The most important process for the immune control and inactivation of hepatitis B virus (HBV) infection is a robust immune response, either spontaneous or treatment induced (1, 2). However, in chronic active infection (chronic HBV hepatitis, CHB), the impaired and/or unbalanced T cell responses are unable to control viral replication but are sufficient to cause chronic liver damage. The latter is initially dependent on viral antigens expressed on hepatocytes and anti-HBV specific CD8⁺-cytotoxic T-lymphocyte (CTL) responses; afterward, the chronic liver damage is amplified by non-specific liver infiltrating cells and CD4⁺-T cell interaction pathways (1, 2).

Among the most powerful mechanisms for achieving a balanced immune response are the expression of programed death 1 (PD1) molecule by T cells (3) and the inhibition of effector T cells (Teffs) by CD4⁺-T regulatory cells (Tregs) (4). Models of viral infection have indicated that the interaction between the inhibitory receptor PD1, expressed in high levels on lymphocytes, and its ligands program cell death 1 ligand (PDL)-1 and PDL2, plays a critical role in T cell exhaustion by inducing T cell inactivation (3, 5). In CHB patients, high PD1 levels are expressed by virus-specific T cells and improvement of the T cell function has been obtained in vitro by inhibition of the PD1/PDL1 interaction (3, 5). Particularly, the PD1/PDL1 blockade increased CD8⁺ T cell proliferation, as well as the production of interferon-gamma (IFN-γ) and interleukin (IL)-2 production by intrahepatic lymphocytes, inducing variable levels of functional T cell restoration both in the liver and in peripheral blood, with a better functional improvement among intrahepatic T cells (5). Moreover, Tregs are important mediators of immune suppression and their presence prevents reactions against self by inducing regulatory signals to antigen presenting cells (APCs) and/or Teffs (6, 7). Their ablation increases the risk of autoimmunity (8) whilst, on the contrary, their signals could also affect non-autoreactive clones, leading to inhibition of antineoplastic, antimicrobial, antiparasitic, and antiviral immune responses (7, 9).

Previous studies have indicated that patients with chronic viral hepatitis display increased numbers of Tregs (both natural and inducible) in peripheral blood (10– 12) or liver (13, 14), which, in turn, exert a suppressive function against specific HBV- or hepatitis C virus (HCV)-Teffs in vitro (10– 14). Interestingly, Aoki et al. reported that the loss of natural Tregs (characterized by the constitutive expression of FOXP3 gene) induces fatal autoimmune hepatitis (AIH) in neonatal thymectomized (NTx)-PD1^−/− mice, due to migration of dysregulated follicular T helper (Tfh) cells from the spleen (15). In this context, we have recently demonstrated that the FOXP3 expression in liver is positively correlated with the intensity of liver inflammation along with a specific pattern of mRNA expression of the apoptosis mediators FAS, FASL, and TRAIL, irrespective of the cause of tissue damage (viral, toxic, autoimmunity), suggesting that might represent a bystander effect and not a causative event of chronic inflammation (16). Considering also that our findings were in line with the attractive view of Zheng and Rudensky (7) claiming that Tregs “have a vital role in preventing autoimmunity and pathology inflicted by uncontrolled immune responses to infections,” we suggested a comprehensive protective model of Tregs to prevent catastrophic pathology on apoptosis-induced inflammation (16).

The aim of this study was to explore whether the long-term antiviral treatment in patients with HBeAg-negative CHB affects the abovementioned model, investigating also another important apoptosis pathway implicated in Teffs dysfunction in liver, namely the PD1/PDL1. Thus, the expressions of FOXP3, characterizing mainly nTregs (7), as well as those of IL10 (encodes IL-10) and TGFB1 (encodes TGF-β1), characterizing type I (Tr1) and T helper type 3 (Th3) inducible Tregs (iTregs) (17), respectively, were examined at the same time with the PD1/PDL1/PDL2 pathway, in relation to the expression of major apoptosis mediators, namely TNFRSF6/FAS (encodes FAS), TNFSF6/FASL (encodes FASL), TNFA (encodes TNF-α), and TNFSF10/TRAIL (encodes tumor necrosis factor related apoptosis inducing ligand, TRAIL). Furthermore, the expression of the inflammatory cytokine IL-1β (encoded by IL1B gene) and cytokines of the immune effector T cell restoration (IL-2, encoded by IL2 gene and IFN-γ, encoded by IFNG gene), together with the expression of CD4 and CD8 were explored. Our data provide clear evidence that in CHB HBeAg-negative disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state and correlates with the intensity of liver inflammation, but not with liver T cell restoration.

Our study provides clear evidence that in the CHB HBeAg-negative disease model, the expression of FOXP3, characterizing mainly nTregs, as well as those of IL10 and TGFB1, characterizing Tr1 and Th3 iTregs (6), are down-regulated in the liver in the maintained on-treatment long-term remission state, as compared with cases histologically, biochemically, and virologically active at diagnosis, before any treatment. In addition, mRNA levels of liver FASL and PD1 (mainly expressed by CTLs, characterized also by the expression of CD8), and PDL1 (mainly attributed to infected hepatocytes and infiltrating lymphocytes) are concomitantly down-regulated in the maintained long-term remission state. However, the down-regulation of CD8, with no up-regulation of IL-2 (encoded by IL2) and IFN-γ (encoded by IFNG), is not in favor of restoration of T cell immune-responsiveness, but rather indicates reduction of CTLs and hepatocyte cytolysis when liver inflammation subsides on long-term antiviral treatment. These findings are also supported by our immunohistochemical findings (Figure 3). As mentioned above, the decrease of FOXP3 expression was not followed by a commensurate decrease of CD4 mRNA levels in human liver tissues. Obviously, this may reflect that not only Tregs are CD4⁺ but also other T cell subtypes, such as Th17 cells (6). However, a more specific analysis of T cell subpopulations by FCM was not available in our human liver tissues, and this is one of the limitations of our study. Consequently, the alterations of the frequency of CD4⁺-T cells identified in our study could not confidently be attributed to a specific T cell subpopulation.

Our data further support the notion that the PD1/PDL1 pathway (elevated levels of PD1 on T cells and increased expression of PDL1 on hepatocytes) is associated with T cell dysfunction in chronic HBV and HCV infections (3). In this context, it has been suggested that the disruption of this pathway is a logical therapeutic strategy to rescue the dysfunctional T cells, aiming to restore HBV/HCV-specific T cell responses. Fisicaro et al. (5) have also reported in short term experimental ex vivo CHB models that the functional recovery of HBV-specific T cells following PD1/PDL1 blockade was more pronounced for liver-resident T cells rather than peripheral T cells, and was characterized by CD8⁺ cell proliferation and the production of IFN-γ and IL-2 by intrahepatic lymphocytes. However, it is still uncertain whether the expression of PDL1 on hepatocytes truly contributes to the development of T cell exhaustion or if it is a homoeostatic mechanism that dampens the inflammatory reaction (3). Kassel et al. reported that the hepatic expression of PD1/PDL1 molecules links more directly with the degree of inflammation than with the underlying etiology of liver damage, concluding that the PD1 pathway may assist the liver in protecting itself from immune-mediated destruction (22). Accordingly, our findings did not support an antiviral Teffs function restoration in long-term maintained remission of chronic HBV infection, since no significant differences of the expression of CD4, IL2, and IFNG were observed. Interestingly, the abovementioned findings, considering Teffs function at remission, are in line with the findings of Nan et al. suggesting that the impaired immune responses of CHB patients are not fully restored by therapy, since no significant differences in the expression of IFN-gamma were found (23). On the other hand, the expressions of PD1 and PDL1 were significantly associated with the intensity of histological liver inflammation. Thus, we further support the conclusions of Kassel et al. suggesting that the down-regulation of PD1 and PDL1 molecules on maintained remission represents an epiphenomenon, contributing to, or resulting from, the resolution of an active liver inflammation.

Furthermore, we observed a down-regulation of the apoptosis mediators FAS and FASL in the maintained long-term remission state in CHB patients. Considering that previous studies, including ours, have demonstrated that the contribution of Fas/FasL pathway in CHB is of utmost importance, closely related to the degree of liver inflammation (16, 24), our findings further confirm the notion that it represents the most common and efficient pathway to kill virally infected cells in liver (25). On the other hand, we unexpectedly observed an inverse correlation of TRAIL expression with the intensity of liver inflammation and the disease stage (active vs. remission), since patients on maintained remission displayed an up-regulation of its mRNA levels in liver. TRAIL is a newly characterized TNF family member, triggering apoptosis in various tumor and virus-infected cells, by binding to certain death receptors, namely DR4 and DR5 (26–28). However, TRAIL can also bind to the decoy receptors DcR1, neutralizing its downstream effect, and DcR2 causing activation of NFkappaB, leading to transcription of genes known to antagonize the death-signaling pathway and/or to promote inflammation (29, 30). As a result, the increased levels of TRAIL are capable of not only inducing apoptosis but also reducing inflammation, as it has already been shown in a rabbit knee model of inflammatory arthritis (31). Considering that we have not investigated the activation cascades of TRAIL in our disease model, further studies are required in order to shed light on the precise role this protein plays in the pathogenesis and/or restoration of liver inflammation.

Likewise, we observed a significant reduction of mRNA levels of genes, which are indicative of T cell mediated immunosuppression, namely FOXP3, IL10, and TGFB1. Moreover, the expression pattern of FOXP3 was identical with those observed by PD1 and PDL1 genes, characterized by a significant positive correlation with the intensity of liver inflammation (Figure 4). Although, Foxp3⁺-Tregs seemed to protect the liver from immune damage and compromise virus control during acute experimental HBV infection (5, 32), their role in chronic viral infections, both HBV and HCV, has been shown to range from suppressing T cell responses directed against viruses to down-regulating the immune responses causing the liver damage (5). Thus, the initial expansion stage of the adaptive immune response against viruses is followed by a contraction stage, during which Tregs might play a prominent role in maintaining a delicate balance between a robust immune response to clear the infection and the immunopathological consequences of sustained immune activation and inflammation (5).

Furthermore, recent data suggest that CD4⁺CD25⁺-Tregs play an active role in CHB not only in modulating effectors of immune response to HBV, but also in influencing the disease prognosis. Several groups have reported that the frequency of Foxp3⁺-Tregs in liver is significantly increased in patients with severe CHB compared to healthy controls (1, 5, 10–14, 33, 34), while their frequency in peripheral blood is significantly correlated with serum viral load (13, 33). Interestingly, in such patients the depletion of circulating Tregs led to an increase of IFN-γ production by HBV-Ag-stimulated peripheral blood mononuclear cells (PBMC). In addition, CD4⁺CD25⁺-Tregs were capable of suppressing the proliferation of autologous PBMC mediated by HBV antigens, probably reflecting the generation of HBV-Ag-specific tissue and circulating Tregs (13). In this context, Stross et al. have recently demonstrated that Tregs significantly delayed the clearance of HBV from blood and infected hepatocytes in a mouse model of acute HBV infection, by down-regulating antiviral activity of Teffs through limiting cytokine production and cytotoxicity (32).

However, we recently observed that accumulation of Foxp3⁺-Tregs takes place in patients with chronic liver inflammation independently of the initial inducer of liver injury (toxic, autoimmunity, and viral, including HBV infection), and it is correlated with elevated expression of apoptosis mediators FAS, FASL, and TRAIL (16). As a result, we have suggested a protective role of Tregs expansion in chronic liver inflammation, in order to prevent self-tissue damage and to avoid catastrophic pathology (16). Should this be the case, the described suppression of virus-specific T cells could be considered as a bystander effect of the nTregs that have been expanded due to the persistent apoptosis-induced inflammation. In favor to our hypothesis, Peiseler et al. have recently reported the presence of normal frequencies and function of Tregs in patients with type 1 AIH; indeed, they found higher Treg frequencies in blood and liver tissue during active disease, correlated with the inflammatory activity of the liver, compared with remission (35). Moreover, Otano et al. have recently demonstrated an increase of hepatic Tregs accompanied by a significantly high expression of anti-inflammatory cytokines, such as TGF-β1 and IL-10, and immunosuppressive molecules, such as PD1/PDL1, in WHV-chronically infected woodchucks (36). Thus, similarly to chronic HBV infection, persistent WHV infection is associated with a strong immunosuppressive environment within the liver. We consider that the results presented herein, including the study of PD1/PDL1 pathway, although correlative rather than conclusive, further support the abovementioned proposed model.

As mentioned above, our CHB HBeAg-negative patients on maintained on-treatment long-term remission displayed a down-regulation of the hepatic expression of FOXP3, PD1, and PDL1 that was also correlated with a minimal intensity of liver inflammation. However, these patients did not exhibit immune restoration phenomena, as they are evident in the ex vivo human HBV infection (5, 37) and the animal models of acute (32) and chronic liver viral infections (36). Therefore, the targeting of Tregs and/or PD1/PDL1 pathway in the acute, or the early chronic HBV infection setting, should be carefully considered as a therapeutic strategy, since their depletion may trigger autoimmune phenomena or increase immune-mediated liver damage.

In conclusion, our data indicate that in the CHB HBeAg-negative disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state, as compared with cases histologically, biochemically, and virologically active at diagnosis, before any treatment. In addition, the contraction of the inhibitory pathways, as measured by the down-regulation of their liver mRNA expression in long-term remission, is possibly a mere consequence of the diminution of liver inflammation, after being hyper-expressed, in order to counterbalance excessive allo- and/or auto-reactive Teffs clones.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.