Expression And Clinical Significance Of Th17/IL-17 In Peripheral Blood In Patients With Chronic Hepatitis B

Over 20 years ago,it was first demonstrated by Mosmann that after antigenic stimulation,naive CD4⁺ T cells could be differentiated into two distinct subsets on the basis of their cytokine production profile.Cells of the T helper(Th)1 lineage produce interferon(IFN)_γand mediate cellular immunity,whereas cells of the Th2 lineage produce interleukin(IL)-4,IL-5,and IL-13 and mediate humoral immunity.IL-12,constructed of IL-12P35 and IL-12P40,could stimulate downstream T-bet expression through activation of STAT4 and drive Thl differentiation,whereas IL-4,could stimulate GATA-3 expression through activation of STAT6 and drive Th2 differentiation.Recent studies have defined a previously unknown arm of the CD4+T-cell effector response—the Th17 lineage—that promises to change our understanding of many diseases. Th17 lineage,distinct from Th1 and Th2,as a new Th cell lineage produce IL-17A, IL-17F,IL-6,TNF-αand GM-CSF,but not IL-4,IFN-γ.TGF-β,IL-6,IL-1βand TNF-αinduce the differentiation of Th17,whereas IL-23 plays an important role in the generation and survival of Th17.The key transcription factor in Thl7 differentiation is RORγt.Recent researches have demonstrated that the main effector factor IL-17 could recruit neutriphil and monocyte into inflammation locus by stimulating the production of chemokines such as IL-8,MCP-1,Gro-α,and IL-17 could also aggravate local Inflammatory reaction by inducing the expression of IL-6,NO,PGE2,IL-1β,TNF-α,IFN-γand CD40L.Certainly,as a bridge between inherent immunity and adaptive immunity,IL-17 also plays a part in the activation of T,B cell,modulation of Inflammatory reaction and autoimmunologic injury.Chronic Hepatitis B(CHB) is very popular in china.There are at least 130 million Hepatitis B virus(HBV) carriers and 30 million Hepatitis B patients.The mechanism of HBV persistence is not fully understood but is likely multifactorial.HBV is a non-cytopathic hepatotropic virus that can lead to a series of complex immunological reactions.Successful clearance of the virus as well as the establishment of liver disease is largely driven by a complex interaction between the virus and the host immune response. At present,cellular immunological response is deemed to be the most important factor in body's clinical process and prognosis after HBV infection.A great many studies have proposed that the unbalance of Th1 and Th2 be one reason in the chronicity after HBV infection,and Th1 could be dominant in the patients recovered from acute HBV infection, whereas Th2 be dominant in the patients recovered from chronic HBV infection.However, whether the new Th cell -Th17 cell lineage produces a marked effect in the clinical process and liver's inflammatory damage of CHB has not been reported.In our research,we collected each sample of 7ml peripheral blood from 93 CHB(21 mild,37 moderate,35 severe CHB) and 28 healthy control.We detected the expression of IL-17-producing T cells in peripheral blood of CHB patients and healthy control,and investigated the relationship between Th17 and liver damage in patients of CHB.Firstly,three methods were applied to research the expression of Thl7 cell / IL-17 in peripheral bloods in distinct degrees of CHB patients and healthy controls.These methods included flow cytometry,Realtime PCR and ELISA.1,Th17 cells in peripheral blood were detected by flow cytometry,we mixed 100μl EDTA-anticoagulant blood with 100μl RPMI1640 medium(containing 10%calf serum) in the presence of 50ng/ml PMA,1μg/ml Ionomycin and 1μl/ml Golgi plug.After 5-hours incubation at 37℃in the circumstance containing 5%CO₂,blood cells were stained by human monoclonal antibody(mAbs) CD4-FITC.Then,according to the protocol of intracellular staining(from BD pharMingen),IL-17,IFN-γwere stained with PE-labeled anti-human IL-17 and APC-labeled anti-human IFN-γrespectively for flow cytometric analysis.The results show:The proportion of CD4⁺IFN-γIL-17⁺T cells (Th17 cells ) in CD4⁺T cells was obviously increased in the peripheral blood of severe and moderate CHB patients as compared with that of healthy control(P＜0.05).This did not yet differ significantly between moderate and mild group,mild and control group. Both the proportions of CD4⁺IFN-γ⁺IL-17^-T cells(Th1 cells) and CD4⁺IFN-γ⁺IL-17⁺T cells(double-positive cells) in CD4⁺T cells differed insignificantly among all groups in statistics,although CD4⁺IFN-γ⁺IL-17⁺T ceils(double-positive cells) seems even higher in severe CHB patients than in healthy control.2,Gene expressions encoding IL-17,RORγt in CD4⁺Tcell were detected by Realtime PCR.In this part,we separated CD4^T cells from blood and divided them into 3 parts.Cells from the first part were cultivated for 4 hours in the presence ofα-CD3 and PMA,and that from the second part were not given any treatment.The third part was used for latter ELISA.Then,Realtime PCR was used to determine the expression of mRNA encoding IL-17,RORγt in these two parts.The results show:After cultivation,the gene expression encoding IL-17,RORγt in CD4⁺T cell were both higher in severe CHB patients than in mild CHB patients and healthy control(P＜0.05).Nevertheless,the differences between severe and moderate,mild and control group were not obvious. However,when CD4+T cells were not stimulated with a-CD3 and PMA(from the second part),there were no significant differences among all groups in their expressions of mRNA encoding IL-17,RORγt.3,Levels of IL-17 in serums and cultivation supernants were detected by ELISA.we collected the supernatants of cells(from the third part),which had been cultivated for 72 hours in the presence ofα-CD3 andα-CD28;And detected the levels of IL-17 in the supernatants and serums by ELISA.Compared with moderate,mild CHB patients and control,the cultivation supernatant of CD4⁺T cells from the severe CHB patients could express much more IL-17(P＜0.05) through ELISA,correlating with the results of flow cytometry and Realtime PCR.Likewise,the levels of IL-17 differed little among all groups in peripheral serums.Secondly,Realtime PCR was used to determine the mRNA expression of TGF-β,IL-6,IL-1β,related to the differentiation of Th17 cell;and IL-22,coexpressed in Th17 cell with IL-17.Compared with healthy control,the expressions of TGF-β,IL-6,IL-1βwere increased in severe CHB patients,although there were no significance in statistics. After stimulation withα-CD3 and PMA,we found that the differences between severe and moderate,mild CHB patients,healthy control were enlarged(P＜0.05).Nevertheless, there were no obvious differences among other groups.The changes of IL-22 among groups were not obvious no matter whether stimulation.Lastly,we detected the levels of ALT and DNA quantities of HBV in peripheral serums.And then we analyzed the relationship between the portions of Th17 cell in CD4⁺T cells and the concentration of ALT in peripheral serums.We found that there were direct correlation in these two indexes(P＜0.01).Furthermore,in this part,we found that there were no direct correlation between the portions of Th17 cells in CD4⁺T cells and the DNA quantities of HBV.In these studies,firstly,three methods were applied to research the expression of Th17 cell/IL-17 in peripheral bloods in distinct degrees of CHB patients and healthy controls.Our studies showed that following the exacerbation of this disease,the portions of CD4⁺IFN-γ-IL-17+T cell(Th17 cell),the mRNA levels of IL-17,RORγt in CD4⁺T cells stimulated withα-CD3 and PMA,the protein levels of IL-17 in supernatants of stimulated CD4⁺T cell were all up-regulated.Secondly,when we detected the mRNA expression of cytokines related with differentiation of Th17 such as TGF-β,IL-6,IL-1β,we found that after stimulation withα-CD3 and PMA,the mRNA levels of TGF-β,IL-6,IL-1βin CD4⁺T cells were much higher in severe CHB patients than in control.But there were no differences among groups in their expressions of IL-22 which had been considered as coexpressed in Th17 cells.Lastly,the relationship among the portions of Th17 cells in CD4⁺T cells,the concentration of ALT and the DNA quantities of HBV in peripheral serums was evaluated.We found that there were direct correlation in the portions of Th17 ceils in CD4⁺T cells and the concentration of ALT(P＜0.01).Furthermore, in this part,we found that there were no direct correlation between the portions of Th17 cells in CD4⁺T cells and the DNA quantities of HBV.Our studies suggested that Th17 cell might play a role in the pathogenesis of CHB. There were no significant differences between mild CHB patients and healthy control(it may be solved by enlargement of the cases),however,in the total tendency,the portions of Th17 cells in CD4⁺T cells,levels of IL-17 in cultivation supernant of CD4⁺T cell and mRNA expression of IL-17,RORγt were gradually increased following the exacerbation of this disease.TGF-β,IL-6 and IL-1βhad been important factors inducing the differentiation of Th17,according to this,in our results,the mRNA levels of these cytokines higher in severe CHB patients than in control.Likewise,it had been reported that there were an up-regulations of TGF-β,IL-6,IL-1βin serums of CHB patients.But it would be also deserved our further research whether there were a direct link between the up-regulation of Th17 cells and TGF-β,IL-6,IL-1β.In our studies,we couldn't obtain the differences among groups before stimulation.This phenomenon may be the result of lower immune reaction in vivo of CHB patients.When stimulated in vitro,these differences were reinforced and could be detected by us.Otherwise,in our studies,we couldn't explain well why IL-22 didn't appear to differ correlating with IL-17.If there were a kind of Th17 cells highly expressing IL-17 alone? And because of the existed phenomenon that the direct correlation between the portion of Th17 cell in CD4⁺T cell and the concentration of ALT in peripheral serum of CHB patients,it would be also deserved our study whether the up-regulations of IL-17 and Th17 cell induced by direct infection of HBV or liver damage after HBV infection.The mechanism of HBV persistence is not fully understood but is likely multifactorial including the interaction of virus components,liver damage and immune system.Current treatments are effective in suppressing HBV replication but in most cases failed to clear the virus.A combined effect of viral suppression and HBV-specific immune reconstitution is needed for effective long -term clearance of infection.Thus, further studying the role of Th17 and IL-17 in the pathogenesis of CHB might become a new target for helping CHB patients to reconstitute immune system.