Role and recruitment of Th9 cells in liver cirrhosis patients

Shan-Yu Qin,Jia-Xu Wang,Mei Chen,Xian-Wen Yang,Hai-Xing JiangDepartmentof Gastroenterology,the First Af fi liated Hospital of Guangxi Medical University,Nanning 530021,Guangxi,PR China



Role and recruitment of Th9 cells in liver cirrhosis patients

Shan-Yu Qin,Jia-Xu Wang,Mei Chen,Xian-Wen Yang,Hai-Xing Jiang*
Departmentof Gastroenterology,the First Af fi liated Hospital of Guangxi Medical University,Nanning 530021,Guangxi,PR China

Original article http://dx.doi.org/10.1016/j.apjtb.2015.11.010

ARTICLE INFO

Article history:

Received in revised form 23Oct2015 Accepted 1Nov 2015

Availableonline 14 Jan 2016

Th9 cells

Cirrhosis

Interleukins-9

CCR6/CCL20 axis

ABSTRACT

Ob jective:To investigate the role of T helper 9(Th9)cells in liver cirrhosis(LC)patientsandwhetherchemokine receptor type6(CCR6)/chemokine ligand 20(CCL20)axis is involving in the recruitment of Th9 cells into liver.

M ethods:Peripheral blood and liver tissue from 30 LC patients and 18 normal controls were recruited.The frequency of Th9 cellsand CCR4,CCR6 in the peripheralblood was tested by fl ow cytometry.Serum interleukin(IL)-9 and CCL20 levels were tested by enzyme-linked immunosorbentassay.Immunohistochem ical staining was used to detect α-smoothmuscle actin,CCR6 and CCL20 expression in liver tissue.

Resu lts:The frequency of Th9 cells in LC patientswassigni fi cantly increased compared w ith controls(P＜0.05).The serum IL-9 level and CCL20 level increased markedly in LC patients comparedw ith controls(P＜0.05),and IL-9 waspositively correlated to Th9 cells and CCL20.Furthermore,the frequency of Th9 cellswas correlated to prothrombin time,total bilirubin level,hyaluronic acid and type IV collagen in LC patients.We also found that Th9 cells in LC patients expressed higher frequency of CCR4+,CCR6+(P＜0.05).Compared w ith normal controls,the expression of CCR6 and CCL20 in LC tissue were signi fi cantly elevated(P＜0.05).The expression ofα-smoothmuscle actin was correlated to the CCR6 and CCL20 in liver tissue of LC patients.

Conclusions:This study suggests that Th9 cellsmay participate in the pathogenesis of LC,and the recruitment of Th9 cells into liver tissuem ight be through CCL20/CCR6 axis.

1.Introduction

Livercirrhosis(LC)is theend stageof chronic liver injury,and most commonly caused by viral infections,alcoholism and fatty liver disease[1,2].Like the spleen,the liver isoneof the important immune organs,to whichmany immune cells are recruited under both physiologicaland pathological conditions[3].Accumulating data have indicated the key role of in fi ltrating CD4+T cells inthe progression of liver in fl ammation and fi brosis[4,5].Currently, CD4+T helper(Th)cells can be subdivided into severalmajor subsets,such as Th1,Th2,Th17 and regulatory T cells(Tregs), which have been reported to be involving in the pathogenesis of liver diseases[6].

Recently,Th9 cells have gained renewed interest because of their prominent roles in the regulation of host responses via interacting w ith the previously Th subsets.Th9 cells characteristically produce interleukin(IL)-9,which exerts proin fl ammatory or anti-in fl ammatory activities by modulating Treg and/or Th17 cell developmentand function[7–9].Th9/IL-9 appears to function in a broad spectrum of autoimmune diseases and allergic in fl ammation.In addition,IL-9 also promotes the maintenance of tolerant environment by enhancing both Tregs andmast cell-mediated immunosuppressive functions[10–12].To date,IL-9 has been implicated in some fi brosis diseases,such as pulmonary fi brosis and cystic fi brosis[13,14].However,the role of IL-9 in liver fi brosis or LC has not been reported.Furthermore,little is known about the role of Th9 cells in the pathogenesis of LC.A lthough chemokine receptor usage by na¨ıve,Th1,Th2,Th17 and Treg cells is well documented[15,16],the m igratory capacity of the functionally dynam ic Th9 cells into liver tissue remains unknown.Therefore,in the present study, we evaluated the role of Th9/IL-9 in LC patients and determ ined thehom ing receptors involved in their recruitment to liver tissue.

2.M aterials and m ethods

2.1.Patients and controls

The study protocol was approved by the Review Board at Guangxi Medical University for human studies,and w ritten informed consent was obtained from each participant.A ll patients were either hospitalized or treated at the First A f fi liated Hospital,Guangxi M edical University,between December 2013 and June 2014.Liver tissue and corresponding peripheral blood samples were collected from 30 LC patients w ith hepatitis B.Patients who w ere co-infected w ith HIV or other hepatitis viruses and autoimmune diseases were excluded.No one received anti-hepatitis B virus agents or steroids six months before sam pling.Peripheral blood and liver tissue samples from 18 normal subjectswere collected as controls.Patients w ith viral hepatitis,autoimmune hepatitis and alcoholic liver diseases were excluded from the normal controls.

2.2.Flow cytometry

Intracellular cytokines were studied by fl ow cytometry to re fl ex the cytokine-producing cells.The expression of markers on T cells from peripheral blood was determ ined by fl ow cytometry,as previously described[17].After surface or intracellular staining w ith speci fi c anti-human antibodies,the cellswere conjugated w ith allophycocyanin,A lexaFluor 647, or phycoerythrin.These human antibodies included anti-CD4, anti-IL-9,and anti-IL-22 monoclonal antibodies,w hich were purchased from BD Biosciences(Franklin Lakes,NJ,USA). The T cell subsetswere incubated in Roswell Park Memorial Institute1640 medium for 5 h at 37°C in the presence of 5% CO2and stimulated w ith phorbol myristate acetate(50 ng/ m L;Sigma–A ldrich,MO,USA),ionomycin(1μg/m L; Sigma–A ldrich),and Golgi-Stop(1.7μg/m L;BD Biosciences).Phorbol m yristate acetate and ionomycin are pharmacological T-cell-activating agents that m im ic signals generated by the T-cell receptor complex and have the advantage of stimulating T-cells of any antigen speci fi city. M onensin was used to block intracellular transport mechanism s,thereby leading to accumulation of cytokines in the cells[17,18].A fter incubation,the cells were stained at room temperature in the dark for 30 m in w ith allophycocyaninconjugated anti-CD 4 monoclonal antibodies.The cells were then stained w ith A lexaFluor 647-conjugated anti-IL-9 monoclonal antibodies and phycoerythrin-conjugated anti-IL-22 monoclonal antibodies(Cat.No.560436)at 4°C for 30 m in after fi xation and permeabilization.A ll the antibodies were from BD Bioscience PharM ingen.Isotype controlsw ere analyzed to ensure correct compensation and con fi rm antibody speci fi city.Stained cells w ere analyzed by fl ow cytometry using a FACScan Cytometer equipped w ith the CellQuest softw are(BD Bioscience PharM ingen).

2.3.Immunohistochemistry

Immunohistochem ical staining was performed by the streptavidin–biotin complex method according to themanufacturer's instructions.Paraf fi n-embedded,formalin-fi xed liver tissues were cut into 4-μm sections and placed on polylysine-coated slides.Each paraf fi n section was deparaf fi nized and rehydrated through a graded series of ethanol.Endogenous peroxidase was blocked using a 3%H2O2methanol solution.Goat serum album in(5%;Zhongshan Golden Bridge Biotech,Beijing,China) was applied to block nonspeci fi c staining.Primary antibodies [chemokine receptor type 6(CCR6;Origene,USA),chemokine ligand 20(CCL20;Bioss,Beijing,China)andα-smoothmuscle actin(SMA;Sigma–Aldrich,USA)]were added and then incubated overnight at 4°C.After washing w ith phosphate buffer saline(PBS)three times,incubation of biotinylated secondary antibodies was performed at room temperature.Slides were treated w ith streptococcus avidin–peroxidaseand placed in the incubator at37°C for 30m in afterwashing w ith PBS three times.The slides were visualized by light m icroscopy after diam inobenzidine reaction.Pieceswere sealed w ith neutralgum after counterstaining w ith hematoxylin.Positive tissue sections were used as a positive control and PBSwas taken as anegative control in place of primary antibodies.Ten high-power fi elds of view were random ly selected for each slides.Image analysis of graphicswasmeasured by Image Pro-Plus 6.0 software.

2.4.Measurement of cytokines and chemokine

The concentrations of cytokines IL-9 as well as chemokine CCL20 in serum weremeasured by ELISA kits according to the manufacturer'sprotocols(allkitswere purchased from R&D Systems,M inneapolis,USA).A llsampleswereassayed in duplicate.

2.5.Statistical analysis

Data are expressed as themean±SD.The statistical significance of Th9,IL-9,and CCL20 among the subjects was determ ined by Student's t-testorW ilcoxon's rank sum testwhere appropriated.The Pearson correlation test was used for correlation analysis depending on the data distribution.Analysiswas completed w ith SPSS version 16.0 by Statistical Software (Chicago,IL,USA),and P value＜0.05 were considered to be statistically signi fi cant.

3.Resu lts

3.1.Clinical data of the subjects

The demographic and clinical information of the LC patients and the normal controls are summarized in Table 1.The data showed no signi fi cant difference between cirrhotic patients and normal controls in term of age and gender.However,the liver function indexesof LC patientswere deteriorated comparedw ith controls.

3.2.Comparison of Th9 cells frequency in LC and normal control patients

We performed fl ow cytometry on mononuclear cells from blood to identify Th9 cells(Figure 1A–F).Th9 cells werede fi ned asCD4+IL-9+IL-22−cells.As shown in Figure 1G,the frequency of Th9 cells represented the higher values in LC patients compared w ith controls(3.55%±1.69%vs. 0.97%±0.24%,P＜0.05).

Table1 Clinical characteristics of subjects.

3.3.Serum IL-9 and CCL20 levels in LC patients

The ELISA test showed that,compared with controls,signi fi cant elevation of serum IL-9 and CCL20 levels were observed in the LC patients[IL-9:(25.55±4.62)vs. (16.18±1.32)pg/m L,P＜0.05;CCL20:(224.77±33.73)vs. (34.58±3.72)pg/m L,P＜0.05].

Correlation analysis revealed that the frequency of Th9 cells was positively correlated to the serum IL-9 levels in LC patients (r=0.413,P=0.023)(Figure 1H).The sim ilar resultwas found between serum IL-9 and CCL20(r=0.417,P=0.015).However,no signi fi cant correlation was found between Th9 cells and IL-9,and between IL-9 and CCL20 in normal controls (P＞0.05).

3.4.Expression of chemokine receptors on Th9 cells in the peripheral blood

The results of fl ow cytometry revealed that,most Th9 cells expressed high levels of CCR4(85.8%±12.5%vs. 56.4%±2.36%,P＜0.05),and CCR6(78.1%±10.6%vs. 56.4%±9.04%,P＜0.05)compared w ith controls.

Comparedw ith controls,theexpression of CCR6 and CCL20 in liver tissue of LC were signi fi cantly increased(CCR6: 0.027±0.021 vs.0.006±0.001,P＜0.05;CCL20: 0.033±0.011 vs.0.017±0.006,P＜0.05).In addition,a signi fi cant increased expression ofα-SMA was detected in the LC patients compared to controls(0.054±0.018 vs.0.007±0.001, P＜0.05).Moreover,we also observed thatα-SMA was positively related to the CCR6,CCL20 in the liver tissue of LC (r=0.492,0.516,P＜0.05).

Figure 1.Representative fl ow cytometry dot plots demonstrate each group.A:Lymphocytesweregated by fl ow cytometry;B:CD4+T lymphocytesweregated by fl ow cytometry;C–F:The representative fl ow cytometric dot-plotsof isotype control in healthy control(HC)and LC group;G:Comparison of Th9 cells frequency between LC patients and HC.Horizontal bars indicate mean±SD.Comparisonsweremade using aW ilcoxon signed-rank test;H:Correlation of Th9 cells frequency and serum IL-9 levels.Correlationswere determined by Spearman rank correlation coef fi cients.

3.5.Expression of CCR6,CCL20 andα-SMA in liver tissue

CCR6 and its ligand,CCL20,have been reported w ith highly expression in a variety of human diseases[19,20].The immunohistochem ical staining images showed that CCR6 was expressed in the membrane of in fi ltration of lymphocytes in the portal area,the expression of CCL20 in the cellmembrane of dendritic cells,hepatic stellate cells,fi broblasts,endothelial cells and cytoplasm of epithelial cells of bile duct in the portal area(Figure 2).

Figure 2.Immunohistochemicalstaining of CCR6,CCL20 andα-SMA in liver tissue.A&B:Representative immunohistochem ical staining of CCR6 in liver tissue of normal control and LC patients,respectively,the CCR6 was expressed in the cellmembrane;C&D:Immunohistochemical staining of CCL220 in normalcontrolgroup and cirrhosis liver tissue,respectively,the expression of CCL20 was in cell membrane and cytop lasm;E&F: Representative immunohistochemicalstaining ofα-SMA in normal control and cirrhosis liver tissue,the expression ofα-SMA was in portal area and fi ber interval area.Positive expression is brown color(400×).

3.6.Correlation analysis between Th9 cells and liver function

Correlation analysis showed that the frequency of Th9 cells had a positive correlation w ith prothrombin time(PT),band total bilirubin level(TBIL)in LC patients(P＜0.05);the sim ilar correlation also observed Th9 cells and hyaluronic acid(HA) and type IV collagen(IV-C)(P＜0.05)(Figure3).However,no signi fi cant correlations were found between IL-9 and liver function and fi brosis indexes(P＞0.05).

Figure 3.Correlation analysis between Th9 cells and liver function.A:Correlation analysis between Th9 cells and HA;B:Correlation analysis between Th9 cells and IV-C;C:Correlation analysisbetween Th9 cells and PT;D:Correlation analysis between Th9 cells and TBIL.

4.Discussion

The role of Th9 cells have been implicated inmany in fl ammatory and immune diseases.In allergic asthma patients,the Th9 cells frequency in peripheralblood was increased compared w ith allergic rhinitisand controlsubjects,the same tendency was observed for IL-9,and the serum IL-9 levelswas correlatedw ith frequency of Th9 cells[21].In the context of experimental autoimmune encephalomyelitis(EAE),adoptive transfer of in vitro-generated encephalitogenic Th9 cells into na¨ıve hosts led to development of neuroin fl ammatory central nervous system lesions and induction of severe EAE[22];while inhibition of IL-9,using IL-9-de fi cientm ice or IL-9 antibody, has been shown to alleviated EAE[23,24].Collectively,these studies suggest that Th9 cells can contribute to the pathogenesis of autoimmunity in multiple organ systems.

In our study,we observed that the frequency of Th9 cells and serum IL-9 levels was signi fi cantly elevated in LC patients compared w ith that of normal controls.We also found that frequency of Th9 cells was positively related to the PT,TBIL, HA,IV-C in LC patients,suggesting that Th9 cells participated in the pathogenesis of LC.However,we did not observe signi fi cant correlation between IL-9 and liver function indexes, suggesting that the role of Th9 cells in LC can notequal to that of IL-9.The reasons of insigni fi cant correlation is not known, we speculated that there were follow ing reasons:fi rst,besides Th9 cells,other Th cells,such as Th2,Th17 and Treg cells can also produce IL-9;second,the small sample size lead to a relatively statistical power.

M igration of Th cells to the lesion sites is essential for the execution of their effector function,where the chemokines and their receptors are orchestrating this process.Chemokines are a fam ily of low molecular weight pro-in fl ammatory cytokines, which bind to chemokine receptors and sustain them igration of neutrophils,lymphocytes,monocytes and dendritic cells. Currently,the role of CCR6 and its ligand CCL20 is well documented in several diseases,including in fl ammatory bowel disease[20]and liver cancer[19].In malignant pleural effusion, there was study reported that Th9 recruited to the pleural space during pleuralmetastasis by pleuralmesothelial cells via a CCR6/CCL20 axis[25].The CCR6/CCL20 axismediates the m igration of circulating Tregs into tumor m icroenvironment, which in turn results in tumor progression and poor prognosis in liver cancer patients[19].

A lthough Th9 cells have been implicated in both allergy and autoimmunity,the m igratory properties of Th9 cells remain enigmatic.In the contextof LC,m igration of Th9 cells into the liver is important to the development and progression.In this study,we found that Th9 cells expressed a high level of CCR6 and CCR4 on their surface compared w ith normal controls. Because an increaseofα-SMA was indication of severe LC,the correlation of CCR6,CCL20 andα-SMA in liver tissue suggested that CCR6 and CCL20 may participate in the pathogenesis of LC.In addition,the increased expression of CCR6 and CCL20 in serum also further con fi rmed this role.Taken together,these data suggested that the CCR6/CCL20 axism ight be related to the in fi ltration of Th9 cells into liver tissue.The mechanism of CCR6/CCL20 axis mediating Th9 cells to the liver remains to be elucidated.According to the previous report,we speculated that IL-9 can induce the expression of CCL20 w ithin the lesions site and CCR6 expression on leukocytes to facilitate tumor in fi ltration[26].However,a chemotaxis assay is necessary in order to further con fi rmed this role.

The presentstudy gives a prelim inary insight into the role of Th9 cells in the pathogenesis of LC,and suggests that the CCR6/CCL20 axism ight recruited Th9 cells into liver tissue to execute their effector function.However,some lim itations should be noted.First,we observed the role of Th9 cells in LC, but themechanism of this role remains to be elucidated,especially how the Th9 cellsact this role via IL-9 and the interaction between other IL-9 producing Th cells.Second,in this study, we only investigated CCR6/CCL20 axis,other chemokine axis, such as chemokine receptor-4/Stromal cell-derived factor 1[27], CCL21/CCR7[28],which have been reported in several immune diseases,were not investigated in our study.Thus,these chemokine axis should be investigated in further study.Third, the number of included subjects was small,which may be the reasons of insigni fi cant correlation between Th9 cells and other indexes.Therefore,in order to enhance the statistical power a larger sample size is need in further study.

In conclusion,the presentstudy suggests that Th9 cellsparticipate in the pathogenesisof LC,and the recruitment Th9 cells into liver tissuemightbe through CCR6/CCL20 chemokineaxis.

Con fl ict of interest statement

We declare thatwe have no con fl ictof interest.

Acknow ledgm ents

Thisworkwassupported by grants from the NationalNatural Scienti fi c Foundation of China(81260083;31360221)and GuangxiNatural Science Foundation(2014GXNSFAA118203). The funders had no role in study design,data collection and analysis,decision to publish,or preparation of themanuscript.

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12 Oct 2015

*Corresponding author:Hai-Xing Jiang,Department of Gastroenterology,the First A f fi liated Hospital of Guangxi Medical University,Shuang-Yong Road 6, Nanning 530021,Guangxi,PR China.

Tel/Fax:+86 771 5356725

E-mail:gxjianghx@163.com

The study protocol was performed according to the Helsinki declaration and approved by Review Board at Guangxi M edical University for human studies. Informed w ritten consentwas obtained from each participant.

Foundation Pro ject:Supported by grants from the National Natural Science Foundation o f China(81260083;31360221)and Natural Science Foundation o f Guangxi Province(2014GXNSFAA 118203).

Peer review under responsibility o f Hainan Medical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.

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