The immune response has a dual role during the course of HCV infection, so in this manuscript we intended to characterize the fine tuning of the interplay between the virus and the liver immune microenvironment in CHC. Both intrahepatic and peripheral immune response component were analyzed in relation to virological and histological parameters to assess its possible role in the pathogenesis. In order to know if the inflammatory process was localized, or if immunological alterations occurred at the systemic level, the results of the evaluated parameters in both compartments were integrated.
Regarding liver infection we demonstrated that most cases displayed NS3 expression, but in a very low number of hepatocytes. The scarce number of HCV+ hepatocytes observed reinforces previous findings of our group (10) and agrees with other authors that also observed low frequencies of infected hepatocytes using different labeling techniques (25, 26) (27). According to this, liver parenchymal affection seems not to be very extensive since a high proportion of the hepatocytes remains uninfected. In agreement with Liang et al, it could be hypothesized that the immune response participates in liver infection control (27). In this regard our previous findings support this assumption since pediatric patients, who showed a less preponderant hepatic immune response than adults, displayed the highest frequencies of infected hepatocytes (10). Consistent with this hypothesis, CTL and Th1 lymphocytes, central components of the antiviral immune response(9), displayed a lobular predominance and negative correlation with viral load, which could indicate a possible control of liver viral replication. However, a lack of correlation between liver CTL frequency and the number of infected hepatocytes was observed which may have two different explanations. First, considering that viral replication is a dynamic process, the detection of viral antigens indicates that the cells are infected but does not take into account how many viral particles contains each cell. Second, it could be possible that CTLs are not all virus-specific, so they could also eliminate uninfected hepatocytes, which is known as 'Bystander killing' (28). The whole CTL population might influence liver damage generation, which could be suggested by the positive correlation between CTL frequency and transaminase levels. Therefore, although the immune response participates in viral replication control, it seems to be also implicated in liver damage generation. In the present cohort intrahepatic inflammatory cytokines, especially IL-1β, IL-8 and TGF-β, presented a profile tending to higher values in cases with most severe hepatitis, together with positive correlations of TGF-β, TNF-α, IL-1β and IL-8 with transaminases levels denoting the idea of the involvement of the immune response in liver damage. Furthermore, the total portal lymphocyte count, which accounts for the magnitude of the infiltrates, correlated with the inflammatory activity, which in turn showed significantly higher values in cases with advanced fibrosis. Consequently, these observations all together support the idea that the immune response is involved in generating liver damage and particularly the inflammation would contribute to liver fibrogenesis.
Regarding the role of different studied lymphocyte populations in relation to liver damage, only Th17 lymphocytes seem to have a particular involvement in CHC pathogenesis. In spite of its known inflammatory potential and its participation in various autoimmune and liver pathologies (24, 29), the role of Th17 lymphocytes in CHC pathogenesis has not been yet fully clarified. In this series we described a low frequency of intrahepatic Th17 lymphocytes, compared to the other Th subpopulations evaluated; however, portal Th17 lymphocytes were associated with biochemical and histological parameters of liver damage, namely plasma AST levels and increased severity of fibrosis. Additionally, intrahepatic Th17 lymphocyte frequency showed a direct correlation with IL-8 liver expression and both of them displayed an association with fibrosis severity, reinforcing their involvement in liver fibrogenesis. In this sense, it has been described that the main function of both IL-17A and IL-17F is the recruitment of neutrophils and macrophages (24). It has been demonstrated in vitro that these cytokines are capable of inducing the secretion of chemokines such as CCL7 and CXCL8 (IL-8) (24). In turn, IL-8 has been widely related to various liver pathologies, and it is postulated that, in addition to promoting the recruitment of neutrophils, it would contribute to the activation of stellate cells, and consequently to liver fibrogenesis (30, 31). Regarding the role of Th17 cells at the peripheral compartment, they were also underrepresented, likewise IL-17 was undetectable in most studied samples as described by other authors(32, 33). Besides, their peripheral frequency did not displayed association with liver damage parameters, supporting the idea of a localized contribution to the pathogenesis.
As previously mentioned, Th17 lymphocytes represents the Treg counterpart since they share differentiation process mediators and these Th subsets could be also repolarized (18). In the studied cohort, TGF−β, IL−21, IL−6 and IL−1β combined hepatic expression favors a Th17 scenario, although the individual role of each cytokine as well as their effect on the Treg lymphocytes remain to be elucidated. This is important since the hepatic cytokine milieu could generate a bias towards a certain lymphocyte profile, which in turn favors or controls liver damage, hence delineating the course of the disease.
In relation to Treg lymphocytes, although its role is still controversial (21, 34), their significant presence, especially at portal tracts, in CHC liver biopsies could denote their involvement in immune response modulation. The correlations between Treg with both CTL and Th1, at portal and lobular areas, could suggest that there could be an increase recruitment or differentiation towards a Treg profile induced by the inflammatory state established in the liver; and their presence could favor the control of effector lymphocytes (CTL and Th1). In this sense, the lack of association between CTL and Th1 with hepatitis or fibrosis severity could be interpreted as a consequence of Treg modulation by inhibiting Th1 and CTL actions and in turn preventing liver damage generation. Further, supporting this theory, the negative correlations between hepatic IL-10 expression and both portal Th and lobular CTL, would denote their participation in the control of inflammation (18, 20). Interestingly, this modulation is not observed in the peripheral blood compartment, reinforcing the idea of an action that occurred at the infection site but not in a generalized manner.
In a comprehensive approach of the liver microenvironment, it is important to consider the interplay between intrahepatic cytokines and the hepatic producer lymphocyte populations. First, the absence of a quantitative relationship between IFN-g or TNF-α expression levels and the CTL or Th1 frequency, could indicate that these cells may have variable activation status and consequently an uneven level of cytokines production. Furthermore, it is important to highlight that during the chronic stage of the infection, various authors have reported an exhaustion of both CTL and Th, so their functions could be compromised (35). However, it may be also possible that the observed discrepancy is based on the fact that CTL or Th1 are not the main source of IFN-g or TNF-α production in the liver. In addition, the absence of correlation between IL-10 and TGF-β liver expression and Treg frequency could be due to diverse activation status in cases with different liver damage severity, or due to the contribution of other cell types not evaluated in this study, such as macrophages and Kupffer cells (36).
At last, it is interesting to discuss the intrahepatic expression of the pleiotropic cytokine IL-21, mainly produced by Th17 and Th follicular (Thf) lymphocytes (37). Based on peripheral blood assays, it was proposed that IL-21 in CHC would be protective since it stimulates CTL activity promoting viral elimination and limiting liver damage (38, 39). However, there are no previous studies evaluating intrahepatic IL-21 in CHC. In this cohort an inverse relationship between IL-21 liver expression and frequency of infected hepatocytes was described, which would support the hypothesis of the protective role of IL-21. In turn, IL-21 showed negative correlations with most of the evaluated lymphocyte populations, namely B lymphocytes, Th, CTL, Treg and Th17 indicating that it would participate in some way limiting inflammation. Likewise, IL-10 would also participate in infection control given its inverse correlation with infected hepatocytes, although the underlying mechanism is not clear from the global analysis of our results.
Even though CHC is mainly a liver disease, it has also been described that adult patients might present extrahepatic manifestations, which could be consequence of immunological disorders. Advanced CHC could be accompanied by the presence of cryoglobulins that cause renal, dermatological, hematological and rheumatic complications (7). Interesting in the peripheral blood assays performed in this series, no differences in T and B lymphocyte counts were observed between patients and non-infected donors, which would indicate at first glance that the infection does not alter the distribution of lymphoid populations. However, the ability of the virus to infect lymphocytes (40) and the persistent antigenic stimulation might cause alterations in T lymphocytes differentiation status (41). In this sense, a decrease in both Th and CTL naïve lymphocytes as well as an increase in EM phenotype and activated lymphocytes (DR+) were described in our cohort. Yet, if it is considered that the number of peripheral HCV-specific lymphocytes is extremely low (42), this alteration in the distribution could be a consequence of a non-specific activation. Hence, in accordance with Alanio et al, one plausible explanation could be that peripheral lymphocytes would have a lower threshold for TCR activation due to HCV persistent stimulation which generates a hyperactivation of non-HCV specific naïve lymphocytes (42). These alterations would have implications in the immune response not only against HCV, but also against other pathogens or vaccines (43), and could even be related to the presence of extrahepatic manifestations associated with chronic HCV infection (42). To reinforce activated peripheral status observed, the evaluation of circulating cytokines in this series showed an altered profile compared to non-infected donors, with an increase in both pro- and anti-inflammatory cytokines that could also condition the establishment of an adequate immune response, even against other stimuli. Although peripheral immune response seems to be altered, neither lymphocyte populations’ frequency nor cytokines levels displayed association with liver damage parameters. The only exception was the association of TGF-β with less severe fibrosis stages in CHC patients, which was previously described by our group (44). TGF-β exerts fibrogenic effects on stellate cells as well as modulatory effects on the immune response. Since fibrogenesis is considered a long process and fibrosis the final picture, a higher level of TGF-ß in lower liver fibrosis stages may reflect fibrogenesis rather than fibrosis (45, 46).
To deepen in the peripheral CTL role, given that many authors have described a dysfunctional IFN-g production in HCV-specific CTLs (47-49), IFN-g secretion activity was explored, but no differences between HCV patients and non-infected donors arose and no relation with liver damage severity was evidenced, perhaps due to the assessment of the total CTL instead of the HCV-specific CTL IFN-g secretion. Considering CTL degranulation activity, it did not seem to be impaired in HCV patients, moreover, the response (as the delta) was even greater in patients. In addition, this difference was accentuated in those cases with more severe fibrosis. Therefore, the inflammatory context may predispose CTLs to trigger an exaggerated degranulation activity against stimuli. The above results would indicate that CTL functionality was not impaired in HCV infection.
Concerning peripheral NK cells, a significant decrease was observed in CHC patients’ samples, in accordance to many authors (50-52). It is proven that NK cells can be classified into two subpopulations with complementary mechanisms, the NK Bright and the NK Dim, and an imbalance between them could affect liver damage generation (53). In this study an altered balance between peripheral NK subpopulations, with an increase of NK Bright and a decrease in NK Dim was found as described by other authors (50, 51, 54). Given different functions of these two subpopulations, we evaluated whether the observed altered proportion resulted in a diminished cytotoxic activity and an augmented IFN-g production capacity or if their functions were conserved. Both total NK cells and NK subpopulations showed conserved IFN-g production capacity and degranulation activity in CHC patients according to previously described by Varchetta et al (55). Interestingly, those cases with more severe fibrosis presented lower basal degranulation percentages interpreted as fewer cells that spontaneously degranulate, but they showed higher basal levels of CD107a expression intensity perhaps as a compensatory mechanism.