The balance between intrahepatic IL-17⁺ T cells and Foxp3⁺ regulatory T cells plays an important role in HBV-related end-stage liver disease

Chronic hepatitis B (CHB) infection affects over 350 million individuals worldwide. It has become a global health problem due to its manifestation as chronic liver failure (CLF), acute on chronic liver failure (ACLF), liver cirrhosis (LC), and primary hepatocellular carcinoma (HCC)[1, 2]. Hepatitis B virus (HBV) is preferentially hepatotropic, not directly cytopathic, and elicits liver diseases of different severity [3]. HBV may also cause sustained liver tissue damage through different pathways, including perforin-mediated cytotoxicity and Fas ligand/Fas-mediated apoptosis, when antiviral immunity is not vigorous enough to clear the virus [4]. CD8 ⁺ T cells are the main effector cells for the elimination of HBV [5]. Thus, the hepatocellular injuries caused by HBV infection are predominantly immune-mediated [1, 2]. Studies investigating the frequency of intrahepatic virus-specific CD8⁺ T cells have shown that the non-virus-specific infiltrating CD8⁺T lymphocytes participate in liver damage in HBV infection [6]. The CD4/CD8 T cell ratio is 1:3.5 for the liver versus 2:1 for blood lymphocytes [7], suggesting that the intrahepatic CD4 ⁺ and CD8 ⁺ T cells may have a specialized role in the pathogenesis of liver disease through an immune response.

CD4 ⁺ T helper cells perform critical immune functions via the production of distinct cytokine profiles. Two new subsets of CD4 ⁺ T cells, IL-17⁺ T helper cells (Th17) and Foxp3⁺ regulatory T cells (Treg), have been described [8, 9]. Evidence has shown that circulating IL-17⁺ cells are largely accumulated in the livers of CHB patients and that their frequency increases with progression from CHB to ACLF [10, 11]. It has been reported that the increased levels of Treg cells in the blood and liver of CHB patients correlated with suppression of HBV antigen-specific T cell effects or responses in vitro[12]. Treg cells play a part in the control of chronic inflammatory responses and contribute to pathologic events in the liver during HBV infection. Th17 cells are implicated in host defense against a number of microorganisms [13, 14], while Treg cells display a suppressive function in immune responses and inflammatory diseases [15, 16]. A balance between Th17 and Treg cells is crucial for immune homeostasis. However, the variations in Th17 and Treg cells in the progression of HBV- related liver failure is still not clear.

Here we considered whether the frequency and ratio of intrahepatic Th17 and Treg cells were changed or disordered during the development of HBV-related ACLF. In this work, we investigated the frequency of IL-17⁺ and Foxp3⁺ T cells in the liver tissue of patients with HBV-related ACLF and CLF by immunochemistry and analyzed the possible association between the frequencies of IL-17 and Foxp3 positive cells with various clinical parameters, aiming to understand the mechanism underlying the effects of Th17/Treg cells on the development of ACLF.

IL-17 is mainly produced by a distinct subset of CD4⁺ T helper cells called Th17 cells. IL-17 may be a new marker for the severity of acute hepatic injury since levels increase in patients with severe acute hepatic injury or fulminant hepatic failure [17]. Liver infiltration of IL-17⁺ T cells has also been found to be positively associated with the grade of liver inflammation in CHB patients [11]. In fact, most experimental evidence to date suggests a role for IL-17 family members in the coordination of local tissue inflammation, mainly via the induced release of pro-inflammatory and neutrophil-mobilizing cytokines [18]. It has been found that the frequency of circulating IL-17⁺ T cells increases with disease progression from CHB to ACLF [10].

To understand whether the frequency of intrahepatic IL-17⁺ T cells increases during the course of HBV-related ACLF just as the frequency of circulating IL-17⁺ T cells does, we performed immunochemistry staining of IL-17 and CD4 in liver biopsies from 57 patients with HBV related end-stage liver diseases. The frequency of intrahepatic IL-17⁺ T cells and the IL-17⁺/CD4⁺ ratio increased significantly in HBV-related ACLF and CLF patients, and the increase in IL-17⁺ T cells positively correlated with Tbil, CHOL and MELD score. Therefore, the increase in IL-17⁺ T cells can reflect the grade of hepatic injury during the progression of liver disease. Inappropriate, excessive, or non-specific IL-17⁺ T cell effector responses may be involved in the pathogenesis of HBV-related ACLF. However, little is known about the regulatory role of IL-17⁺ T cells in HBV infection. An in vitro study by Li, J et al. has demonstrated that HBcAg can stimulate the production of IL-10, which negatively regulates HBcAg-specific Th17 cell responses in CHB patients [19]. Similar findings in a study of chronic hepatitis C virus (HCV) has also revealed that HCV-specific Th1 and Th17 cells are suppressed by HCV nonstructural protein 4 (NS4) -induced production of IL-10 and transforming growth factor (TGF)-β [20].

Th17 and Treg, which are two subsets of CD4⁺T cells, share reciprocal developmental pathways in immune responses. The generation of Treg cells is dependent on a critical differentiation factor named TGF-β [21]. An experiment in mice demonstrated that the generation of Foxp3⁺ Treg cells can be completely inhibited by IL-6 induced during inflammation, while the differentiation of pathogenic Th17 cells from naive T cells is induced by IL-6 plus TGF-β[22]. Notably, a more recent study has suggested that Th17 cells play a crucial role in the mediation of airway inflammatory responses and that antigen-specific Treg cells suppress Th17-mediated lung inflammation [23].

Immunochemistry staining of Foxp3 was used to determine the number of Treg cells infiltrating the liver tissue since Foxp3 is not only a transcription factor but also a specific marker for Treg [24]. Our result showed that the frequency of Foxp3⁺ T cells increased significantly in HBV-related ACLF and CLF patients compared to NC, but there was no significant difference between the CLF and ACLF patients. Xu et al. have found that the frequency of Foxp3⁺ Treg cells increased dramatically in the circulation and liver of 9 patients with chronic severe hepatitis B, and that the increase in Treg at the inflammatory site is associated with the chronicity and severity of liver inflammation [12]. Our finding that Foxp3⁺ T cells in the ACLF and CLF patients were similar is in contrast to that study, probably because the patients enrolled in our study suffered from severe and end-stage liver diseases. As a result, there was a small increase in Foxp3⁺ T cells but they were unable to suppress the inflammation, leading to massive hepatic necrosis and thereafter, to poor prognosis.

The IL-17⁺/Foxp3⁺ ratio in the ACLF patients was higher than that in CLF patients, which suggests that the number of IL-17⁺ T cells increased and caused immune hepatic injury in the progression of ACLF. However, although the numbers of suppressive Foxp3⁺ T cells increased a little, they did not reach the same proportion as the IL17⁺ T cells, leading to a relative shortage of suppressive factors. This suggests that the imbalance of the IL-17⁺/Foxp3⁺ ratio in the liver infiltrating lymphocytes may be the key factor for the development of fatal ACLF.

CD8⁺ T cells participate in the elimination of HBV and may cause sustained liver damage [5] while Treg cells have a negative regulatory function. Previous studies on chronic hepatitis C have found that human CD4⁺ CD25⁺ T cells can cause pronounced and sustained inhibition of CD8⁺ T cell proliferation [25, 26]. Franzese et al [27] have found that the frequencies of CD4⁺CD25⁺Treg cells showed no significant difference in patients with immunotolerant, chronic active and asymptomatic HBV infections, while the decrease in CD4⁺ CD25⁺ T cell frequency was found in patients with a flare-up of chronic hepatitis B. An in vitro study showed that depletion of the CD4⁺ CD25⁺ T cell population affected not only the expansion of HBV-specific CD8⁺ T cells but also their function [27]. This confirms the ability of circulating CD4⁺ CD25⁺ T cells to suppress antiviral immune responses mediated by CD8⁺ T cells [25, 28]. CD4⁺ CD25⁺ T cells are activated to suppress the expansion of HBV-specific CD8⁺ T cells, thus precluding HBV clearance but limiting excessive immune-mediated liver damage. This regulation is clearly non-antigen specific [27].

Our study suggests that intrahepatic IL17⁺ T cells played an important role in the development of chronic hepatitis B and that an imbalance between IL-17⁺ and Foxp3⁺ T cells in the liver might lead to progression of disease. Because the increase in Foxp3⁺ T cells in the liver-infiltrating lymphocytes of ACLF and CLF patients was not proportional with the increase in IL-17⁺ T cells, the Foxp3⁺ T cells could not negatively regulate the immune system and ultimately, this would cause liver failure. We did not study the frequencies of T cell subsets infiltrating into the liver by using more precise methods such as flow cytometry or study the proportions of these subsets in the peripheral blood simultaneously or dynamically. Thus, we do not know the exact mechanism underlying the effects of the Treg cells on the Th17 cells and CD8⁺ T cells. The function of IL-17⁺ T cells and Foxp3⁺ T cells in the progression of HBV-related ACLF should be further explored.