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+CD45R−Foxp3highcells), 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-3−Tregs[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.