Introduction
Hepatitis B virus (HBV) is a member of the Hepadnaviridae family and specifically infects hepatocytes[1], leading to the development of acute or chronic hepatitis B infections. A staggering 250 million individuals worldwide are chronic carriers of this virus[2], experiencing either asymptomatic persistence or progression to severe outcomes, such as liver cirrhosis, liver failure and hepatocellular carcinoma (HCC)[3]. Alarming global statistics report 799 000 annual deaths attributed to HBV infection, with a significant proportion of cirrhosis and liver cancer cases being HBV-associated [4] . Particularly in China, a region with high hepatitis B prevalence, over 80% of HCC cases are linked to HBV infection[5].
HBV infection constitutes a highly intricate disease that involves intricate immune mechanisms, encompassing both innate and adaptive immune responses, with particular emphasis on the latter, which plays a pivotal role in controlling acute HBV infection. In contrast, chronic HBV infection is characterized by weakened T-cell response to HBV antigens[6]. It is evident that host immune response assumes a critical function in the pathogenesis of liver inflammation, liver fibrosis, and HCC[7]. Collectively, HBV infection is accompanied by a compromised immune system function, leading to hightened viral replication and progression of liver disease, ultimately culminating in the development of HCC as a long-term complication[8].
Tregs represent a distinct subset of CD4+ T lymphocytes that serve as key guardians of immune tolerance and are essential in controlling excessive immune activation[9-11]. Tregs can be classified into two subsets based on their origin: natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs)[12-15]. While nTregs arise in the thymus iTregs are derived from naive CD4+ T lymphocytes under the influence of tolerogenic conditions and various factors, such as IL-10 and TGF-β [16]. Initially, the surface marker CD25 (IL-2 receptor α chain) was identified as a hallmark of Tregs[17]. Later, the transcription factor Foxp3 was recognized as a specific marker of Tregs in both rodents and humans[18-20]. These CD4+CD25+Foxp3+Tregs constitute approximately 5-10% of the total CD4+ helper T cells[21].The expression of Foxp3 confers functional and phenotypic distinctions between Tregs and non-Tregs[22]. Notably, ectopic expression of Foxp3 in conventional T cells (Tconvs) bestows them with Treg-like suppressive function in vivo and in vitro, indicating that the FOXP3 gene is a master regulator of Treg inhibitiory function [18, 20]. In human studies, Tregs have been identified through the use of cell surface markers CD127 and CD45RA (the naive T cell marker), in addition to CD25 and Foxp3[23-25]. Upon stimulation, naive Tregs (CD4+CD45RA+Foxp3lowCD127low cells) can differentiate into effector Tregs (CD4+CD45RFoxp3highcells), which exhibit hightened suppressive capabilities in vitro[11]. Tregs’ impairment has been implicated in various autoimmune diseases, including type 1 diabetes[26], arthritis[27],thyroiditis[28]. However, in HCC patients, both circulating and tumor infiltrating FoxP3+ Tregs were found to be enriched[29], thus exerting suppressive effects on antitumor immunity. Given the dual role of Tregs in autoimmune diseases and HBV-related HCC (HBV-HCC), this review aims to comprehensively explore the associations between Tregs and HBV-related liver diseases. Acronyms and abbreviations used throughout this work are defined in Table 1.
Nature history of HBV infection
Patients infected with HBV can develop acute hepatitis, ranging from subclinical to icteric hepatitis, and even acute liver failure, which develops in approximately 1% of patients with acute hepatitis B(AHB) and jaundice[30]. In most cases, acute HBV infection is self-limited, and the risk of chronicity in immunocompetent adults is not more than 5%. In recovery, HBV surface antigen (HBsAg) is cleared followed by undetectable HBV DNA from serum[31]. However, the risk of chronic hepatitis B(CHB) is remarkably increased in newborn (up to 90%), whose immune system is thought to be immature[32]. The clinical course of CHB can be classified into four stages, reflecting the dynamic relationship between the host immune system and viral replication. The immune-tolerant (IT) or HBeAg-positive chronic infection phase is the first phase of CHB, characterized by high HBV DNA, positive HBeAg, normal alanine aminotransferase (ALT), and minimal or no liver damage. The phase is more common and prolonged in perinatally infected subjects. Studies have revealed that events involving the initiation and promotion of HCC, such as HBV integration and clonal hepatocyte expansion, may start in this early phase of chronic infection[33]. The second phase is named “Immune active or clearance HBeAg positive phase”. During this phase, liver fibrosis can develop rapidly, with some progressing to cirrhosis or even liver failure. The third phase is characterized by the absence of HBeAg and the presence of anti-HBeAg, low serum HBV DNA levels, and normal ALT. This phase was termed “Inactive HBeAg negative phase”. The final phase is the “Reactivation phase”. The low replication phase can persist lifelong, but some patients may subsequently develop HBV DNA replication either spontaneously or triggered by active immunosuppression, with or without e-antigen seroconversion, elevated HBV DNA load, and persistent ALT levels or relapses[6, 8].
Suppressive mechanisms of Tregs
Tregs play a key role in maintaining peripheral tolerance, limiting inflammatory response and preventing autoimmune diseases. However, they also restrict beneficial responses such as anti-tumour immunity[34]. In the past few years, there has been a lot of research about Tregs. The mechanisms by which Tregs are involved in the immune response can be divided into two main categories, contact-dependent and contact-independent. The former include the following: (a) Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) is expressed on Tregs and effector CD4+ and CD8+ T cells, which is a co-inhibitory molecules. It interacts with B7 of antigen-presenting cells (APC), especially dendritic cells(DCs), with a higher affinity than CD28[35]. Its interaction with these ligands induces their trans-endocytosis which can downregulate the expression of B7 by DCs[36], and lead to the production of Indoleamine 2,3 dioxygenase (IDO), which can affect the function and activation of effector T cells (Teffs)[37-39]. A previous study showed that Treg-specific CTLA-4 deficiency is able to produce autoimmune disease[40]. (b) Just like CTLA-4, programmed death-1 (PD-1) delivers a negative signal when interacting with its ligands (PD-L1 and PD-L2), blocking T cell activation and function. Even more interestingly, it has been shown that PD-L1 ligation can induce Foxp3 expression and pTregs generation, and loss of PD-1 expression on Tregs is conducive to Tregs phenotype unstability[41, 42]. (c) CD73 and CD39 have been identified as novel immune checkpoint targets. They are responsible for hydrolyzing extracellular ATP and ADP to AMP (by CD39) and converting AMP to adenosine (by CD73), which binds to the cell surface A2A receptor of effector cells and thereby suppresses a T cell response[43-45]. There has been a study indicating that Foxp3 can upregulate CD73/CD39 in Tregs and thses two molecule are highly expressed on about 80% of Foxp3+Tregs [46, 47]. (d) Lymphocyte activation gene-3 (LAG-3) expressed on the surface of Tregs, with a high homology with CD4, binds to MHC II molecules on DCs with a higher affinity. This interaction suppresses DCs maturation and function. In addition, LAG-3 may also interfere with TCR signaling[48]. T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is highly expressed on Tregs, and it competes with CD226 for binding to common ligands CD112 and CD155 with higher affinity[49-51]. The interaction suppresses both the function of DCs and the activation of T cells. Further research demonstrated that the interaction of TIGIT with CD155 on human DCs inhibits IL-12p40 production and potentiates IL-10 secretion by regulating the phosphorylation of p38 and Erk[52]. T-cell immunoglobulin-3 (TIM-3) alongside TIGIT, LAG-3 represents the next generation of immune checkpoints in cancer immunotherapy. Studies demonstrate that Tim-3 has four different ligands : Galectin-9 (Gal-9), high-mobility group protein B1 (HMGB1), phosphatidylserine (PtdSer), and carcinoembryonic antigen cell adhesion molecule 1 (CEACAM-1)[53]. Tim-3+ Tregs are related to more immuno-suppressive activity compared to Tim-3Tregs[54]. In addition to those molecules, Tregs can secret extracellular vesicles(EVs). EVs are considered mediators of intercellular communication, containing a range of contents, such as lipids, proteins, and nucleic acids and Tregs have been proven to release immunosuppressive EVs[55]. A few undiscovered genes might be important. However, there remain many challenges about whether a few mechanisms are important or whether different mechanisms are required in different diseases. On the other hand, Tregs can secret soluble mediators that contribute to their suppressive activity, such as, IL-10, TGF-β, and IL-35, has also been evidenced[56-59]. Tregs constitutively express CD25, the α-chain of the IL-2 receptor with a higher affinity for IL-2, which is crucial for the proliferation of T cells. Thus, Tregs can cause cell death[60]. In addition, Tregs can release cytotoxic granules (perforin and granzymes), leading to apoptosis of the target cell. Other mediators also play a role in the inhibitory function of Tregs, such as neuropilin-1, Amphiregulin, and interleukin-34 (IL-34)[61].(Figure.1)
Regulatory T cells in acute HBV infection
HBV does not directly cause liver cell injury. The outcomes after infection are closely related to the host immune response. Appropriate immune response can lead to viral clearance and recovery, excess immune response can lead to liver failure and inadequate immune response will result in sustained HBV infection[62]. During the acute phase of infection, the majority (>95%) of adult patients are able to clear HBV spontaneously. Understanding the mechanisms of successful host immune responses leading to viral clearance and the mechanism of immune failure in persistent infections, is of great importance. Innate immunity is the host’s first line of defense against HBV infection. After infection, HBV employs immune evasion strategies to induce little or no innate immune responses[63]. Many studies demonstrated that adaptive immune response significantly affects HBV infection clearance during AHB, especially HBV-specific effector CD8+ T cells[64]. An early CD4 T-cell response to HBV infection may be necessary to induce the CD8 T-cell response required to clear the infection[65]. During acute resolving hepatitis B, there is a robust, coordinated adaptive immune response with virus-specific CD8+ T cells mediating clearance of infected cells, B cells secreting neutralizing antibodies against HBsAg that block further infection and CD4+ T cells supporting effective viral clearance[66]. In chimpanzees, at least 90% of the viral DNA is eliminated from the liver during a typical HBV infection by noncytolytic processes such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)[67, 68]. However, CD4+CD25+ Tregs suppress the activation, proliferation, and IFN-γ production of CD4+ and CD8+ T cells in chronic HBV infection[69, 70].
Nirupma revealed that the frequencies of peripheral blood Tregs are higher in AHB patients than CHB patients and healthy control (HC)[71]. Peng reported that the frequency of CD4+ CD25+ Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients[72]. Xu revealed that in AHB patients, circulating CD4+CD25+Tregs frequency was initially low, and with time, the profile reversed to show an increased number of circulating Tregs during the convalescent phase and restored to normal levels after resolution[73].
Characteristics of the intrahepatic specific T-cell response against HBV in AHB patients have not been studied due to the risk of complications associated with liver biopsies. Therefore, research using animal models is required. Stross et al. found that the numbers of Tregs increase in the liver rapidly after the early stage of HBV replication in a mouse model. Tregs alleviated immune mediated liver damage by attenuating the antiviral activity of effector T cells, but did not affect the development of HBV-specific CD8+T cells [74]. Kosinska et al. reported that male mice showed functionally suppressed woodchuck hepatitis virus-specific CD8+ T-cell responses in the liver and notably higher numbers of intrahepatic Tregs than females. Subsequently, they found that functional blockade of Tregs by anti-CD25 antibody during transient HBV infection contributed to increased numbers of CD8+T cells. This was related to a significantly reduction in HBV/WHV viral loads. Highly effective depletion of Tregs in DEREG mice also improved HBV-specific T-cell responses and accelerated virus clearance, although at the expense of increased liver damage[75, 76]. A similar result was found in another study. The article reported that male mice were treated with a neutralizing anti-CD25 antibody to deplete Tregs. As expected, serum HBsAg levels were significantly decreased after Tregs depletion, showing that Tregs depletion enhanced HBsAg clearance[77]. The immunomodulatory role of Tregs during acute HBV infection is a double‐edged sword. Tregs prevent severe inflammation and immunopathology but also interfere with viral clearance[78, 79].
As opposed to Tregs, Th17 cells can produce proinflammatory cytokine IL-17, and can participate in liver damage and viral clearance after HBV Infection[80]. Another study revealed that the dynamic changes in the frequencies of Th17 and Tregs and the Th17/Treg ratio may be associated with the outcome of AHB patients. Compared with the control group, the acute stage group showed significant increases in the frequencies of Th17 and Tregs. Compared with the acute stage group, the early recovery group showed a significant reduction in the frequency of Th17, a substantial increase in the frequency of Tregs, a significant decrease in the Th17/Treg ratio. Compared with the early recovery group, the full recovery group showed a slight increase in the frequency of Th17, a significant reduction in the frequency of Tregs, but which was significantly higher than that in the control group, and a slight increase in the Th17/Treg ratio, which showed no significant difference between this group and the control group. In the acute stage of AHB, HBsAg ,and HBeAg levels were positively correlated with Th17/Treg ratio [81].
Tregs in chronic hepatitis B virus infection
The role of Tregs in AHB seems different from CHB. Scientists have explored the number of Tregs in HBV-related liver disease, summarized in Table 2. Previous studies showed that circulating Tregs level was elevated and positively correlated with serum HBV load and HBsAg levels in CHB patients[56, 82, 83]. In most studies, elevated Tregs is relevant to persistent HBV infection, but the regulatory mechanism of Tregs levels remains unclear. In addition, Patients with hepatitis B envelope antigen (HBeAg)-positive CHB harbored a higher percentage of peripheral blood and intrahepatic Tregs than those with HBeAg-negative CHB[84, 85]. HBeAg has been considered to promote chronicity, this function might be partially accomplished by inducing Tregs. Compared with HC, Tregs were also accumulated in the liver tissues and peripheral blood of CHB patients and were associated with the severity of liver inflammation in CHB patients[73] [86, 87]. A recent study found that HLA-DQ+ Tregs have more robust inhibitory functions than HLA-DQ- Tregs. Reducing the HLA-DQ+Tregs can enhance the antiviral immune response to clear the virus[88]. Others did not find difference in Treg frequencies between patients with chronic and resolved HBV infection[89]. Similarly, Feng et al. found that Tregs increased significantly in the HBV group, but no relationships existed between Tregs and HBV-DNA load[90]. Discrepant findings might be due to different cell surface markers of Tregs, different stages of HBV infection, and patient heterogeneity.
The exact mechanisms of Tregs upregulation in CHB is still obscure. Recently, in vitro experiments suggested that Furin and transforming growth factor-β1 (TGFβ1) formed a positive feedback loop to activate Tregs, Furin or TGFβ1 knockdown in Tregs promoted Teff cell proliferation, stimulated IL-2 and IFN-γ secretion, and inhibited HBV replication. Furthermore, furin or TGFβ1 depletion in Tregs can enhance the killing activity of cytotoxic T lymphocytes (CTLs) against HBV1.3P-HepG2 cells[58]. A study showed that the significant source of TGF-β in the liver is hepatic stellate cells (HSCs), which are activated during chronic inflammation[91]. Therefore, persistent infection with HBV might contribute to TGF-β production from HSCs and result in the differentiation of conventional CD4+ T cells into iTregs[92]. Liu et al. reported that Inhibitors of DNA-binding 3 (Id3) are favorable for Tregs differentiation, which could further inhibit antiviral immunity to promote the chronicity of infection. Id is natural negative regulators of E proteins, which can initiate the transcription of many genes [93]. Autophagy, an intracellular process, plays a role in maintaining cell homeostasis and survival[94]. Enhanced autophagy was shown to favor Tregs expansion and function[95]. It has been shown that HMGB1 and its receptors was significantly upregulated in both peripheral blood and intra-hepatic of CHB patients and HMGB1-induced autophagy could maintain cell survival and functional stability of CHB-Tregs[96].In addition, another study showed that Galectin-9 contributes to the expansion of the Tregs through galectin-9/Tim-3 interaction[97]. Additionally, plasmacytoid dendritic cells (pDC) from HBV-infected patients induced the generation of a higher proportion of CD4+CD25+ Tregs compared with healthy patients[98]. It was recently shown that 5-Aza-2’-deoxycytidine (5-aza-CdR), the general methylation inhibitor, can induce naive T-cells differentiate toward Tregs by mediating demethylation[99]. (Figure. 2) The mechanism by which Tregs are regulated still needs to be further explored.