Background
Liver fibrosis is the wound-healing response of the liver to many causes of chronic injury, of which hepatitis B virus (HBV) infection is the most common in China [
1]. Hepatic stellate cells (HSC) have dominated studies exploring mechanisms of liver fibrosis over the last two decades [
2]. Characterizing the interaction of HSC with immune cells is a research priority, yet has been largely overlooked until recently [
3]. Moreover, chronic liver injury is associated with varying degrees of hepatic fibrosis, yet its relationship to immune cells status is unknown. In recent studies, we have established that activated HSC from patients suffering from hepatitis B can modulate the phenotype and function of monocytes and NK cells [
4‐
6]. These findings support the hypothesis that HSC from hepatitis B patients plays an important role in regulating the immune status of hepatic fibrosis.
IL17-producing CD4+ T (Th17) cells and regulatory T cells (Tregs) have been recognized as unique subsets of effector T cells that are distinct from the Th1 and Th2 subsets [
7‐
9]. Moreover, they have been implicated as potent effectors of autoimmune disorders and inflammation [
10‐
12]. Particularly, it is found that they were all involved in the pathogenesis of liver fibrosis [
13,
14]. It is argued that IL-17 was upregulated in fibrotic liver tissue, which promoted pro-inflammatory cytokine expression, neutrophil influx, liver injury, inflammation, and fibrosis [
14‐
17]. Similarly, the enrichment of Tregs in the livers of patients with advanced fibrosis was found and these Tregs might enhance fibrosis by releasing IL-8 [
18]. Both Th17 and Tregs were enriched in tumors or marginal region of HCC and increased intratumoral IL-17-producing cell or Tregs density predicted poor prognosis in HCC patients [
19‐
21]. However, the relationship between Th17 cells, Tregs and different stages of liver fibrosis remains elusive and further investigation is warranted and basically no research available so far to detect the effect of HSC on the status of Th17 cells as well as Tregs in HBV-related liver fibrosis (HBV-LF).
In previous studies, we found that HSC can secrete many kinds of cytokines and chemokines to affect the immune status of hepatic fibrosis, therefore we propose an hypothesis that HSC may also modulate the phenotype and function of Th17 cells and Tregs. To this end, we compared the levels of Th17 cells and CD4+ CD25+ Foxp3+ Tregs in patients with different stages of HBV-LF by flow cytometry and immunohistochemistry. Afterwards, we explored how HSC regulate Th17 cells and Tregs in vitro, attempting to support some available evidences for the future study of mechanisms in fibrosis persistence and hepatocellular carcinoma (HCC) pathogenesis following cirrhosis.
Discussion
Liver fibrosis mainly occurs upon chronic hepatitis virus infection and potentially leads to portal hypertension, hepatic failure and HCC [
29]. However, the relationship between the immune cells and HBV-LF has remained elusive. Here, we observed significantly higher percentages of hepatic Th17 cells and CD4+ CD25+ Foxp3+ Tregs in advanced HBV-LF. PGE2 secreted by HSC acted on its receptors EP2 and EP4 to perform this function. These data suggest a latent mechanism in which human HSC are equipped to simultaneously upregulate the levels of Th17 cells and Tregs via the PGE2/EP2 and EP4 pathway to affect the development of HBV-LF and even HCC.
Increased expression of IL-17 was detected in livers from patients with severe liver fibrosis or cirrhosis (Group 2) (Fig.
1c, d and g). Considering pro-inflammation function of IL-17 and the close relationship between cancer and inflammation [
30], the increased hepatic Th17 cells in advanced HBV-LF might effectively explain the continued inflammation and HCC pathogenesis. However, the accurate molecular mechanisms underlying LF and Th17 are still poorly defined. Although some studies have reported that IL-17 is involved in the pathogenesis of LF [
15,
31], few studies focus on the role of HBV-LF on Th17 cells. In our study, increased hepatic Th17 cells in advanced HBV-LF effectively revealed the immune status in hepatic microenvironment. So far, the role of IL-17 in tumourigenesis remains controversial. While some studies demonstrate that IL-17/Th17 cells promote tumor development by various ways [
32,
33], others revealed that IL-17 is involved in tumor surveillance in immunocompetent mice and has anti-tumor ability in mice [
34,
35]. It is necessary to ascertain whether Th17 cells predominantly skew towards pro-tumor or anti-tumor functions. Critical evaluation in appropriate mouse models of cancer using genetically altered animals lacking specific IL-17/IL-17R isoforms should address this issue.
Tregs can impair innate and adaptive immune responses by suppressing the responses of antigen presenting cells (APC), natural killer (NK) cells, NKT cells, T and B cells [
36‐
40]. While abundant data demonstrate that there is a very close relationship between increased Tregs and tumor progression, impaired anti-tumor immunity or poor survival [
21,
41,
42], the role of Tregs in the development of fibrosis remains controversial. Some studies find that Tregs can promote fibrosis. In hepatitis C, Tregs modify the interaction between NK cells and HSC in a cell-contact-dependent manner and by secreting soluble factors, resulting in the altered control of hepatic fibrogenic activity [
43] and contributing to fibrogenesis via inducing the upregulation of profibrogenic markers in pHSC [
18]. TGF-β produced by CD4+ Foxp3+ Tregs may worsen fibrosis by activating HSC. However, Tregs are likely to be only a minor source of free active TGF-β in the liver, and TGF-β bound to the membrane of Tregs only inhibits other immune cells in close proximity [
13,
44]. IL-10 produced by Tregs can inhibit collagen matrix deposition by HSC [
45], and CD4+ Foxp3+ Tregs may inhibit the effector functions of other intrahepatic T cells, thereby indirectly inhibiting HSC activation [
46]. The different Tregs identification, species (human/mouse) and disease models might partly account for the different roles of Tregs in the development of fibrosis. Limited information concerns the function of hepatic Tregs in HBV-LF. In our study, we found a significant increase hepatic CD4+ CD25+ Foxp3+ Tregs in advanced HBV-LF compared to early HBV-LF. Researches have demonstrated that CD4+ CD25+ Foxp3+ Tregs inhibited proliferation, activation, degranulation, and production of granzyme A, granzyme B, and perforin of CD8+ T cells induced by anti-CD3/CD28 antibodies [
21] and are potent suppressors of autologous tumor-specific T cell responses [
47]. Briefly, these results demonstrates a very close relationship between Tregs and liver fibrosis, which likely accounts for the development of HBV-LF and HCC following LF considering the immune suppression function of Tregs. In the future, more studies are necessary to ascertain the exact function of Tregs, especially in the liver microenvironment.
Notably, HSC activation dominates in the process of LF and cirrhosis. Most stromal cells can produce large quantities of PGE2, and our previous study reported that HSC secrete higher concentration of PGE2 than normal skin fibroblasts [
4]. In our study, we found the concentration of PGE2 in Group 2 patients increased significantly compared to Group 1 patients (Additional file
4: Figure S3). Cyclooxygenase-2(COX-2) is the rate-limiting enzyme in PGE2 synthesis. However, the role of COX-2/PGE2 in the development of LF remains ambiguous. Elevated COX-2 in fibrosis/cirrhosis suggests a potential role of COX-2/PGE2 signaling in hepatic fibrogenesis. Furthermore, many investigators have reported that COX-2/PGE2 signaling accelerates LF by promoting HSC cell line proliferation [
48] and upregulating α-SMA on HSC cell lines [
49]. However, a separate study showed that PGE2 inhibit collagen I synthesis in a HSC cell line [
50], suggesting that PGE2 may inhibit HSC activation to limit LF. Existing studies dispute the role of PGE2 in HSC cell lines and LF. Moreover, few studies focus on the relationship among PGE2, HSC (especially pHSC) and immune cells in HBV-LF. We demonstrated that PGE2 secreted by not only LX-2 but also pHSC can simultaneously augment the percentages of Th17 cells and Tregs via its receptors EP2 and EP4, which is similar with previous reports [
28,
51]. PGE2 has both pro- and anti-proliferative effects depending on cell type [
52,
53], pro- and anti-inflammatory effects depending on the context and target of its action [
54,
55], and pro- and anti-apoptotic effects depending on the maturation and activation state of T cells [
43,
48]. Our data show that PGE2 secreted by HSC act on EP2 and EP4 to increase synchronously the levels of Th17 cells and Tregs, providing novel support for the dual function of PGE2 from the perspective of inflammatory response and immune suppression and potentially accounting for the continuous LF.
Some data show that IL-17/Th17 cells and Tregs can induce HSC activation to accelerate the pathogenesis of LF [
13,
14,
31,
44,
50]. In turn, in our study, these activated HSC upregulate the levels of Th17 cells and Tregs via the PGE2/EP2 and EP4 pathway. This pathway forms a detrimental loop, leading to the deterioration of LF and the pathogenesis of HCC probably. Furthermore, the Th17/Tregs balance is important in inflammatory diseases and autoimmune diseases. In the current study, both hepatic Th17 cells and Tregs were increased in advanced HBV-LF, which is similar to existing studies in the tumor environment [
56]. This imbalance further contributes to the development of LF, cirrhosis and even HCC.
There are some limitations in our research. The isolation of “functional” Tregs in vitro is difficult because traditional Tregs isolation depends on CD25 expression, but CD25 can be expressed on activated T cells upon activation. Thus, it is difficult to study the phenotype and function of “functional” Tregs in vitro. In addition, studies targeting the intrahepatic lymphocytes are rare, especially in fibrotic liver. Therefore, the presence and action of Th17 cells and Tregs in the liver, the site of virus replication and chronic inflammation, should be analyzed specifically.
Authors’ contributions
XYL, YJS, XL and ZLG designed the research study, acquired and analyzed the data and wrote the paper; XFH, CX, JL, XL and ZLG analyzed and interpreted the data; YHH, LZ and MZ revised the paper and language wrongs. All authors read and approved the final manuscript.