| Backgroud and aimsHepatitis B virus (HBV) is a non-cytopathic, hepatotropic DNA virus that is capable ofinducing necro-inflammatory liver disease with varying severity. Persistent infection by HBVis often associated with chronic liver disease, which can further lead to the development ofcirrhosis and hepatocellular carcinoma. While many of the underlying mechanisms of HBVinfection progression have been described, the complex array of pathogen-host interactions isnot yet fully understood. A growing body of recent evidence has suggested thatCD4~+CD25~+Foxp3~+regulatory T cells (Tregs) may play an important role in the suppressionof antiviral T cell responses during the chronic phases of HBV infection. Some studies havedemonstrated that HBV carriers have a higher frequency of Tregs in peripheral blood andliver than healthy controls or individuals with resolved infection, indicating that Tregs maycontribute to HBV persistence. However, other studies have failed to detect any differences inTreg frequencies between asymptomatic carriers (AsCs) and healthy controls. Thus, the roleof Tregs in the progression of liver disease remains controversial, and little is known aboutwhether their potential roles in viral hepatitis pathogenesis differ according to their functionalactivities.The largely discordant results among the studies that have assessed the role of Tregs inHBV infection may reflect variation in study design and model systems. For example, thedefinition of Tregs varied between studies, and in many was based solely upon theco-expression of CD4~+and CD25hiFoxp3~+. Unlike the Tregs in mice, human Tregs areheterogeneous and the population exhibits considerable diversity. Phenotypically andfunctionally distinct subsets of Tregs can mediate immune suppression through distinctmechanisms, including specific profiles of immunomodulatory factors, such as IL-10, TGF-β,granzyme B, perforin, CTLA-4, GITR, and LAG-3. In addition, CD39-mediated interactionswere recently implicated in Treg activities during different stages of viral infection.CD39is a recently described molecule with immunomodulatory properties, and is expressed on human and murine Tregs. Several studies have identified the CD39/NTPDase1molecule as a useful biomarker of a CD4~+FoxP3~+regulatory suppressor T cell population withpotent immunosuppressive activity in humans and mice. Notably, a significant increase ofCD39expression on Tregs has been observed in cancer patients and patients with humanimmunodeficiency virus (HIV) infection, and a strong association was found between CD39expression on Tregs and tumor or AIDS disease progression. Decreased frequency andfunction of CD39~+Tregs have been reported in multiple sclerosis, ryegrass allergy, andvascular inflammation after transplantation.Although the critical roles of CD39in some diseases have been described, the potentialroles of CD39-expressing Treg cells in hepatitis B pathogenesis have yet to be elucidated.Therefore, this study was designed to investigate the phenotype characteristics and frequencyof peripheral and intrahepatic Tregs and CD39~+Tregs in hepatitis B patients, and to determinewhether the characteristics of this subset are related to the HBV disease process.Results1. CD39defines a discrete subset of CD4~+Foxp3~+T cells and is preferentially expressedby CD45RA-FoxP3hiTreg cellsIn this study, FCM analysis revealed a high variance in the frequency of CD4~+T cellsexpressing CD39, which ranged from1%to20%among the healthy donors andHBV-infected subjects. Based on the distribution shown in the scatter diagram for thefrequency of CD4~+T cells expressing CD39, we subdivided the total cohort into three groupsaccording the frequency of CD39-expressing CD4~+T cells: CD39low (<5%), CD39int(5~10%) and CD39high (>10%), in order to characterize the CD39expression with diseasestatus in more detail. Initial characterization studies of healthy peripheral CD39~+FoxP3~+CD4~+T cells revealed that the majority were CD45RA-FoxP3hiactivated Treg cells. We consistentlyobserved an increasing trend in the proportion of CD39-expressing Foxp3~+Tregs in CD39low(17%), CD39int (45%) and CD39high (65%) groups. The results showed that the majority ofCD39~+CD4~+T cells were CD45RA-. Specifically,>70%proportions of CD39~+CD4~+T cells(denoting memory phenotype) were found in each of the CD39low, CD39int and CD39highgroups.Sakaguchi and colleagues showed that human FoxP3~+CD4~+T cells were composed ofthree phenotypically and functionally distinct subpopulations: CD45RA~+FoxP3lowresting Treg cells (Fr.I, rTreg cells), CD45RA-FoxP3hiactivated Treg cells (Fr.II, aTreg cells), bothof which demonstrated suppressive functions in vitro, and cytokine-secretingCD45RA-FoxP3lownon-suppressive T cells (Fr. III, non-Treg cells). In our study, thefrequency of CD39gated on each fraction was analyzed and the results showed that CD39accounted for the vast majority of aTreg cells (94%in CD39high,89%in CD39int, and36%in CD39low groups). In contrast, non-Tregs and rTregs contained much lower CD39expression in each of the CD39-defined groups. Assessment of co-expression of CD39andother Treg functional makers, such as CTLA-4, Ki-67, and HLA-DR, on FoxP3~+CD4~+T cellsrevealed that the indicated markers expressed on CD39~+Tregs was higher than that on theCD39-Tregs (by~1-fold), suggesting that CD39~+Treg cells might be stronger suppressorsthan CD39-Tregs. Functional assays indicated that CD39~+Treg cells exhibited strongersuppressive ability to inhibit the proliferation of CD4~+CD25-T cells at different S/R ratios, ascompared to the CD39-Treg cells. In addition, we also observed that the CD39moleculecould be expressed on Th1and Th17cells, as indicated by the CD39int and CD39high groupsdisplaying a small fraction of CD39~+CD4~+T cells capable of secreting IFN-γ and IL-17A,respectively.2. CD39~+FoxP3~+Treg populations correlate with hepatitis B progressionResults showed that there was no difference in the proportion of CD39expression onCD4~+T cells in AsCs, CAH and ACLF patients, as compared to the healthy control. Wefurther found that the frequency of total Treg cells (CD4~+FoxP3~+T cells) was significantlyhigher in CAH and ACLF patients than in normal controls. However, the proportion ofCD39-expressing Tregs was more dynamic. The CD39~+Treg proportion was higher in AsCspatients than in healthy controls (P<0.05). However, this proportion decreased significantly(nearly to the level in healthy controls) in the CAH and ACLF patients as compared with thatin AsCs patients (P<0.05, respectively). Thus, distinction of Treg cell subsets based on thecombination of CD39and FoxP3expression is a highly informative approach for assessingthe dynamics of Treg cell differentiation under HBV disease conditions.3. Circulating CD39~+Treg cell level is correlated with HBV copy number and ALT level,but not with HBeAg expressionWe analyzed the correlation between Treg subsets and HBV copy number by measuringCD39expression, serum ALT levels, HBV DNA load, and HBeAg in AsCs, CHB and ACLF patients, and comparing the results with those from total FoxP3~+Treg cells. Results showedthat there were no significant correlations between total FoxP3~+Tregs and HBV viral copies.In contrast, when the frequency of Treg cells was analyzed on the basis of CD39expression,it was significantly and positively correlated with the collective serum HBV viral copies in alltested hepatitis B patients, especially in AsCs patients. Moreover, when analysis wasperformed with the CD39high/int/low groups, the HBV viral copies were found to besignificantly increased in the CD39high group, as compared with the CD39low group in allHBV-infected subjects.Next, the correlation between Treg subsets was analyzed based on CD39and serumALT. Similar with the results described above for the HBV viral copies, there were nosignificant correlations found between total FoxP3~+Tregs and serum ALT. In contrast, thefrequency of CD39~+FoxP3~+Treg cells was significantly negatively correlated with serumALT in total hepatitis B patients, and a strong correlation was also observed in CAH patients.Further analysis indicated that the serum ALT levels were increased in the CD39low group,as compared with the CD39int/high groups in CAH patients (P<0.05), but not in the ACLFpatients.In addition, we observed that there were no marked differences in the frequencies oftotal FoxP3~+Tregs or CD39~+FoxP3~+Tregs between hepatitis B patients with or without serumHBeAg expression. Similar results were observed in the CD39high/int/low groups. It ispossible that the data in our study, which was obtained from routine clinical tests, did notrepresent an absolute quantification of the HBeAg protein expressions.4. The distribution of CD39~+Treg cells was examined in HBV-infected livers by FCManalysis and morphological observation.The LILs of distal liver tissue from the liver hemangioma patients with HBV infectionwere isolated and stained with various antibodies. The CD39-expressing Foxp3~+Tregsaccounted for about46%of the total Foxp3~+Tregs, similar to our previous observations. Wefurther examined the in situ distribution of the CD39~+Treg subset in the livers of CHBpatients by immunohistochemical and immunofluorescence staining techniques. Resultsshowed that the CD39~+and Foxp3~+cells were mainly localized near the hepatic portal tract.Using triple fluorescence staining, we confirmed that CD39and FoxP3expressionsco-localized within regions with partial CD4~+T cells, indicating that the CD4~+FoxP3~+CD39~+ Treg cells did exist in the portal area of liver tissue and might be involved in hepatitis Bpathogenesis. Quantitative analysis of the positive cells per hpf in liver portal areas in HBVpatients demonstrated that about50%of total Foxp3~+Tregs expressed CD39, in agreementwith the observations from the above-described FACS assay.Conclusions:In this study, we investigated the phenotype and the suppressive function of the newlyidentified CD39~+Tregs in details. In HBV infected cases, the CD39~+Tregs proportion wasfound to be increased in AsCs patients, but decreased in CAH and ACLF patients.Importantly, the CD39~+Treg proportion is positively correlated with HBV load but inverselycorrelated with serum ALT level. These findings indicate that CD39expression on FoxP3~+Treg cells is involved in disease progression of HBV infection, suggesting that CD39~+Tregpopulations are dynamic and can be followed longitudinally, thereby opening a new avenuetowards therapeutic interventions aimed at modulating Treg functions. |
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