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1、Research ArticleDeregulation of Regulatory T Cells in Acute-on-ChronicLiver Failure:A Rat ModelShunlan Ni,1Shanshan Li,2Naibin Yang,2Xinyue Tang,2Shengguo Zhang,2Danping Hu,3and Mingqin Lu21Department of Infectious Diseases,Jinhua Municipal Central Hospital,Jinhua,Zhejiang,China2Department of Infect

2、ious Diseases,The First Affiliated Hospital of Wenzhou Medical University,Wenzhou Key Laboratory of Hepatology,Hepatology Institute of Wenzhou Medical University,Wenzhou,Zhejiang,China3Department of Infectious Diseases,Ruian City Peoples Hospital,The Third Affiliated Hospital of Wenzhou Medical Univ

3、ersity,Wenzhou,Zhejiang,ChinaCorrespondence should be addressed to Mingqin Lu;Received 14 April 2016;Revised 20 October 2016;Accepted 26 October 2016;Published 15 January 2017Academic Editor:Alex KleinjanCopyright 2017 Shunlan Ni et al.This is an open access article distributed under the Creative Co

4、mmons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original work is properly cited.Aims.Acute-on-chronic liver failure(ACLF)and acute liver failure(ALF)are similar in many respects during their acuteexacerbation;however,ACLFgenerallyhasa

5、poorerprognosis.WeaimedtoinvestigatetheroleanddynamicchangesofregulatoryT cell(Treg)and T helper 17(Th17)cell proportions during ACLF progress.Methods.All rats were classified into two groupsrandomly:ACLF group and ALF group(control group).The rat model of ACLF was preestablished by intraperitoneal

6、injection ofcarbon tetrachloride for 2 months.Then acute liver injury was induced by combined D-galactosamine and lipopolysaccharide.Six time points were examined before or after acute induction.Liver samples were performed with hematoxylin-eosin andMasson staining;circulatory Treg and Th17 cell fre

7、quencies were determined using flow cytometry assays;serum levels of alanineaminotransferase,aspartate aminotransferase,interleukin-10(IL-10),and interferon-(IFN-)were examined.Results.In groupACLF,both Th17 cell proportion and IFN-level presented upgrade firstly and then descend latter tendency;the

8、 trends of Treg cellproportion and IL-10 level were observed to gradually decrease and became stable.Conclusion.The Treg cells played an importantrole in the immunologic mechanism during the process of ACLF.And the function of Treg cells in ACLF was defective.1.IntroductionLiver failure is a serious

9、 condition that consists of jaundice,coagulopathy,encephalopathy,and metabolic derangement.Acuteliverfailure(ALF)developsintheabsenceofanypreex-isting liver injury,and acute-on-chronic liver failure(ACLF)develops with preexisting known or unknown chronic liverdisease 1,2.ALF and ACLF show similar sy

10、mptoms andsigns which are difficult to distinguish each other duringthe acute phases.However,the prognosis is always poorerand mortality is higher in ACLF than in ALF 3.Early-to-mid mortality of ACLF was found to be as high as 50%90%4,5.Recently,numerous clinical studies showed thatimmuneresponsepla

11、yedanimportantroleintheoccurrenceand development of ACLF 68.Wasmuth et al.demon-strated that ACLF shows a similar immune paralysis as insevere sepsis 9.However,the accurate pathogenesis andmechanisms remain to be elucidated.Astoimmunity,theregulatoryTcells(Tregcells)andIL-17-producing helper T cells

12、(Th17 cells)received increasingconcern in recent years 1012.Many studies demonstratedthat Treg and Th17 cells were tightly associated with manyhuman diseases,including the ACLF 13,14.The primaryfunction of Treg cells is immune suppression.Deficiency ordisruptionofTregcellswasreportedtoresultinautoim

13、muneand inflammatory diseases in both humans and animals15,16.Previous studies demonstrated that Treg cells canaccumulate and expand at infection site where they exertimmune suppression activity 17,18.Treg and Th17 cellsshare the same naive T cells and similar signaling pathwaysof cell differentiati

14、on.TGF-,IL-6,IL-21,IL-23,INF-,andothercytokinesplaypositiverolesinpromotingtheprocessofHindawi Publishing CorporationMediators of InflammationVolume 2017,Article ID 1390458,10 pageshttp:/dx.doi.org/10.1155/2017/13904582Mediators of Inflammationn=80n=60(9 died)ACLFLPS+D-GalN(29 died)LPS+D-GalN(19 die

15、d)ALFCCl4for 2 monthsn=6 6n=6 696h48h24h12h6h0h96h48h24h12h6h0hFigure1:TheprocessofmakingmodelsofACLFandALF.Allratswereclassifiedintotwogroupsrandomly:ALFgroupandACLFgroup.(1)ACLFgroup:theratsweretreatedwithCCl4dissolvedinpeanutoil(volume,1:1)1.5mL/Kgweightinthe1stmonthand2.0mL/Kgweightinthe2ndmonth

16、onceeverythreedays;sixratswereselectedasthebaselinecontrolandnamed“0h”;therestratsweretreatedwith500mg/Kgweight D-GalN and 80g/Kg weight LPS,and five time points were selected.(2)ALF group:no treatment was received for two months;subsequent treatments of ALF group were the same as ACLF group(ALF:acu

17、te hepatic failure;ACLF:acute-on-chronic liver failure;CCl4:carbon tetrachloride;D-GalN:D-galactosamine;LPS:lipopolysaccharide).Th17cellsdifferentiationandtheirfunction.Th17cellsprotectagainst extracellular pathogens and induce tissue inflamma-tion in host 19.In addition,IFN-is a proinflammatorycyto

18、kine associated with accelerating hepatic inflammationand aggravating liver parenchymal damage 20.The Treg and Th17 cells participate in the progression ofhepatic failure.Niu et al.found that both circulating Tregand Th17 cell frequencies increased in hepatitis B virus-(HBV-)ACLF patients compared t

19、o the healthy ones 7.However,others showed that the circulating Th17 cell pro-portionincreasedsignificantlyandTregcellchangedwithoutstatistical significance 8,21.An interesting study demon-strated that the peripheral Th17 cell proportion increased inboth acute hepatitis B virus(AHB)and HBV-ACLF pati

20、entscompared with healthy controls(HC),and the Treg cellproportion significantly increased in AHB while it decreasedwith no statistical significance in HBV-ACLF compared withHC 6.Combined,the clinical data of Treg and Th17 cells inACLF are not consistent,and related experiment researchesare absent.T

21、herefore,we established an ACLF rat model andexamined the frequencies of circulating Treg and Th17 cell atdifferent time points after acute induction to investigate thedynamic changes of the two immunoregulation cells duringthe process of ACLF.2.Materials and Methods2.1.Animals.Male Sprague-Dawley r

22、ats weighing 160180gwere purchased from Shanghai Laboratory Animal Center(Shanghai,China)and maintained in a 12h light/dark cycleroom and freely got food and water in animal centre ofWenzhou Medical University.All experimental procedureswere conducted under the guidelines of Ethics Committee ofAnima

23、l Care and Usage of Wenzhou Medical University.Thelicense of rats was SYXK(zhe)2014-0006.2.2.Experimental Protocols.The rats were classified into twogroups randomly:ACLF group(=80)and ALF group(control,=60).In ACLF group,chronic liver disease waspreestablished using CCl4(Bo Di,Tianjin,China)dissolve

24、din peanut oil(volume,1:1)by intraperitoneal injection onceevery three days for two months.The dosage regimen is1.5mL/Kg weight in the 1st month and 2.0mL/Kg weight inthe 2nd month.No treatment was received in ALF group.Aftertwomonths,nineratsweredeadinACLFgroupduringthe chronic process while all ra

25、ts were alive in control group.Then,six rats in each group were selected as the baselinecontrol and named“0h”subgroup.Next,the rest rats weretreated with 500mg/Kg weight D-GalN(Sigma-Aldrich,St.Louis,MO,USA)and 80g/Kg weight LPS(Sigma-Aldrich)by intraperitoneal injection to induce acute liver failur

26、e.To examine the dynamic changes of immunity,five timepoints after injection were selected:6h,12h,24h,48h,and96h.Ineachtimepoint,sixratsineachgroupwererandomlyselected from the remaining live rat,respectively(=6).Then the rats were sacrificed for subsequent experiments.Approximately 1.5mL blood samp

27、le was collected from cau-dal vein and liver tissues were isolated for later analysis.Theserumwascollectedfromthefreshbloodaftercentrifugationat 3000rev/min for 10min.Of note,there were 29 rats inACLF group and 19 rats in ALF group that died from liverfailure in the first 96 hours after acute induct

28、ion.The flowchart of operation procedures is showed in Figure 1.2.3.Histology.Liver tissues were fixed in 4%paraformalde-hyde(Solarbio,Beijing,China)for 48h,embedded withparaffin,and cut into 4m thick sections.Then,the slideswere treated with hematoxylin and eosin(H&E)or Massonstainingaccordingtothe

29、manufacturersprotocols(Solarbio).Pathological changes were observed under an optical micro-scope.Mediators of Inflammation3NormalHE50m(a)ALF(b)ACLF(c)Masson100m(d)(e)(f)Figure2:PathologicalfeaturesoflivertissueunderlightmicroscopebyHE(magnification400 x)andMassonstaining(magnification200 x).Liver se

30、ctions of normal,ALF,and ACLF group were stained with HE(a,b,and c)and Masson(d,e,and f),respectively.(a,d)Normal:therats were from 0h subgroup in ALF model;(b,e)“12h”subgroup in ALF group;(c,f)“12h”subgroup in ACLF group(HE:hematoxylin andeosin staining;ALF:acute hepatic failure;ACLF:acute-on-chron

31、ic liver failure).2.4.Determination of Laboratory Data and ELISA.Theserumlevelsofaspartateaminotransferase(AST)andalanineaminotransferase(ALT)were measured by Backman Kurtau5800 automatic biochemical analyzer(Beckman Kurt Co.Ltd.,USA).Quantitativeanalysisofinterleukin-10(IL-10)andinterferon-gamma(IF

32、N-)levels in serum was performedusing enzyme-linked immune sorbent assay(ELISA)kitsaccording to the manufacturers instructions(RB,USA).2.5.FlowCytometry.Theperipheralbloodmononuclearcells(PBMC)were isolated from whole blood using the lympho-cyte isolation Kit(Hao yang BM,Tianjin,China),and 2 106cell

33、s were aliquoted to each tube.For Th17 cells analysis,cells were incubated in RPMI 1640 medium containing 10%FBS(Gibco,USA),50ng/mL phorbol 12-myristate 13-acetate(PMA),1g/mL ionomycin,3g/mL brefeldin A(BFA),and 1.4g/mL monensin(Multi-Sciences,China)at 37Cwith 5%CO2for 6 hours.Cells were firstly sta

34、ined withanti-CD4-FITC.Next,the cells were permeabilized usingthe Fix/Perm solution(eBioscience,USA)and stained withanti-IL-17-PE after fixation.For analysis of Treg cells,cellswere firstly stained with anti-CD4-FITC and anti-CD25-APC synchronously and then stained with anti-Foxp3-PEafter fixation a

35、nd permeabilization as described above.Allthe antibodies used above were purchased from eBioscience(USA)and incubated for 30min at 4C in the dark.Finally,cellswereresuspendedin1%paraformaldehydeandanalyzedusing a FACSCalibur(BD,USA)within 1 hour.Appropriateisotype controls and single staining contro

36、ls were used.Thedata were analyzed using FlowJo7.6.1 software.2.6.Statistical Analysis.Statistical analyses were performedusing IBM SPSS Statistic v21.0(IBM Co.,Armonk,NY,USA).To show the relative changes of cell proportions andassociated factors after acute insult,the normalized valuerelative to“0h

37、”was approved and calculated as follows:normalized value(%)=(time point 0h)/0h.Data areshownasmeanstandarddeviationandcomparedusingtheindependent-test and analysis of variance(ANOVA).Valueof 0.05 was considered to be a significant difference.3.Results3.1.Liver Histopathology.To examine the validity

38、of ACLFmodels and observe the histopathological changes in acutephase after induction,liver tissues were examined withhematoxylin-eosin(HE)and Masson(M)staining,and theALF models were used as controls.We observed hard texture and nodular appearance of theACLFlivers.Theslicesshowedcellswelling,necros

39、is,inflam-matorycellsinfiltration,double-nucleuslivercellsincreasing,and multiple pseudolobule formation(Figures 2(c)and 2(f)show the tissues of“12h”subgroup in ACLF group).Thesurfaces of livers in normal rats(from 0h subgroup in ALFmodel)were smooth;hepatic cells were integral and hepaticlobulesstr

40、uctureswereclearinthespecimens;fibroplasiawas4Mediators of Inflammation0100200300400500ALFACLF(U/L)ALT(0h)AST(0h)(a)ACLF:ALTACLF:ASTALF:ALTALF:AST010002000300040005000(U/L)96h48h24h12h6h0h(b)ACLF:ALTALF:ALTRelative changes of ALT10010203040(folds)96h48h24h12h6h(c)ACLF:ASTALF:ASTRelative changes of A

41、ST0102030(folds)96h48h24h12h6h(d)Figure 3:Serum ALT and AST levels.(a)The levels of ALT and AST at 0-hour point in ACLF group and ALF group;results were shown asmeans SD,0.001.(b)The dynamic changes of ALT and AST in ACLF group and ALF group.(c,d)The relative changes of ALT andAST levels(ALT:alanine

42、 aminotransferase;AST:aspartate aminotransferase).not observed under light microscope(Figures 2(a)and 2(d).The liver tissue sections in group ALF showed necrosis andinflammatory cells infiltrating in portal duct areas withoutfibroplasia(Figures 2(b)and 2(e)showed the tissues of“12h”subgroup in ALF g

43、roup).3.2.Laboratory Data.As serum biochemical changes reflectliver function,levels of ALT and AST were determined.Theresults are shown in Figure 3.ALT and AST levels signifi-cantlyincreasedinACLFgroupafterCCl4administrationfor2 months compared with the control group at 0-hour timepoint(220.847.0 ve

44、rsus 66.212.8,0.001;333.355.2versus 146.3 38.7,0.05).Figure 6(b)shows the relative changes of Treg/Th17 ratio.3.4.Immunologic Cytokines.IFN-and IL-10 played a role indifferentiation of Th17 and Treg cells.Here,serum levels ofIFN-and IL-10 were measured by ELISA kit and data weresummarizedinFigure7.O

45、urdatashowedthelevelofplasmaIFN-increased while plasma IL-10 level had no significantdecreaseinACLFgroupcomparedtocontrolgroupat0-hourpoint(96.876.84versus31.293.28,0.001;32.845.48versus 35.33 5.75,=0.459).6Mediators of Inflammation01234Th17 cell proportion(%)ACLFALF96h48h24h12h6h0h(a)0246810Treg ce

46、ll proportion(%)ACLFALF96h48h24h12h0h6h(b)Relative changes of Th175051015cells(folds)ACLFALF96h48h24h12h6h(c)Relative changes of Treg1.00.50.00.51.01.52.0cells(folds)ACLFALF96h48h24h12h6h(d)Figure 5:The changes of Th17 and Treg cell proportions.(a,b)Th17 and Treg cell proportions;results were shown

47、as means SD.(c,d)Relative changes of Th17 and Treg cells.05101520Treg/Th17ACLFALF96h48h24h12h0h6h(a)Relative changes of Treg/Th17ACLFALF96h48h24h12h6h1.00.50.00.5(folds)(b)Figure 6:Change of Treg/Th17 ratio.(a)Treg/Th17 ratio in ACLF group and ALF group.(b)Relative changes of the ratios;0.05.Mediato

48、rs of Inflammation7ACLFALF96h48h24h12h0h6h0100200300400IFN-(pg/ml)(a)01020304050IL-10(pg/ml)ACLFALF96h48h24h12h0h6h(b)ACLFALF96h48h24h12h6hRelative changes of IFN-051015(folds)(c)Relative changes of IL-100.60.40.20.00.2(folds)ACLFALF96h48h24h12h6h(d)Figure 7:Serum levels of IFN-and IL-10.(a,b)Change

49、s of IFN-and IL-10 level in serum in ACLF group and ALF group.(b)IFN-andIL-10 levels were normalized by“0h”subgroup,respectively;0.01.(IFN-:interferon-;IL-10:interleukin-10).Our results also showed plasma IFN-level significantlyincreased first(0.05)in ACLF group.Meanwhile,plasma IFN-level in control

50、group had a similar trend with ACLF group,but it decreasedsignificantly(0.05)with the peak advanced to 12-hourpoint(Figure 7(a).There was an unconspicuous peak at 12-hour point and a subsequently significant decrease of plasmaIL-10 level in control group,while plasma IL-10 level slowlydecreased duri