Reply to: “Controversy on the role of FoxP3+ regulatory T cells in fibrogenesis in chronic hepatitis C virus infections”
Authors: Bettina Langhans, Ulrich Spengler
Journal: Journal of Hepatology (2013)

Controversy on the role of FoxP3+ regulatory T cells in fibrogenesis in chronic hepatitis C virus infectionsJournal of HepatologyVol. 60Issue 1PreviewTo the Editor: Full-Text PDF Open AccessIntrahepatic IL-8 producing Foxp3+CD4+ regulatory T cells and fibrogenesis in chronic hepatitis CJournal of HepatologyVol. 59Issue 2PreviewRegulatory CD4+ T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions. Full-Text PDF To the Editor: We are grateful that Claassen et al. draw attention to several issues in our manuscript on "Intrahepatic IL-8 producing Foxp3+CD4+ regulatory T cells and fibrogenesis in chronic hepatitis C" [[1]Langhans B. Krämer B. Louis M. Nischalke H.D. Hüneburg R. Staratschek-Jox A. et al.Intrahepatic IL-8 producing Foxp3+CD4+ regulatory T cells and fibrogenesis in chronic hepatitis C.J Hepatol. 2013; 59: 229-235Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar] which had not been discussed in depth and thus might lead to misleading conclusions. We agree, that IL-8+Foxp3+CD4+ T cells comprise only a small population of intrahepatic immune cells and that other types of intrahepatic cells also produce IL-8. However, in line with the report by Zimmermann et al. [[2]Zimmermann H.W. Seidler S. Gassler N. Nattermann J. Luedde T. Trautwein C. et al.Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis.PLoS One. 2011; 6: e21381Crossref PubMed Scopus (210) Google Scholar], macrophages were the only other cells in hepatitis C, which in addition to CD4+ T cells produced sufficient IL-8 as to enable in situ detection by immunofluorescence. Unlike IL-8+CD4+ T cells, which were located preferentially at the boundaries of fibrotic areas, macrophages formed cell clusters at regions of necrosis and inflammation (see Fig. 1). Furthermore, IL-8+CD4+ T cells can be triggered by HCV antigens [[3]Langhans B. Braunschweiger I. Arndt S. Schulte W. Satoguina J. Layland L.E. et al.Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C.Clin Sci (Lond). 2011; 119: 97-109Crossref Scopus (44) Google Scholar] and thus are probably activated continuously by antigen in chronic hepatitis C. Taken together these findings suggest a regulatory role of IL-8+CD4+ T cells in HCV-infected livers. In support of this hypothesis we directly demonstrated induction of fibrogenesis in primary human hepatic stellate cells by in vitro co-culture experiments. As reported previously the same type of IL-8 producing CD4+ regulatory T cells also exerts other functions, e.g., our regulatory T cell clones inhibit activity of CD4+ effector T cells by releasing the cytokines IL-10 and IL-35 [[3]Langhans B. Braunschweiger I. Arndt S. Schulte W. Satoguina J. Layland L.E. et al.Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C.Clin Sci (Lond). 2011; 119: 97-109Crossref Scopus (44) Google Scholar]. Concerning interferon-γ the comment of Claassen et al. illustrates that data from microarray analysis must be interpreted with caution. As can be seen in the supplementary file to our previously published data [[3]Langhans B. Braunschweiger I. Arndt S. Schulte W. Satoguina J. Layland L.E. et al.Core-specific adaptive regulatory T-cells in different outcomes of hepatitis C.Clin Sci (Lond). 2011; 119: 97-109Crossref Scopus (44) Google Scholar], the regulatory T cell clones studied by microarray analysis (T1_7, H7_3, H7_16) only produced negligible amounts of interferon-γ even upon stimulation with mitogen (<5 pg/ml), whereas bona fide TH1 cells produced 800–6000 pg/ml under the same conditions. Of note, the regulatory T cell clones even suppressed interferon-γ production of TH1 reporter cells in co-culture experiments (see Fig. 2). This constitutes further experimental evidence suggesting a dual role of Foxp3+CD4+ regulatory T cells in both regulation of fibrosis and inhibition of inflammatory activity. To guard against false interpretation of our microarray data, we have confirmed at the protein level that regulatory T cell clones as well as freshly isolated Foxp3+CD4+ T cells produce IL-8 in chronic hepatitis C. These findings indicate that IL-8 production of regulatory T cells may reflect distinct T cell differentiation associated with chronic hepatitis C (and possibly also some other infections). Indeed several in vitro studies have demonstrated that HCV proteins, HCV transfection, and experimental infection are associated with activation of the IL-8 promoter and IL-8 production (see references 30 to 44 in our publication). Concerning the putative relationship between regulatory T cells and stages of fibrosis, a closer look reveals that our data are rather similar to those reported previously in the Journal of Hepatology by Wards et al. and Sturm et al. [4Ward S.M. Fox B.C. Brown P.J. Worthington J. Fox S.B. Chapman R.W. et al.Quantification and localisation of FOXP3+ T lymphocytes and relation to hepatic inflammation during chronic HCV infection.J Hepatol. 2007; 47: 316-324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 5Sturm N. Thelu M.A. Camous X. Dimitrov G. Ramzan M. Dufeu-Duchesne T. et al.Characterization and role of intra-hepatic regulatory T cells in chronic hepatitis C pathogenesis.J Hepatol. 2010; 53: 25-35Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar]: both authors stated that the "relative proportion of Foxp3+ cells remains similar in both mild and severe fibrosis". This statement holds also true in our study, where the relationship between numbers of Foxp3+CD4+ regulatory T cells and fibrosis indicated only a small non-significant trend when IL-8 secretion was not taken into account separately (rS = 0.274, p = 0.185). Thus, it is not the number of regulatory T cells but their fraction with differentiation towards IL-8 production that increases with progressive fibrosis. Moreover, the apparent discrepancy of these previous reports (including our own) to the data of Claassen et al. [[6]Claassen M.A. de Knegt R.J. Tilanus H.W. Janssen H.L. Boonstra A. Abundant numbers of regulatory T cells localize to the liver of chronic hepatitis C infected patients and limit the extent of fibrosis.J Hepatol. 2010; 52: 315-321Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar], probably reflects a difference in the reference frame for reporting numbers of regulatory T cells: frequencies of regulatory T cells were given with respect to numbers of total liver leucocytes by Claassen et al., whereas in our study numbers of IL-8+Foxp3+CD4+ regulatory T cells were reported as fraction of CD4+ T cells. Thus, it might be quite possible that inflammatory cells other than T cells, which accumulate in liver tissue during progression of chronic hepatitis C, may have "diluted" the analysis of regulatory T cells with respect to total leucocytes resulting in an apparent decline in advanced fibrosis. We did not study IL-8+ regulatory T cells under treatment with interferon and ribavirin combination therapy and thus cannot comment on persistence of these cells after therapeutic clearance of HCV infection. However, we want to draw attention to a previous brief report by Bahra et al. who studied evolution of fibrosis progression in hepatitis C when patients had achieved sustained viral clearance after therapy [[7]Bahra M. Neumann U.P. Jacob D. Langrehr J.M. Berg T. Neuhaus R. et al.Fibrosis progression in hepatitis C positive liver recipients after sustained virologic response to antiviral combination therapy (interferon–ribavirin therapy).Transplantation. 2007; 83: 351-353Crossref PubMed Scopus (41) Google Scholar]. In serial biopsies taken before 1, 3, and 5 years after sustained response they observed a delayed reduction of fibrosis scores over the first 3 years. This finding might be well compatible with persisting of profibrotic regulatory cells after viral clearance as reported by Claassen et al. [[8]Claassen M.A. de Knegt R.J. Jannssen H.L.A. Boonstra A. Retention of CD4+CD25+Foxp3+ regulatory T cells in the liver after therapy-induced hepatitis C virus eradication in humans.J Virol. 2011; 85: 5323-5330Crossref PubMed Scopus (34) Google Scholar]. We hope that the additional data provided in our response helps to clarify the invaluable comments of Claassen and Boonstra, which highlight the complexity of regulatory T cell involvement in the pathogenesis of chronic hepatitis C. Part of regulatory T cell heterogeneity probably results from the fact that a uniform marker to identify this population has not yet been established, making it necessary to identify regulatory T cells by a panel of multiple features. Thus, it is difficult to ascertain if regulatory T cells in hepatitis C reported by different authors actually refer to the same T cell population. By demonstrating that Foxp3+CD4+ regulatory T cells in hepatitis C can exert dual functions – both inhibition of T effector cells and activation of hepatic stellate cells – our manuscript illustrates that functional heterogeneity of regulatory T cells exists and must be considered in future studies. Multiple issues remain to be resolved. Thus, we do not know if dual functionality is acquired sequentially at different stages of hepatitis C or if exists in HCV-associated regulatory T cells right from the beginning. To be frank we fear that current knowledge on the heterogeneity of regulatory T cells may only reflect the tip of the iceberg. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.