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Interleukin-17–Producing CD4؉T Cells Increase with Severity of Liver Damage in Patients with ChronicHepatitis BJi-Yuan Zhang,1*Zheng Zhang,1*Fang Lin,2Zheng-Sheng Zou,2Ruo-Nan Xu,1Lei Jin,1Jun-Liang Fu,1Feng Shi,1Ming Shi,1Hui-Fen Wang,2and Fu-Sheng Wang 1Interleukin-17(IL-17)-producing CD4؉T cells (Th17)-mediated immune response has been demonstrated to play a critical role in inflammation-associated disease;however,its role in chronic hepatitis B virus (HBV)infection remains unknown.Here we characterized peripheral and intrahepatic Th17cells and analyzed their association with liver injury in a cohort of HBV-infected patients including 66with chronic hepatitis B (CHB),23with HBV-associated acute-on-chronic liver failure (ACLF),and 30healthy subjects as controls.The frequency of circulating Th17cells increased with disease progression from CHB (mean,4.34%)to ACLF (mean,5.62%)patients versus healthy controls (mean,2.42%).Th17cells were also found to be largely accumulated in the livers of CHB patients.The increases in circulating and intrahepatic Th17cells positively correlated with plasma viral load,serum alanine aminotransferase levels,and histological activity index.In vitro ,IL-17can promote the activation of myeloid dendritic cells and monocytes and enhance the capacity to produce proinflammatory cytokines IL-1␤,IL-6,tumor necrosis factor (TNF)-␣,and IL-23in both CHB patients and healthy subjects.In addition,the concentration of serum Th17-associated cytokines was also increased in CHB and ACLF patients.Conclusion:Th17cells are highly enriched in both peripheral blood and liver of CHB patients,and exhibit a potential to exacerbate liver damage during chronic HBV infection.(H EPATOLOGY 2010;51:81-91.)More than 350million people worldwide suffer from persistent infection with hepatitis B virus (HBV)and are at risk for developing liver cir-rhosis and hepatocellular carcinoma.1HBV itself is non-cytopathic,but immune-mediated liver damage often occurs in patients with both acute and chronic HBV in-fection.Such damage has conventionally been attributed to killing of infected hepatocytes by virus-specific cyto-toxic CD8ϩT cells.2-4Increasing evidence,however,sug-gests that non-HBV-specific inflammatory infiltration into the liver is likely responsible for hepatic pathology in patients with chronic hepatitis B (CHB).5,6For example,in HBV infection activated HBV-specific CD8ϩT cells are often present at high levels in the livers of patients without evident liver inflammation;by contrast,nonan-tigen-specific lymphocytes were found to be massively infiltrated into the livers of patients with hepatic inflam-mation.7An HBV transgenic mouse model further rein-forced the concept that liver inflammation initiated by virus-specific CD8ϩT cells is amplified by other lympho-cytes.4,8Indeed,a large number of immune cells,includ-ing myeloid dendritic cells (mDCs),plasmacytoid dendritic cells,and FoxP3-positive regulatory T cells canAbbreviations:ACLF,acute on chronic liver failure;ALT,alanine aminotrans-ferase;CBA,cytometric bead array;CHB,chronic hepatitis B;HAI,histological activity index;HBcAg,hepatitis B core antigen;HBV,hepatitis B virus;HC,healthy control;IFN,interferon;IL,interleukin;mDC,myeloid dendritic cell;MFI,mean fluorescence intensity;PBMC,peripheral blood mononuclear cell;Th17,interleukin-17–producing CD4T cells;TNF-␣,tumor necrosis factor al-pha.From the 1Research Center for Biological Therapy,Beijing,China;2Department of Infectious Diseases,Beijing 302Hospital,Beijing,China.Received April 21,2009;accepted August 30,2009.Supported by grants from the National Grand Program on Key Infectious Disease (No.2009ZX10004-309,No.2008ZX10002-007,and No.2008ZX10002-005-6),the National Key Basic Research Program of China (No.2007CB512805),the Key Program of National Natural Science Foundation of China (No.30730088),and the National Laboratory of Biomacromolecules,In-stitute of Biophysics,China Academy of Sciences.*These authors contributed equally to this work.Address reprint requests to:Prof.Fu-Sheng Wang,Ph.D.,M.D.,Research Center for Biological Therapy,Beijing 302Hospital,100039,China.E-mail:fswang@;fax:86-010-********;and Prof.Hui-Fen Wang (wanghuifen@).Copyright ©2009by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience ().DOI 10.1002/hep.23273Potential conflict of interest:Nothing to report.Additional Supporting Information may be found in the online version of this article.81be observed in the livers of mildly and severely affected CHB patients.9-12Thesefindings,therefore,suggest that multiple types of immune cells may actively participate in HBV-associated liver pathogenesis.Understanding which types of immune cells contribute to liver damage during chronic HBV infection is a prerequisite for discov-ering effective treatment strategies.Human interleukin-17(IL-17)-producing CD4ϩT cells(Th17)comprise a newly identified proinflamma-tory T-cell subset.Several studies have demonstrated that several key cytokines,including IL-1␤,IL-6,tumor ne-crosis factor alpha(TNF-␣),and IL-23create a cytokine milieu that regulates the differentiation and expansion of human Th17cells.13Th17cells can also produce a cock-tail of cytokines such as IL-17A,IL-17F,IL-21,IL-22, IL-6,and TNF-␣,of which IL-17A is characterized as a major effector cytokine.IL-17A can mobilize,recruit,and activate neutrophils,leading to massive tissue inflamma-tion,and promote the progression of autoimmune dis-ease.14In alcoholic liver disease,activated liver-infiltrating Th17cells are also responsible for neutrophil recruitment into the liver.15Furthermore,serum IL-17levels are in-creased and serve as a marker of the severity of acute hepatic injury.16These studies all provide evidence link-ing Th17cells with immune-mediated liver injury.Th17cells also play a protective role in the host’s de-fense against some bacterial and fungal infections in mice.14The Th17response can be induced by virus anti-gens,17-23and the virus-induced Th17cells may regulate local antiviral immune responses by secreting inflamma-tory cytokines,which may in turn mediate the tissue dam-age in humans.22,24A recent study indicated that Th17 cells up-regulated antiapoptotic molecules and thus in-creased persistent infection by enhancing the survival of virus-infected cells,suggesting a novel pathogenic role of Th17cells during persistent viral infection.25These stud-ies suggest that Th17cells may contribute to the immu-nopathogenesis induced by persistent viral infection; however,the role of Th17cells in liver damage of CHB patients remains unknown.The present study characterized Th17cells in CHB patients and found that the peripheral and intrahepatic Th17population was selectively enriched and subse-quently exacerbated liver damage.Thesefindings may allow the development of rational immunotherapy for enhancing viral control,while limiting or blocking liver inflammation.Patients and MethodsPatients.Blood samples were collected from66CHB patients and23HBV-associated acute-on-chronic liver failure(ACLF)who were diagnosed according to the de-scribed criteria.10-12,15,26CHB patients were adults with no evidence of liver cirrhosis based on liver biopsy(nϭ21),unequivocal clinical and biochemical data(nϭ36), or compatiblefindings on imaging techniques(nϭ32). Individuals with concurrent HCV,hepatitis G virus,and human immunodeficiency virus(HIV)-1infections and autoimmune liver diseases and who met clinical or bio-logical criteria of bacterial or fungal infection were ex-cluded.Thirty age-and sex-matched healthy individuals were enrolled as controls.The study protocol was ap-proved by the ethics committee of our unit and written informed consent was obtained from each subject.The basic characteristics of these subjects are listed in Table1. Liver biopsies from47CHB patients undergoing di-agnosis and12healthy liver transplant donors were col-lected for immunohistochemical analysis.The degree of hepatic inflammation was graded using the modified his-tological activity index(HAI)described by Scheuer.27 Flow Cytometric Analysis.All antibodies were pur-chased from BD Biosciences(San Jose,CA)except for phycoerythrin(PE)-conjugated anti-IL-17A andfluores-Table1.Clinical Characteristics of the Populations Enrolled in the StudyGroup CHB ACLF Healthy Control Case662330Sex(male/female)46/2017/618/12 Age(years)31(16–48)32(21–46)29(16–45) ALT(U/L)258(44–1561)614(120–1,700)21(8–37) TBIL(␮mol/L)21.3(7.1–119.2)313(186–595)8(4–16) PTA90.9%(65.4%–129.2%)29.1%(20.3%–39.6%)NDHBV DNA(copies/mL)23,000,000(4540–74,0000,000)610,000(1,358–43,220,000)ND HBsAg positive66230 HBeAg positive66230 HBeAb positive000 HBcAb positive66230 HBcAb IgM positive000Data are shown as median and range.ND,not determined.82ZHANG,ZHANG,ET AL.HEPATOLOGY,January2010cein isothiocyanate(FITC)-conjugated anti-FoxP3fromeBioscience(San Diego,CA).For intracellular IL-17staining,fresh heparinized peripheral blood(200␮L)was incubated with phorbol12-myristate13-acetate(PMA,300ng/mL,Sigma,St.Louis,MO)and ionomycin(1␮g/mL,Sigma-Aldrich)in800␮L RPMI1640medium supplemented with10%fetal calf serum(FCS)for6hours.Monensin(0.4␮M,BD PharMingen)was added during thefirst hour of incubation.The blood was then lysed withfluorescence-activated cell sorting(FACS)lys-ing solution(BD PharMingen)and further permeabil-ized,stained with the corresponding intracellular antibody,fixed,and analyzed using FACSCalibur and FlowJo software(Tristar,San Carlos,CA)as previously described.28-30Cell Isolation.Peripheral blood mononuclear cells(PBMCs)were isolated and CD4ϩT cells,CD11cϩDCs,and monocytes were purified by positive or negative se-lection using microbeads according to the manufacturer’sinstructions(Miltenyi Biotech,Bergisch-Gladbach,Ger-many).The isolated CD4ϩT cells were further labeledwith PE-conjugated anti-CD45RO,allophycocyanin-conjugated CD45RA,or FITC-conjugated anti-CCR7antibodies.CD45RA high CCR7pos CD45RO neg(naive)and CD45RA neg CCR7pos/neg CD45RO pos(memory)cellswere sorted using FACSAria(Becton Dickinson,SanJose,CA).The purity of the mDCs,CD4ϩT-cell subsets,and monocytes were eachϾ95%.Unless otherwisestated,freshly isolated cells were incubated in completeRPMI1640medium containing10%FCS,2mM L-glutamine,20mM HEPES,100U/mL penicillin,100␮g/mL streptomycin,and5ϫ10Ϫ5M2-mercaptoetha-nol.Cell Stimulation.Isolated CD14ϩmonocytes andmDCs were incubated with medium in a96-well platewith or without IL-17(1ng/mL or3ng/mL;PeproTech,Rocky Hill,NJ)for24hours.Then the cells were har-vested for evaluating the expression of B7-H1,B7-DC,CD86,and CD83.The supernatants were collected todetect cytokine production.For measurement of antigen-specific cytokine production,PBMCs and CD4-deletedPBMCs were cultured with medium alone or with hepa-titis B core antigen(HBcAg;2␮g/mL)in96-well plates in duplicate for3days.Then the cells were collected for messenger RNA(mRNA)quantification and the super-natants were collected for IL-17A detection.RNA Extraction and Real-Time Reverse-Transcrip-tase Polymerase Chain Reaction(RT-PCR).TotalRNA was extracted from sorted CD4ϩT cells andHBcAg-stimulated cells using the RNeasy Mini Kit(Qia-gen,Santa Clarita,CA)according to the manufacturer’sinstructions.The RNA was reverse-transcribed to com-plementary DNA(cDNA)using oligo(dT)primers at 42°C for30minutes and at95°C for5minutes.Quanti-tative expressions of the ROR␥t and IL-17A transcripts were determined by staining with thefluorogenic dye SYBR Green using the reported primers and methods.15 GAPDH was used to normalize the samples in each PCR reaction.12The absence of nonspecific primer-dimer products was verified by melting-curve and gel-migration analyses.Results are expressed in terms of relative mRNA quantification calculated by using the arithmetic formula 2Ϫ⌬Ct.Multiplex Cytometric Bead Assay.A cytometric bead assay(Bender Medsystems,Copenhagen,Denmark) was employed to measure levels of IL-17,IL-23p19,IL-1␤,IL-6,IL-12p35,interferon(IFN)-␥,IL-22,IL-8,and GRO-␣of plasma and supernatants according to de-scribed protocols.30Immunohistochemical Staining.Paraffin-embedded, formalin-fixed liver tissue(5␮m)was incubated with an-ti-IL-17(AF-317-NA,R&D Systems,Minneapolis, MN)antibody overnight at4°C after blocking endoge-nous peroxidase activity with0.3%H2O2.3-Amino-9-ethyl-carbazole(red color)was used as the substrate followed by counterstaining with hematoxylin for single staining.Double staining was performed by using the avidin-biotin-peroxidase system with two different sub-strates:vector blue(blue color)for IL-17,and3-amino-9-ethyl-carbazole for CD4.Positively stained cells were counted at high-powerfield(hpf,ϫ400)according to described protocols.10-12Virological Assessment.The virological assay was performed according to our described protocols.10-12The limit of detection of the assay was500copies/mL. Statistical Analysis.All data were analyzed using SPSS software(Chicago,IL)and are summarized as means and standard parison between var-ious individuals was performed using the Mann-Whitney U parison between the same individual was per-formed using the Wilcoxon matched-pairs T test.Corre-lation analysis was evaluated by the Spearman rank correlation test.For all tests,two-sided PϽ0.05was considered statistically significant.ResultsIdentification of Th17Cells in CHB Patients.We first identified peripheral IL-17–producing cells in vitro by way of PMA/ionomycin stimulation.IL-17–produc-ing cells were mainly comprised of CD4ϩT cells;in con-trast,CD8ϩT cells,monocytes,natural killer(NK)cells, B cells,mDCs,and␥␦T cells expressed low levels of IL-17(Fig.1A).Phenotypic analysis indicated that IL-HEPATOLOGY,Vol.51,No.1,2010ZHANG,ZHANG,ET AL.8317ϩCD4ϩT cells expressed high levels of the memory marker CD45RO,but low levels of CD45RA,CD57(a senescence marker),and Ki67(a proliferation marker)(Fig.1B).The expression levels of these markers by Th17cells were seen at approximately the same levels in all enrolled subjects (data not shown).ROR ␥t is a unique marker that is restricted primarily to Th17cells.31We therefore measured ROR ␥t and IL-17mRNA expression in various subsets of memory CD4ϩT cells in CHB patients and found that ROR ␥t and IL-17mRNA expression levels were 8-fold higher in memory CD4ϩT cells than that in naive CD4ϩT cells (Fig.1C).These data further suggest that IL-17–produc-ing CD4ϩT cells can be considered Th17cells that dis-play memory properties.Th17Cells Are Enriched in the Peripheral Blood of CHB Patients.We then determined the frequencies of Th17cells,IFN-␥–producing CD4ϩT cells (Th1),IL-4–producing CD4ϩT cells (Th2),and FoxP3-positive CD4ϩT cells (Tregs)in peripheral blood from healthy controls (HCs),CHB,and ACLF patients.All subjects clearly displayed all four of the CD4ϩT-cell subsets (Fig.2A).Notably,the distribution of these subsets in HBV-infected subjects differed from that of HC subjects.We found that the percentage of Th17cells was significantly increased in CHB patients as compared to HC individu-als (P Ͻ0.01;Fig.2B).Particularly in ACLF patients,the Th17frequency was further increased over that in CHB patients (P Ͻ0.01).In contrast,there was no significant difference in the frequency of Th1or Treg between CHB patients and HC subjects,but there was a slight increasein the frequency of the Th2subset in CHB patients versus HCs (P Ͻ0.05;Supporting Fig.1A).In ACLF patients the Treg frequency was increased relative to that in CHB patients or HC subjects (both P Ͻ0.05),and no signifi-cant alteration was observed in the frequency of Th1or Th2cells between ACLF and CHB patients or HC sub-jects.In addition,we further investigated the activity of Th17cells through measurement of IL-17production from purified CD4ϩT cells in response to plate-coated anti-CD3and soluble anti-CD28.CD4ϩT cells from CHB patients produced more IL-17than those of HC subjects under anti-CD3and anti-CD28stimulation (Fig.2C).Thus,these data indicate that Th17cells were preferentially increased in the peripheral blood of CHB patients and simultaneously displayed increased activity.Interestingly,we found that a minority of Th17cells secreted IFN-␥or IL-4,or simultaneously expressed FoxP3,regardless of disease status (Supporting Fig.1B).The frequencies of these double-positive (IL-17ϩIL-4ϩ,IL-17ϩIFN-␥ϩ,or IL-17ϩFoxP3ϩ)CD4T subsets were also significantly increased in CHB and ACLF patients compared with HC subjects,whereas their frequencies were similar in CHB patients and ACLF patients.These data indicate that in HBV-infected patients some Th17cells may have properties of Th1,Th2,or Treg cells.We also detected the frequency of IL-22–producing Th17cells,which have been shown to protect against T-cell–induced hepatitis.32,33Surprisingly,only a few Th17cells have the capacity to produce IL-22in response to PMA/ionomycin stimulation in HC subjects,CHB,and ACLF patients (Supporting Fig.2A).There were nosignificantFig.1.Identification of Th17cells in CHB patients.(A)Screening IL-17–producing cells from peripheral blood in response to PMA/ionomycin stimulation in CHB patients (n ϭ3).The values in dotplots represent the percentage of IL-17ϩcells.(B)Flow cytometry analysis of phenotypes expressed by IL-17–producing CD4ϩT cells from CHB patients (n ϭ4).The values in the quadrants indicate the percentage of each subset among total CD4ϩT cells.(C)The relative mRNA expression of IL-17and ROR ␥t by isolated total CD4ϩT cells,CD45RA ϩCCR7ϩCD45RO Ϫnaive CD4ϩT cells and CD45RA ϪCCR7ϩ/ϪCD45RO ϩmemory CD4ϩT cells from CHB patients (n ϭ3).84ZHANG,ZHANG,ET AL.HEPATOLOGY,January 2010differences observed in the frequencies of IL-22–produc-ing CD4ϩT cells and IL-22–producing Th17cells among these three groups of subjects (Supporting Fig.2B,C).Antigen-specific Th17cells have been described in HCV infection,23but it is unknown whether HBV-spe-cific Th17cells will be present in patients with CHB.Our data indicated that PBMCs from patients with CHB ex-pressed high levels of ROR ␥t and IL-17mRNA in re-sponse to HBcAg (Fig.2D).Simultaneously,these PBMCs could also produce median amounts of IL-17A after HBcAg stimulation (Fig.2E).These capacities of PBMCs to express ROR ␥t and IL-17mRNA and pro-duce IL-17A in response to HBcAg were largely reduced after deletion of CD4ϩT cells from PBMCs in patients with CHB (Fig.2D,E).These data clearly indicated that in CHB patients there are some HBV-specific Th17cells displaying responsiveness to HBcAg.Increased Th17Population Is Positively Correlated with Liver Injury in CHB Subjects.We analyzed the correlation between Th17frequency and plasma HBV DNA load or serum alanine aminotransferase (ALT)lev-els in these CHB and ACLF patients.There were some significant positive correlations between Th17frequency and both plasma HBV DNA load (r ϭ0.212,P ϭ0.024;Fig.3A)and serum ALT levels (r ϭ0.390,P Ͻ0.001;Fig.3B)in these HBV-infected subjects.Further analysis indicated that these positive associations occurred only in patients with CHB (Fig.3A,B)but not in patients with ACLF.In addition,we also found that CHB patients with high HAI scores (G2-G3)(n ϭ12)had a greater propor-tion of Th17cells than did CHB patients with low HAI scores (G0-G1)(n ϭ9)(Fig.3C).These data suggest that peripheral Th17cell frequency is closely associated with liver injury,indicated by serum ALT levels and liver HAI scores in CHB patients.IL-17–Positive Cells Accumulate in the Livers of CHB Patients.We also examined the distribution of IL-17ϩcells in the livers of CHB patients.As shown in Fig.4A,tonsil tissue from a healthy individual,which served as a positive control,showed obvious IL-17stain-ing,whereas the liver tissue from a healthy donor had few IL-17ϩcells.Interestingly,more IL-17ϩcells were found accumulated in the lobular and portal areas of livers in CHB patients (Fig.4B).The liver-infiltrating IL-17ϩcells were differentially distributed in CHB patients with varying G scores:more IL-17ϩcells were found to be infiltrated in the livers of patients with a G4score than those of patients with G2and G1scores (Fig.4B).Using double immunostaining we confirmed thatintrahepaticFig.2.Th17cells are preferentially increased in peripheral blood of CHB patients.(A)Representative dotplots of IL-17and IFN-␥,IL-4or FoxP3coexpression in peripheral CD4ϩT cells of HC subjects,CHB,and ACLF patients.The values in the quadrants indicate the percentage of each CD4ϩT-cell subset.(B)Pooled data indicate the percentages of Th17cells in HC,CHB,and ACLF groups.(C)IL-17A production by purified CD4ϩT cells in response to anti-CD3and anti-CD28antibody stimulation in HC subjects (n ϭ10)and in CHB patients (n ϭ11).**P Ͻ0.01.Horizontal bars represent the median values of indicated index.(D,E)The relative mRNA expression of IL-17and ROR ␥t,and IL-17A production by PBMCs and CD4-deleted PBMCs stimulated with medium alone or with HBcAg in CHB patients (n ϭ16).**P Ͻ0.01.The bars represent means with standard deviations.HEPATOLOGY,Vol.51,No.1,2010ZHANG,ZHANG,ET AL.85Fig.3.Peripheral Th17frequency is significantly correlated with liver injury.Significant correlations were found between the Th17frequency and plasma HBV loads (A)and serum ALT levels (B)in CHB and ACLF patients.Solid line,linear growth trend;r ,correlation coefficient.P -values are shown.(C)CHB patients with higher HAI scores (G2-G3,n ϭ9)had a higher percentage of Th17cells in their peripheral blood compared with patients with lower HAI scores (G0-G1,n ϭ12).*P Ͻ0.05.Horizontal bars represent the median percentages of Th17frequency.Fig.4.In situ liver infiltration of IL-17–producing cells is correlated with liver injury in CHB patients.(A)Immunohistochemical staining for IL-17in tonsil (positive controls;400ϫ)and in situ liver of healthy controls (400ϫ).(B)Immunohisto-chemical staining for IL-17in lobular area and portal area in CHB patients with various degrees of liver injury (400ϫ).(C)Colocalization of CD4(red,on cell membrane)and IL-17(blue,in cell plasma)in liver of a representative CHB patient was shown with double labeling (400ϫ).(D,E)Numbers of IL-17–positive cells in liver portal (D)and lobular (E)areas are shown in HC subjects and CHB pa-tients with various degree of liver in-jury.Each dot represents one individual.*P Ͻ0.05and **P Ͻ0.01.Horizontal bars represent the median Th17numbers.86ZHANG,ZHANG,ET AL.HEPATOLOGY,January 2010IL-17ϩcells were primarily expressed on CD4ϩT cells (Fig.4C).Quantitative analysis of intrahepatic IL-17ϩcells documented that livers from CHB patients exhibited more IL-17ϩcell infiltration than did livers from HC subjects.In addition,in the lobular area of patients with a G4score the number of IL-17ϩcells per hpf was signifi-cantly more than in patients with G2-G3scores and in HC subjects (Fig.4D;all P Ͻ0.01).In the portal area the number of IL-17ϩcells per hpf was progressively in-creased in patients with various G phases (Fig.4E;all P Ͻ0.01for any two G phases).These data indicate that IL-17ϩcells were markedly accumulated in livers of CHB patients,and this infiltration was closely associated with inflammatory injury.IL-17Activates Monocytes and mDCs of CHB Pa-tients to Produce Proinflammatory Cytokines In Vitro .The immune consequence of the increase in peripheral and intrahepatic Th17cells remains unknown in CHB patients.Previous studies indicate that CHB patients gen-erally display dysfunctional innate immune responses,such as increased release of monocyte-derived proinflam-matory cytokines (IL-1␤,TNF-␣,and IL-6)and mDC-derived cytokines (IL-12and IL-23).6,8To address whether the increase of Th17cells is associated with these dysfunctional responses in CHB patients,we examined the expression of IL-17R (subunit A)in various cell pop-ulations.IL-17R was constitutively expressed by mono-cytes and mDCs in peripheral blood,but could not be observed in CD4ϩT cells,CD8ϩT cells,B cells,and NK cells (Fig.5A).Further analysis indicates that mean fluo-rescence intensity (MFI)of IL-17R on both mDCs and monocytes was slightly down-regulated in CHB patients compared with that in healthy subjects (Fig.5B).These data indicate that mDCs and monocytes are uniquely expressed IL-17R,but the overall expression levels seem to be decreased in CHB patients.Next we detected the responsiveness of mDCs and monocytes to IL-17in vitro .IL-17could significantly up-regulate B7-H1,B7-DC,CD86,and CD83expres-sion on monocytes and mDCs of CHB patients in vitro (Fig.6A).Increasing IL-17doses (up to 3ng/mL)signif-icantly enhanced the expression of these markers,indicat-ing that the effect of IL-17was dose-dependent.Surprisingly,we found that the MFI levels of these mark-ers were significantly decreased in CHB patients com-pared with HC subjects in response to IL-17stimulation in vitro (Fig.6B).These data indicated that IL-17can activate both mDCs and monocytes in vitro,and this promotion seemed poorer in CHB patients than HC sub-jects.IL-17can also significantly stimulate monocytes and mDCs to produce more inflammation-associated cyto-kines,including IL-1␤,TNF-␣,IL-6,IL-23p19,and IL-12p35in a dose-dependent manner;by contrast,unstimulated monocytes and mDCs produced lower lev-els of these cytokines (Fig.6C).Similar to maturation markers,IL-17has a relatively poor capacity to stimulate mDCs and monocytes to produce these cytokines in CHB patients than that of HC subjects.These data indi-cate that IL-17can activate monocytes and mDCs and induced them to produce proinflammatory cytokines,a process that is likely involved in the inflammation-medi-ated liver injury seen in CHB patients.Alteration of Serum Th17-Associated Cytokines in CHB Patients.We also detected the serum concentra-tions of Th17-associated cytokines such as IL-17,IL-23p19,IL-1␤,IL-6,IFN-␥,IL-12p35,IL-22,IL-8,and GRO-␣(Fig.7).It was found that the serum IL-17,IL-23p19,and IL-1␤concentrations were significantly higher in CHB compared to those of HC subjects,whereas other cytokine levels of CHB patients were sim-ilar to those of HC subjects.In ACLF patients nearly allofFig.5.IL-17R expression in monocytes and mDCs.(A)Screening IL-17receptor (IL-17R)-expressing cells from PBMCs in CHB patients (n ϭ6).The values in dot plots represent the mean percentage of IL-17R expression.(B)Pooled data indicate the MFI values of IL-17R on monocytes (left)and mDCs (right)from both CHB patients and HC subjects.The bars represent means with standard deviations.**P Ͻ0.01.HEPATOLOGY,Vol.51,No.1,2010ZHANG,ZHANG,ET AL.87Fig.6.IL-17in vitro induces the activation of monocytes and mDCs.(A)IL-17in vitro up-regulated the expression of the activation markers on monocytes and mDCs.Representative histograms indicate the expression of B7-H1,B7-DC,CD86,and CD83on isolated monocytes and mDCs from a CHB patient.The red histograms represent the staining of activation markers and the green histograms represent the isotype controls.The data represent the MFI of maturation markers.(B)Pooled data indicate the expression levels of B7-H1,B7-DC,CD86,and CD83on monocytes and mDCs from both HC subjects and CHB patients in response to IL-17in vitro .(C)IL-17in vitro induced monocytes and mDCs to produce proinflammatory cytokine including IL-6,IL-1␤,TNF-␣,IL-23p19,and IL-12p35.The bars represent means with standard deviations.*P Ͻ0.05and **P Ͻ0.01.88ZHANG,ZHANG,ET AL.HEPATOLOGY,January 2010these cytokines,except IL-22and GRO-␣,were increased in the serum compared with those of HC subjects;among these cytokines,IL-17,IL-6,IFN-␥,and IL-12p35con-centrations were even higher than that seen in CHB pa-tients.These results suggest that CHB patients had significantly altered Th17-associated cytokine profiles.DiscussionIncreasing evidence suggests that non-HBV-specific inflammatory infiltration into liver is likely responsible for the liver pathology during chronic HBV infection in humans.2-4However,little is known about how Th17cells operate in CHB patients.Here,we characterize Th17cells in CHB patients,and document a significant increase in peripheral and intrahepatic Th17cells.The increased Th17cells may further activate mDCs and monocytes to release inflammatory cytokines,a process likely to be involved in liver injury during chronic HBV infection.These properties of Th17cells may represent an unknown mechanism leading to the pathogenesis of HBV-induced liver disease.We first characterized Th17cells in a cohort of CHB patients and found that Th17cells were mainly enriched in CD4ϩT cells and displayed memory phenotypes.This finding was further supported by the observation of higher levels of IL-17and ROR ␥t mRNA occurring in memory CD4ϩT cells relative to naive CD4ϩT cells in these CHB patients.We also confirmed that both periph-eral and intrahepatic Th17cell number was relativelypreferentially increased in CHB patients compared with other CD4ϩT-cell subsets (including IFN-␥–producing Th1cells and FoxP3-positive Treg cells),suggesting Th17cells might actively participate in immune-patho-genesis of patients with CHB.Recent studies have demonstrated that IL-17from Th17cells may contribute to T-cell-mediated hepati-tis,34,35whereas another report indicated that IL-17did not lead to T-cell hepatitis.33The present study indicates that the preferential skew of the Th17subset is associated with liver injury in CHB patients.There are three aspects of evidence to support this notion.First,the peripheral Th17frequency in these patients with CHB was posi-tively correlated with serum ALT levels,which often serves as a marker of liver injury.1In addition,according to the liver biopsy diagnosis,patients with higher HAI scores have more Th17subsets not only in peripheral CD4ϩT cells but also in liver in situ than do patients with lower HAI scores.Third,ACLF patients also exhibited a considerably greater increase in peripheral Th17cells than did CHB patients.This cohort of ACLF patients often presented clinically exacerbated episodes following certain precipitating events and provide a compatible control for CHB patients with mild liver damage.26No-tably,Th17cells are significantly increased in patients with alcoholic liver disease without HBV or HCV infec-tions.15Our data also indicated that peripheral Th17fre-quency and serum concentrations of Th17-associated cytokines such as IL-17,IL-6,and IL-1␤were bothsig-Fig.7.The concentration of se-rum Th17-associated cytokines is in-creased in CHB patients.Cytometric bead assays were performed to quantify serum IL-17,IL-23p19,IL-1␤,IL-6,IFN-␥,IL-12p35,IL-22,IL-8,and GRO-␣production in HC subjects (n ϭ20)and CHB (n ϭ66)and ACLF patients (n ϭ13).The bars represent means and stan-dard deviations.*P Ͻ0.05and **P Ͻ0.01.HEPATOLOGY,Vol.51,No.1,2010ZHANG,ZHANG,ET AL.89。