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Research ArticleInterleukin 10 Gene-Modified Bone Marrow-Derived DendriticCells Attenuate Liver Fibrosis in Mice by Inducing RegulatoryT Cells and Inhibiting the TGF-/Smad Signaling PathwayYejin Xu,1Xinyue Tang,2Min Yang,3Shengguo Zhang,3Shanshan Li,3Yukai Chen,3Minhui Liu,1Yuxiang Guo,1and Mingqin Lu31Department of Infectious Diseases,Jinhua Municipal Central Hospital,Jinhua,Zhejiang,China2Department of Gastroenterology,the First Affiliated Hospital of Gannan Medical University,Ganzhou,Jiangxi,China3Department of Infectious Disease,The First Affiliated Hospital of Wenzhou Medical University,Wenzhou Key Laboratoryof Hepatology,Institute of Hepatology,Wenzhou Medical University,Wenzhou,325000 Zhejiang,ChinaCorrespondence should be addressed to Mingqin Lu;Received 10 June 2018;Revised 14 September 2018;Accepted 5 November 2018;Published 17 January 2019Academic Editor:Kerstin WolkCopyright 2019 Yejin Xu et al.This is an open access article distributed under the Creative Commons Attribution License,whichpermits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly cited.Aim.To explore the therapeutic effects and mechanisms of interleukin 10 gene-modified bonemarrow-derived dendriticcells(DC-IL10)onliverfibrosis.Methods.Invitro,BMDCsweretransfectedwithlentiviral-interleukin 10-GFP(LV-IL10-GFP)attheMOIof1:40.Then,the phenotype(MHCII,CD80,and CD86)and allo-stimulatory ability of DC-IL10 were identified by flow cytometry,and the levels of IL-10 and IL-12(p70)secreted into the culture supernatants were quantified by ELISA.In vivo,DC-IL10 wasinjected into mice with CCl4-induced liver fibrosis through the tail vein.Lymphocytes were isolated to investigate thedifferentiation of T cells,and serum and liver tissue were collected for biochemical,cytokine,histopathologic,immune-histochemical,and Western blot analyzes.Results.In vitro,the expressions of MHCII,CD80,and CD86 in DC-IL10 weresignificantly suppressed,allogeneic CD4+T cells incubated with DC-IL10 showed a lower proliferative response,and the levels ofIL-10 and IL-12(p70)secreted into the DC-IL10 culture supernatants were significantly increased and decreased,respectively.In vivo,regulatory T cells(Tregs)were significantly increased,while ALT,AST,and inflammatory cytokines were significantlyreduced in the DC-IL10 treatment group,and the degree of hepatic fibrosis was obviously reversed.The TGF-/smad pathwaywas inhibited following DC-IL10 treatment compared to the liver fibrosis group.Conclusion.IL-10 genetic modification ofBMDCs may maintain DC in the state of tolerance and allow DC to induce T cell hyporesponsiveness or tolerance.DC-IL10suppressed liver fibrosis by inducing Treg production and inhibiting the TGF-/smad signaling pathway.1.IntroductionLiver fibrosis is the ultimate pathological consequence ofchronic hepatic diseases and is featured by the formationand deposition of the extracellular matrix(ECM)1.Sustained inflammatory responses lead to liver fibrosis andfinally contribute to cirrhosis,portal hypertension,andhepatocellular carcinoma(HCC).Until now,how toeffectively prevent hepatic fibrosis and the end-stage liverdiseaseisstillanimportantmedicalandbiologicalproblem to be solved due to the lack of effective drugsand transplant donors for the liver.Th17 cells participated in the pathophysiology process ofinflammatory disease,and the proliferation of Th17 cellsleads to the progression of liver fibrosis 2,while regulatoryT cells(Tregs)were mainly responsible for the maintenanceof immune tolerance and homeostasis by releasing anti-inflammatory cytokines 3.The imbalance of Th17 cellsand Tregs is of vital importance in hepatic diseases,such asautoimmune hepatitis and liver fibrosis 2,4.To correctHindawiMediators of InflammationVolume 2019,Article ID 4652596,15 pageshttps:/doi.org/10.1155/2019/4652596the imbalance of Th17/Treg cells may be a key target for thetreatment of liver fibrosis.Dendritic cells(DCs)are professional antigen-presentingcells(APC)whichactasanimportantroleintheactivationofprimary immune responses,while DCs also silenced T cellimmune response and induced Treg development 5.Thefunctional activities of DCs are mainly depending on theirstate of activation and maturation;controlling the state ofDC maturation is a highly promising therapeutic interven-tion of diverse diseases.DCs activated by antigen expresscostimulatory molecules and generate different types ofcytokines which determine whether T cells differentiate intoTh1,Th2,or Tregs 6.Genetically modified DCs exhibitedtolerance which render them immunosuppressive 7 andmaybeapromising methodtoreverse liver fibrosis.Intensiveefforts should be taken to design gene therapy strategies forthe treatment of liver fibrosis.IL-10 is acknowledged as an anti-inflammatory and anti-fibrotic mediator 8.In addition,IL-10 is also a criticalimmunoregulatory cytokine which could negatively influ-ence both T cells and DCs.It regulates the function of DCsby reducing the surface markers as well as the secretion ofinflammatory cytokines 9.Previous studies revealed thatIL-10-modified DCs display hyporesponsiveness to subse-quent lipopolysaccharide(LPS)stimulation,and their allo-geneic stimulating capacity is significantly decreased 10.The TGF-/smad signaling pathway obviously participatesin hepatic stellate cell(HSC)activation which plays animportant role in the pathogenesis of liver fibrosis 11.IL-10 is a pleiotropic cytokine characterized by a broadspectrum of anti-inflammatory activities.There seems tobe some sort of association between the IL-10 gene andTGF-/smad signaling pathway.Given the evidence above,we believed that IL-10 is a pro-tective factor in liver fibrosis.Increasing studies researchingfor the effect of IL-10 gene therapy have been carried out.Considering that IL-10 exerts its influence on the immunesystem largely by suppressing the function of APCs,wetransducedbonemarrow-derivedDCwithlentiviral-interleukin 10-green fluorescent protein(GFP)(LV-IL10-GFP)to explore the changes of DC-IL10 in phenotype andfunction,effects on the T lymphocyte differentiation,andmolecular mechanisms of treatment on liver fibrosis.2.Materials and Methods2.1.Preparation of Bone Marrow-Derived Dendritic Cells.BMDCs were flushed from the femurs and tibiae of 6-8-week-old BALB/c mice.The cells were resuspended at a den-sity of 1106cells/ml in RPMI 1640(Gibco,USA)and 10%fetal bovine serum(FBS,Gibco,USA)complete mediumwith IL-4(PeproTech,USA,10ng/ml)and granulocyte-macrophage colony-stimulating factor(GM-CSF,Pepro-Tech,USA,20ng/ml)on the first day.Every other day,themedium was removed,and a fresh medium containingGM-CSF and IL-4 was added.2.2.Lentiviral Transduction of BMDCs.After 6 days of cul-ture period,BMDC populations were transfected withcontrol lentiviral vector GFP(LV-mock-GFP)at multiplici-ties of infection(MOIs)of 1:20,1:40,and 1:60 for 72h todetermine the optimal conditions for gene transduction.Transduction efficiency was determined by fluorescencemicroscopy on day 9 of culture.After the optimum MOIwas confirmed,day 6 BMDCs were transfected with LV-IL10-GFP or LV-mock-GFP at a MOI of 1:40 for 72 hoursto acquire DC-IL10 and DC-mock cells,respectively.2.3.Identification of DC.For immune-phenotyping,5105cells(DC,DC-mock,and DC-IL10)were resuspended in0.2ml of phosphate-buffered saline(PBS,Thermo Scientific,USA)and incubated with phycoerythrin-(PE-)conjugatedmurine anti-CD80 and MHCII as well as PE-Cyanine7-conjugated murine anti-CD86,at 4C for 30min.All of theseantibodies were purchased from eBioscience.Staining withisotype control antibodies was performed in all experi-ments at the same concentrations as the specific primaryantibodies.The fluorescence intensities of the cells wereevaluated by flow cytometry,and data was analyzed withFlowJo 7.6.1 software.2.4.ELISA Analysis of the Secretion of IL-10 and IL-12(p70).To determine the production of IL-10 and IL-12(p70)secreted by DC,DC-mock,and DC-IL10 treated with orwithout 1g/ml LPS for 48h,the supernatants of the DC cul-tures were collected and the expressions of IL-10 and IL-12(p70)secreted into the culture supernatants were quantifiedby ELISA using reagents purchased from eBioscience accord-ing to the kit manufacturers instructions.2.5.MixedLymphocyteAssay.Tlymphocytecellproliferationwas assessed by mixed lymphocyte reaction(MLR)cultures.Splenocytes were isolated from the cell suspensions by gradi-ent centrifugation at 400g for 25min at 4C with Lympho-prep(TBD Science,LTS1092PK).CD4+T cells(4106/ml)were labeled with 5mol/l Cell Proliferation eFlour 670(eBioscience,USA)and then added into a 24-well plate.Allogeneic DC,DC-mock,and DC-IL10 had been pre-treated with mitomycin C(Dakewe,Beijing,25g/ml)for30 minutes and then cocultured with CD4+T cells asresponders(3105/well)from C57BL/6 mice at ratios of1:5,1:10,and 1:30 for 5 days.T cell proliferation reac-tion was examined by flow cytometric measurement withanti-CD4 mAb(eBioscience,USA).2.6.Administration of DC-IL10 in a Mouse Model of CCL4-Induced Fibrosis.6-8-week-old 1822g weighing maleBALB/c mice and C57BL/6 mice were purchased from theShanghai Laboratory Animal Center(Shanghai,China).They were housed under normal laboratory conditions(212C,12h light-dark cycle).All experiments were con-ducted under the standard procedure set by the Committeefor the Purpose of Control and Supervision of Experimentson Animals and the National Institutes of Health for thespecification use of the experimental animals.The researchprotocol was approved by the Animal Ethics Committee ofWenzhou Medical University,China.Mice were randomlydivided into 4 groups as follows:group 1(G1,control group),2Mediators of Inflammationgroup 2(G2,model group),group 3(G3,DC-mock-treatedgroup),and group 4(G4,DC-IL10-treated group).Liver fibrosis(G2,G3,and G4)was induced in maleBALB/c by twice a week intraperitoneal injection of 0.2ml/100g of 40%carbon tetrachloride(CCl4)as a 2:3 mixturewith olive oil for 8 weeks.The control group was adminis-tered with the same volume of olive oil only,twice a weekfor 8 weeks.In the 6th week of CCl4 injection,the mice inthe G3 and G4 groups were treated with DC-mock or DC-IL10(1106/0.2ml)suspended in RPMI 1640 into the tailvein(twice a week for 2 weeks)till the 8th week,while thecontrol group and model group received equivalent vol-umes of RPMI 1640 tail vein injection instead of DC treat-ment.All mice were sacrificed 72h following the finalCCl4 or olive oil injection at the 8th week,under the con-dition of anesthesia with 10%chloral hydrate.Blood sam-ples were collected,and liver tissue samples were fixed in10%buffered formalin or immediately frozen and storedat 80C for analysis.Splenic tissues were separated forT lymphocyte flow cytometry analysis.2.7.The Differentiation of T Lymphocytes.To analyze theimmune-regulatory effect of DC-IL10 on Th17 and Treg cellsin CCl4-induced liver fibrosis,the percentages of Th17 andTreg cells in the mouse spleen were measured by flow cytom-etry.Splenocytes were isolated from the cell suspensions bygradient centrifugation at 450g for 25min at 4C withLymphoprep.Following surface staining with anti-CD4-fluorescein isothiocyanate(FITC,11-0041;eBioscience,SanDiego,CA,USA)and anti-CD25-peridinin-chlorophyll-pro-tein(APC,45-0251;eBioscience),the cells were fixed,perme-abilized,and stained with anti-FoxP3-phycoerythrin(PE,12-5773;eBioscience)to detect the percentage of Treg cells.ForTh17 cell detection,the lymphocytes from the spleen werestimulated for 5h with 50ng/ml phorbol 12-myristate 13-acetate(PMA),1g/ml ionomycin,3g/ml brefeldin A(BFA),and 1.4g/ml monensin(MULTI SCIENCES,China)in RPMI 1640 containing 10%FBS.The cells were harvestedand stained with anti-CD4-FITC then fixed and perme-abilized with Fix/PERM kit(eBioscience,USA)and stainedintracellularly with anti-IL-17-PE(12-7177;eBioscience).Cells stained with IgG isotype control were used as controls.All antibodies were purchased from eBioscience(USA).Datawere obtained using a FACSCalibur flow cytometer and ana-lyzed using FlowJo 7.6.1 software 12.2.8.Blood Biochemistry.Blood samples were collected fromthe retroorbital cavity(1ml),and serum was centrifugedbyspinning at3500r for10min,after which the samples werestored at 80C.The levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in the serumwere tested byan automatic biochemistryanalyzer(BeckmanKurt Co.Ltd.,USA).2.9.Measurement of Serum Cytokines.Serum cytokine levelswere determined by ELISA according to the manufacturersinstructions.ELISA kits for detecting TNF-and IL-6 wereobtained from eBioscience,USA.2.10.Histological Examination.For histopathological study,specimens of the liver tissues were fixed in 4%formalin,embedded in paraffin wax,and sectioned(5m).Massonstrichrome staining was used for collagen determination.2.11.Immuno-HistochemicalAnalysis.Forimmune-histochemical analysis,5m sections were deparaffnized inxylene and rehydrated in alcohol.A 500W microwave wasused to realize antigen retrieval,and then the sections wereheated in citric saline for 15min.Endogenous peroxidaseswere blocked by 3%hydrogen peroxide for 10min.In orderto block the nonspecific binding of antibodies,sections weretreated with 5%BSA and then incubated with polyclonalrabbit anti-SMA(ab5694;Abcam,Cambridge,MA,USA)at 4C overnight in the concentration of 1:100.Thesections were incubated with HRP-conjugated goat anti-rabbit secondary antibody(1:2000;ab7090;Abcam)for50min at 4C after washing in PBS.A DAB enhancer(ZSGB-BIO,ZLI-9018)was used to visualize the positivestaining.After that,sections were washed with water beforeredyeing with hematoxylin.2.12.Western Blot Analysis.Proteins were separated by 10%SDS-PAGE(Solarbio,S1052)and then transferred onto apolyvinylidene difluoride(PVDF)membrane.The mem-branes were incubated with relevant antibodies in WB pri-mary antibody diluent overnight at 4C,including TGF-1(ab179695),-SMA(ab5694),smad2(ab63576),and smad3(ab40854),which were purchased from Abcam(Cambridge,MA);anti-Smad7(sc-11392)which was purchased fromSanta Cruz Biotechnology Inc.(Santa Cruz,CA),and rabbitanti-GAPDH(AB-P-R-001)which was obtained fromGOOD HERE Technology.Then,membranes were incu-bated with horseradish peroxide-conjugated secondary anti-bodies for 1 hour at room temperature after washing.Proteins were visualized by an enhanced chemiluminescencesystem(Thermo Fisher Scientific Inc.,Waltham,MA,USA).Band intensities were analyzed by Quantity One 1-D Analy-sis software(Bio-Rad Laboratories Inc.,Hercules,CA,USA),and values were normalized against the value of GAPDH.3.Statistical AnalysisStatistical analysis was performed by SPSS 22.0.All data(from at least 3 separate experiments)are presented asmean standard deviation(SD).Statistical analysis wasperformed using Students t-test or one-way ANOVA.p 0 05 was considered significant.4.Results4.1.The Confirmation of Optimum MOI.BMDCs were trans-fected with LV-mock-GFP at different MOIs(1:20,1:40,or1:6