4.2 Tregs response to antiviral therapy
CHB infection is characterized by an impaired antiviral immune response. Tregs positively associated with the levels of serum HBV DNA and HBsAg in CHB patients. At present, antiviral drugs mainly include nucleoside/nucleotide analogs (NAs) and pegylated interferon (PEG-IFN) [109]. Previous studies revealed that IL-2, IFN-γ, TNF-α, and IL-4 increased in CHB patients after NA treatment, and the numbers of Tregs were reduced[110, 111]. However, in contrast to this opinion, Zhang et al. argue that Tregs (CD4 + CD25+) levels were lower in the group that responded to lamivudine treatment than in the group that did not respond. No significant change of Tregs was observed in the entecavir treatment group[112], which may be due to the difference in the specific markers selected for Tregs. Th17 cells mediate non-specific inflammation, and the Th17/Treg imbalance is associated with disease progression in patients with CHB infection[113]. Liu reported that the Th17/Treg(CD4+ CD25+CD127dim) ratio was reduced in the therapeutic response (TR) group which responded to NAs therapy, and no differences were founded between the TR group and HCs[114]. In addition, the frequency of CD25+CD4+ T cells reduced following Peg-IFN-α-2b therapy[115].
Tregs in the progression of HBV-induced liver fibrosis
The global prevalence of HBV infection in patients with liver cirrhosis (LC) was 42%[116]. Cirrhosis imposes a substantial health burden on many countries, which has increased at the global level since 1990, partly due to population growth and ageing[4].Many inflammatory factors were expressed during liver cirrhosis. Tregs suppresses the activation of T and other immune cells and thus inhibits inflammation[117]. Many studies demonstrate a very close relationship between increased Tregs and CD8+T-cell impairment and poor survival in HCC[118]. But the role of Tregs in developing liver fibrosis (LF) remains controversial.
Some studies reported that Tregs can activate HSC to promote LF. In turn, activated HSC upregulate the levels of Tregs via the PGE2/EP2 and EP4 pathway. And TGF-β produced by Tregs may also worsen fibrosis by activating HSC. However, Tregs are likely to be only a minor source of TGF-β in the liver[119, 120]. In addition, IL-10 produced by Tregs has an anti-fibrotic effect by inhibiting the activation and proliferation of HSC[121]. Instead, Deng et al. reported that Tregs had an anti-fibrosis function by improving the anti-cirrhosis activity of human amniotic mesenchymal stem cells (hAMSC) in a mouse model[117]. The different Tregs identification, species (human/mouse), and disease models might partially explain the different roles of Tregs in LF. As discussed above, Tregs and Th17 cells have a common origin and belong to CD4+ T cell subsets. They have antagonistic effects on each other during inflammation. A study revealed that the frequencies of CD4+CD25+ Tregs and Th17 cells significantly increased in mouse liver fibrosis models. Elevated Tregs are induced to antagonize the Th17 cells. And Tregs directly downregulated the pro-fibrotic features of HSC[86]. Lan et al. found that circulating Tregs were reduced in LC compared with HC, Tregs and Th17 cells were negatively correlated[113]. Addtionally, many studies thought that Treg/Th17 ratio was negatively related to the severity of liver fibrosis[122-124]. Currently, it was limited research on the role of Tregs in HBV-LF. In the future, more studies are necessary to ascertain the exact function of Tregs, especially in the liver microenvironment.
Tregs in HBV-HCC
The interaction of Tregs with adaptive immune cells in HBV-HCC
HBV is the most common cause of HCC, with an estimated prevalence of 44%-55% of HCC cases worldwide[125]. Persistent HBV infection can lead to varying liver damage, eventually leading to hepatitis, fibrosis, cirrhosis, and HCC. The mechanism underlying HBV-HCC is not completely clear. As immunosuppressive cells, Tregs and their associated factors, such as metabolites and secreting cytokines, mediate the immune tolerance of the tumor microenvironment. A recent study reported that the HBV-HCC microenvironment is more immunosuppressive and exhausted than the non-viral-HCC microenvironment[126]. It is well-documented that HCC patients with a high lymphocyte infiltration level in the tumors have a lower risk of recurrence and a better prognosis[127]. The percentage of Tregs in the peripheral blood and intra-hepatic is increased in HBV-HCC. Increased Tregs may impair CD8+T cells and be associated with cancer progression and poor survival in HBV-HCC[128-130]. In addition, an elevated level of Tregs is accompanied by reduced infiltration of CD8+T cells in tumor regions compared with nontumor regions. CD8+T cells in HCC tumor tissues can differentiate into CTLs, which then produce cytokines (IFN-γ, TNF-α) and cytotoxic enzymes (perforin and granulosin B) to clear cancer cells[131]. However, the expression of granzyme A, granzyme B, and perforin was decreased significantly in tumor-infiltrating CD8+T cells[128]. The exhausted CD8+ effector T cells eventually weaken the tumor surveillance of the adaptive immune system and lead to the immune escape of tumor cells.
The proportion of Tregs in HBV-HCC microenvironment was significantly higher than that of non-virus-associated HCC, and the expression levels of inhibitory receptors PD-1 and LAG-3 were higher, showing a more potent immunosuppressive capability[126]. Qiu et al. demonstrated that Furin or TGFβ1 knockdown in Tregs promoted Teff cell proliferation and enhanced the killing activity of CTLs against HCC cells and HBV in vitro[58]. Nishida et al. reported that CCR4+ Tregs were the principal type of Tregs in HBV-associated HCC, which were related to sorafenib resistance and HBV load titers. In addition, CCR4+Tregs can express more IL-10 and IL-35 compared with CCR4- Tregs, which enhanced the ability of suppressing CD8+ T cells[132]. CCR4+ Tregs are recruited into tumor microenvironment by binding to CCL22 and CCL17 in HBV-HCC[133]. HBV-encoded gene HBx induces substantial IL8 production through activating MEK-ERK signal. The activity of IL8 is mainly determined by binding to its receptor CXCR1 and CXCR2. The IL8-CXCR1 axis on the hepatic vascular endothelium promotes the growth and dissemination of HCC by recruiting Tregs in the liver microenvironment[134]. Tregs play a vital role in HBV-related diseases. Thus, an in‐depth investigation of factors that regulate Tregs functions is crucial for the better treatment of HBV‐HCC and CHB.
The interaction of Tregs with innate immune cells in HBV-HCC
Natural killer (NK) cells are an essential component of innate immunity in the liver, the site of HBV replication[135]. The study revealed that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through membrane-bound TGF-β (mTGF-β) and OX40/OX40L interaction in HBV-associated liver disease[136]. In a mouse model, Liu et al. found that MicroRNA-15a/16-1 prevented Kupffer cells (KCs) from overproducing CCL22 by inhibiting nuclear factor-κB, which activates the transcription of CCL22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 can impair Treg function[137]. Thus, microRNA-15a/16-1 prevents HCC by disrupting the communication between KCs and Tregs, which provides new ideas for the treatment of HCC. Under the hypoxic state.A large amount of triggering receptor expressed on myeloid cells-1 positive (TREM-1+) tumor-associated macrophages(TAM) were enriched in tumor tissues. Wu et al. reported that TREM-1+ TAMs abundant at advanced HCC indirectly impaired the killing function of CD8+ T cells and induced CD8+ T-cells apoptosis, and recruited CCR6+ Foxp3+ Tregs into tumor tissues by secreting a large number of chemokine CCL20 to promote the progression of HCC[138].
The myeloid-derived suppressor cells (MDSC) is a group of regulatory immune cell population residing in the liver. HBV can enhance the secretion of TGF-β and IL-10 by MDSC, and then induce the formation of Tregs. APCs that play a crucial role in activating T cell-mediated, antigen-specific adaptive immune responses. B7-H4 belongs to B7 family member, which can negatively regulate T cell responses[139]. Kryczek et.al showed that Tregs can induce APCs to produce high levels of IL-10 and, in turn, stimulate APC B7-H4 expression. Thus, APCs can suppress T cell activation via B7-H4 induction[140]. TIGIT was expressed on regulatory, memory and activated T cells. Poliovirus receptor (PVR, also called CD155), which is highly expressed on DCs, bound TIGIT with high affinity. TIGIT interacted with CD155 on DCs to enhance the secretion of IL-10 by DCs, which impedes CD4+ T-cell proliferation and function[141]. In a mouse model of acute HBV infection, NK cells have been shown to be critical for HBV clearance[142]. NK cell dysfunction is associated with impaired CD8+ T cell responses in CHB[143]. Ma et al. demonstrated that HBeAg induces IL-10 production in Tregs, which subsequently upregulates the expression of NKG2A on NK cells, leading to NK cell exhaustion, which in turn suppresses the anti-tumor immunity of organism and evades the monitoring of the immune system in HCC[144, 145]. (Figure.3)
Therapies targeting Tregs in HBV or HBV-HCC
Tregs play a critical role in maintaining peripheral tolerance, thus garnering substantial interest as a potential therapeutic approach for preventing autoimmunity and establishing transplantation tolerance[146]. However, in the context of HCC, immune suppression can facilitate immune escape of tumor cells. Over the past years, Tregs have been implicated in the suppression of virus-specific immune responses, thereby providing a mechanism for the persistence of HBV. Notebly, the depletion of Tregs during AHB has been shown to impede its transition to a chronic state[22]. Moreover, Tregs and associated factors play a key role in immunosuppressive tumor microenvironment of HCC, thus highlighting the potential of targeting Tregs to inhibit HCC progression. Immune checkpoint inhibitors (ICIs) are designed to target immune checkpoints that dampen immune cell activity and exhibit ant-tumor effects. Several immune checkpoints, such as TIM-3, TIGIT, or LAG-3 are expressed in Tregs, presenting promising avenues for future research in HCC treatment aimed at inhibiting Treg function [147, 148]. Non-virally associated HCC generally exhibits higher levels of IFN-γ, IL-17, Granzyme B, and TNFα, whereas virally-associated tumors have increased PD-1 expression on T cells, indicative of a more suppressive environment fostered by HBV infection [126].
Research indicated that Tregs induce immune suppression via the secretion of immunosuppressive cytokines such as IL-35, IL-10, and TGF-β. IL-35 can restrain the proliferation of HBV-specific CTL cells and IFN-γ secretion in vitro. IL-35 is associated with HBV-related hepatic complications, including CHB, cirrhosis, and HCC[59]. An elevated level of IL-10 is linked to HBV serum titers and the degree of liver inflammation[149]. A recent study showed that TGFβ1 knockdown in Tregs can inhibit HBV replication in vitro[58]. Therefore, blocking IL-10, IL-35, and TGFβ1 expression helps restore the impaired function of effector T cells. A recent study reported that CCR4+Tregs were the predominant type of Tregs recruited to HBV-HCC, associating with sorafenib resistance and HBV load titers. Targeting intratumoral CCR4+ Tregs was shown to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer[132]. Herein, severe autoimmunity could be avoided by selectively depleting intratumoral Tregs.
Recent studies demonstrated that blocking glycolysis can promote the generation of Tregs through the hypoxia-inducible factor 1α (HIF1α). The concentration of amino acids can affect the differentiation and function of Tregs. The metabolic pathways and factors influencing Tregs differentiation and function can be considered new therapeutic approaches during viral infection[150, 151]. It has been found that IL-15 were downregulated in CHB patients with type 2 diabetes mellitus. IL-15 can suppress Treg function and inhibit the expression of immune checkpoint molecules in Tregs[152]. Huang et al. showed that exosomal circGSE1 stemming from HCC cells promotes the progression of HCC by inducing Tregs expansion via regulating the miR-324-5p/TGFBR1/Smad3 axis[153]. In the future, exosomal circGSE1 may be used as a promising biomarker for immunotherapy of HCC. Other findings may open new avenues in developing therapeutic strategies to activate specific anti-HBV immunity by diminishing Tregs autophagy[96].
In conclusion, Tregs appear to play a pivotal role in the maintenance of HBV infection, HBV-related cirrhosis, and HCC. AS such, it becomes imperative to explore strategies targeting Tregs molecules and attenuating Treg activity during specific periods of HBV infection and HCC. Novel ICIs targeting molecules such as anti-TIM-3, anti-TIGIT, or anti-LAG-3 have demonstrated the ability to restore the functionality of tumor-infiltrating T cells in vitro[154] and are currently being evaluated in early-stage clinical trials.
Conclusions
Collectively, the role of Tregs in hepatitis viral infections appears to exhibit a dual nature. On one hand, Tregs mediate targeted suppression of T cells in the context of HBV infection, potentially contributing to viral persistence, while simultaneously safeguarding against excessive liver damage. However, it remains uncertain whether these divergent roles can be ascribed to the same Treg populations or if they involve distinct Treg subpopulations. Moreover, the underlying mechanisms governing these distinct mechanisms mediate these disparate functions requires further investigation. A promising avenue for enhancing HBV-related disease management involves the specific targeting of Tregs and a refined understanding of the optimal time frame for such intervention. In addition, Tregs have the ability to shape a tumor-prone immune microenvironment for HCC formation by weakening the immune surveillance function of the innate and adaptive immune systems. Consequently, strategies aimed at inhibiting Treg function and promoting an enhanced immune state within the tumor microenvironment hold considerable promise for the treatment of HCC. Exploring the modulation of Treg activity and its potential impact on HCC treatment merits further research and consideration.