Treg roles in HCV disease progression are not yet clearly established. Reasons could be that Treg are heterogeneous populations and unambiguous Treg markers remain elusive. Consequently, potential Treg subsets are certainly missed, since most Treg studies use phenotypic markers to identify Treg, even if identified cells are then studied functionally. In peripheral blood of subjects with chronic HCV infection, we previously detected TGFβ mediated suppressive activity against HCV-specific effector function and identified a novel population of non-classical human Tregs responsive to HCV that produced the Treg-associated cytokine TGFβ (25
). In this report, we defined the relation of hepatic and peripheral HCV-specific T cell-produced TGFβ to HCV related liver disease.
Blocking Treg-associated cytokines increased effector HCV-specific T cell responses in slow progressing subjects with chronic HCV infection. This suppressive function was detected in both peripheral and liver compartments, suggesting presence of similar T regulatory activity in peripheral blood and liver, at least for certain types of Treg populations. Presence of various hepatic Treg populations have been suggested: CD4+
(by liver histological co-staining assays)(17
) and CD8+
Treg cells (systematic random cloning)(23
). However, it is not clear whether there are differences or similarities in Treg content and function between periphery and liver. Our finding of a strong correlation between HCV-specific PBMC and IHL IFNγ responses revealed upon Treg cytokine blockade support similarities in cytokine mediated Treg activity between these compartments. In addition, revealed PBMC HCV-specific effector responses actually correlated with IHL HCV-specific IFNγ responses assayed without Treg cytokine blockade. It would be ideal if these peripheral responses revealed upon use of Treg cytokine blockade reflect, at least in part, what is occurring in liver, since this would provide a robust surrogate, enabling follow-up longitudinal studies of T cell immunity to HCV.
Importantly, increases of both PBMC and IHL HCV-specific IFNγ responses resulting from Treg-associated cytokine blockade were significantly greater in patients with slow liver fibrosis progression compared with subjects with rapid liver fibrosis progression, suggesting presence of effective suppressive Treg-associated cytokine activity in slow, but not rapid progressors, and therefore involvement of this regulatory activity in protection from destructive inflammation and subsequent liver disease progression. Alternatively, it is possible that Treg-associated cytokine blockade in rapid progressors did not increase effector HCV-specific T cell response because T cells became anergic upon long term immunosuppression. However, no differences in effector HCV-specific T cell responses were observed between the groups without Treg cytokine blockade or in response to mitogen. The immunosuppressive effect appeared to be predominantly mediated by HCV-specific TGFβ rather than IL-10. This is consistent with our previous results with a different cohort of HCV subjects, where blocking TGFβ significantly increased IFNγ response to HCV while IL-10 blockade did not have significant effect (25
), confirming predominant involvement of TGFβ in this suppressive activity, rather than IL-10 .
T cell secretion of TGFβ in response to HCV has been described for CD4+
) and CD8+
) Tregs in subjects with CHC and an anti-inflammatory role for TGFβ during chronic HCV infection has been suggested (22
). Interestingly, in HCV-HIV co-infection, high levels of plasma TGFβ, but not CD4+
cells, is associated with low levels of liver fibrosis (33
). Here, we provide a plausible mechanistic explanation for this observation, since we studied HCV-specific TGFβ T cell production in relation to liver inflammation and fibrosis. Not only do we confirm an inverse correlation of HCV-specific TGFβ (not IL-10) with histological liver inflammation, but we also found significant inverse correlation with liver fibrosis stage and progression. Together, these findings support the hypothesis that locally HCV-specific T cell-produced TGFβ, may play a role in controlling the chronic inflammatory response, and consequently may even have an anti-fibrotic role, thereby attenuating hepatic scarring in chronic HCV infection. Intriguingly our data also suggest involvement of IL-17 as anti-fibrotic in this setting, since we found a strong inverse correlation of liver fibrosis stage with HCV-specific IL-17. Other cytokines which we found at substantial amounts in HCV-stimulated supernatants, IL-1β and IL-6, might be concurrently expressed in vivo
and influence T cell lineage commitment. Together, these observations underline complexity of the system, since IL-17 is a pro-inflammatory cytokine and TGFβ, generally assumed to be anti-inflammatory, can become pro-inflammatory in combination with other cytokines such as IL-1 and IL-6 (26
). In this context ability of TGFβ-producing Treg to readily loose Foxp3 and acquire IL-17 expression in Th17-polarizing conditions has been described (34
Because of lack of definitive surface marker(s) for TGFβ producing Treg, and therefore, currently an absence of methods allowing their depletion, direct demonstration of effect of their elimination was not possible. Since establishing pure co-cultures of the 'Treg' of interest with human HSC is not currently practical, we tested the effect of IHL supernatants in response to HCV peptides on human HSC. Interestingly, factor(s) produced in supernatants in response to HCV from slow progressors, in whom TGFβ blockade increased IHL effector IFNγ response, had an anti-fibrotic effect on human HSC and treating these supernatants with anti-TGFβ antibodies abrogated fibrolytic gene expression by HSC. Conversely, no such effect was observed with rapid progressors for whom TGFβ blockade had no such enhancing effect. Such apparently paradoxical observations for TGFβ, most often considered a profibrogenic cytokine, could be explained since TGFβ is a multifunctional cytokine that modulates its function depending on cell type producing it and other factors present with it or induced by it. In this regard, regulatory IL-10 might also be involved. In a fibrosis mouse model, for example, TGFβ gene therapy lead to appearance of cells producing IL-10 (32
). TGFβ ’gene therapy’ ameliorated fibrosis in wild type, but not IL-10 deficient mice and induced Smad4, which then binds to and activates the IL-10 promoter. In our study, we observed significant production of IL-10 by IHL in response to HCV and peripheral HCV-specific IL-10 data did not exclude its participation in suppressive activity. Although too preliminary to formally conclude, it is possible that when TGFβ is locally produced by HCV-specific Treg, it induces substantial amounts of other cytokines, including IL-10, that participate to counterbalance profibrogenic effect of TGFβ produced by other surrounding hepatic cells. Whether IL-10 and/or other factors contribute to explain the anti-fibrotic effect of Treg TGFβ will be the object of our future studies.
In conclusion, these results suggest that TGFβ produced locally
by Tregs suppresses, rather than enhances, hepatic fibrogenesis. The data also suggest that suppression is in part in concert with other regulatory factor(s) secreted by intrahepatic lymphocytes in response to HCV. Treg have been associated with HCV persistence in chronic HCV infection (13
). However, they may play a more beneficial anti-inflammatory role by locally
protecting against surrounding tissue damage. Failure to develop appropriate effector and regulatory HCV-specific T cell responses presumably serves to drive HCV-related liver fibrosis. A better understanding of opposing effects and roles of different T cell subsets could provide novel tools allowing maintenance of such a beneficial balance, suggesting novel therapeutic approaches to prevent HCV-mediated liver disease progression.