCs-mediated immunosuppres-sion accounts for the ameliorative effects upon NASH.

In summary,MSCs isolated from compact bone have potential clinicalvalue uponNASH(summarized in Fig. 7).MSCs transplantation enhancesthe resistance toMCDdiet-inducedNASH, as indicated by several param-eters including reductions in MCD diet-induced weight loss, steatosis,ballooning and lobular inflammation. As based upon these findings, theameliorative effects upon NASH by MSCs likely results from suppressionof activation of CD4+ IFN-γ+andCD4+IL-6+ lymphocytes. However, theeffectiveness andmechanics of treatment in regards to gender and age ofanimals are unclear and remain to explore in the future.

Author contributions

H.W. and D.W. conceived and designed the experiments. D.W., J.J.,H.C., C.L. and Y. L. performed the experiments. D.N., C.L., Y. W. and L.Y.collected and analyses data. H.W., and L.Y. wrote the manuscript. H.W.and C.L. contributed to the guidance of experiments and contributedto the final manuscript. All authors reviewed the manuscript.

g of mice liver sections. (B) Scale scores of liver fibrosis. (C) Quantification of fibrosis area.b 0.05 with t-TEST.

Fig. 6. Effects of MSCs on CD4+ T cells differentiation. For intracellular cytokine staining, splenocytes were treated with 50 ng/ml PMA, 1 μg/ml ionomycin and 3 μg/ml brefeldin A for 5 h.The cells were stained for surfacemarkers using rat anti-mouse CD4. For intracellular staining, the cells werefixed and permeabilizedwith Cytofix/Cytoperm buffers for 20min at 4 °C andthen washed with permeabilization wash buffer. The Fc receptors were blocked with 2% rat serum and 10% bovine serum albumin prior to intracellular cytokine staining. Next, the cellswere stained with rat anti-mouse IFN-γ, IL-6 and IL-17A. (A) CD4+ IFN-γ+, (B) CD4+ IL-6+ and (C) CD4+IL-17A+ lymphocytes was analyzed by flow cytometry. Representative data areshown for all panels.

Fig. 7. Compact bone-derivedMSCs have potential clinical value uponNASH.MSCs transplantation enhances the resistance toMCDdiet-induced NASH, as indicated by several parametersincluding reductions in MCD diet-induced weight loss, steatosis, ballooning and lobular inflammation. As based upon these findings, the ameliorative effects upon NASH by MSCs likelyresults from suppression of activation of CD4+ IFN-γ+ and CD4+IL-6+ lymphocytes.

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Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Acknowledgements

This workwas supported by One College One Policy Project, ModernCollege of Arts and Science, Shanxi Normal University (2016KJYJ-6);Scientific Research Foundation for Doctor, Shanxi Normal University(0505/02070293); Shanxi Provincial University Science and TechnologyInnovation Project (20161107); The Natural Science Foundation ofShanxi Province (2015021177).

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