Pathological functions of interleukin-22 in chronic liver inflammation and fibrosis with HBV infection by promoting Th17 cell recruitment

Abstract

It is well established that interleukin (IL)-22 has hepatoprotective and anti-fibrotic functions in acute liver injury models; however, its function in patients with liver fibrosis and liver cirrhosis (LC) remains obscure. In the current study, we demonstrated that expression of numerous IL-22 pathway-associated genes was significantly upregulated in hepatitis B virus (HBV)-infected liver tissues compared with normal controls through microarray analysis. In agreement with these findings, liver-infiltrating IL-22⁺ cells were largely increased in HBV-infected patients with LC compared with those without LC or healthy subjects, and were positively associated with liver fibrosis staging scores. Immunohistochemistry and flow cytometric analyses revealed that IL-22 was produced by multiple intrahepatic immune cells, and preferentially by Th17 cells in LC patients. In a HBV transgenic mouse model of T cell-mediated chronic liver inflammation/fibrosis, blockade of IL-22 attenuated hepatic expression of CXCL10 and CCL20, and subsequently reduced Th17 recruitment and liver inflammation/fibrosis progression. In vitro treatment with IL-22 stimulated hepatic stellate cells (HSCs) to secret several chemokines and subsequently promoted Th17 cell chemotaxis. Blocking CXCR3 or CCL20 reduced Th17 cell chemotaxis by IL-22-treated HSCs.

Conclusions

Our findings suggest that IL-22 plays a pathological role in exacerbating chronic liver inflammation and fibrosis by recruiting hepatic Th17 cells in HBV infected patients and HBV transgenic mice.

Introduction

Liver fibrosis and cirrhosis are the major causes of liver disease related morbidity and mortality worldwide.^1,2 Liver fibrosis, occurring as a result of chronic liver inflammation or injuries, is generally resulted from the excessive accumulation of extracellular matrix and eventually progresses to liver cirrhosis (LC). Hepatic stellate cell (HSC) activation is considered to be the key step for liver fibrogenesis through producing a large amount of α-smooth muscle actin (α-SMA) and collagens.^3,4 However, the mechanisms underlying the promotion of liver fibrosis progression remain obscure.

Emerging evidence indicates that host genetic,^5,6 virological^7,8 and immunological factors influence liver fibrotic progression. Particularly, host immune components have been considered to actively modulate HSC activation and be ultimately responsible for liver fibrogenesis.^1,3,4 It is commonly accepted that T helper (Th) 1 cytokines suppress fibrosis; whereas Th2 cytokines promote fibrosis.⁹ The importance of natural killer (NK) cells^10,11 and NKT cells^12,13 in regulating HSC activation and liver fibrosis has been demonstrated by cell depletion or inactivation in animal models. These studies suggest that liver-infiltrating lymphocytes (LILs) orchestrate highly complex immune responses, thereby regulating liver fibrogenesis. However, few studies have reported the essential effects of these important immunological events on liver fibrosis particularly in patients with chronic hepatitis B virus (HBV) infection.

Interleukin (IL)-22 recently has also been demonstrated to play a key role not only in controlling bacterial infection but also in promoting tissue repair via upregulating a variety of antiapoptotic, antioxidant and proliferative genes in various types of epithelial cells including hepatocytes.^14–17 IL-22 is produced primarily by Th17 cells,¹⁸ Th22¹⁹ and activated NK and NKT cells.²⁰ IL-22 receptors (R) including IL-22R1 and IL-10R2 is expressed exclusively on epithelial cells in various organs and ubiquitously expressed in multiple types of cells, respectively.^14,16 By binding to IL-22 receptors, IL-22 appears to control tissue responses to inflammation. Regarding liver diseases, several lines of evidence support that IL-22 plays a beneficial role in preventing acute hepatocellular damage induced by carbon tetrachloride (CCl₄), concanavalin A, Fas ligand21,22 or alcohol in animal models.²³ However, IL-22 was also shown to play a pathological role in exacerbating chronic liver inflammation and injury in a HBV transgenic mouse model.²⁴ Additionally, IL-22 was reported to promote the proliferation of liver stem/progenitor cells and induce HSC senescence in IL-22 overexpressing mouse models with chronic liver injury.^25,26 Recent studies have revealed that IL-22 expression is upregulated in the livers of patients with chronic HBV^24,27,28 or hepatitis C virus (HCV)^28,29 infection. These data suggest that IL-22 exhibits pro-inflammatory and anti-inflammatory actions under different sets of conditions of liver diseases; however, it is illusive that when and why such divergent outcomes occur. Identification of the predominant cell types secreting IL-22 and how it is being regulated are also of importance in determining outcomes of chronic HBV infection.

The present study analyzed the expression profile and functional roles of intrahepatic IL-22 in HBV-infected patients and determined the effects of IL-22 blockade on the progression of liver inflammation and fibrosis in HBV transgenic mice. The findings uncover a pathological role of IL-22 in chronic liver inflammation and fibrosis that may facilitate the rational development of immunotherapeutic strategies targeting IL-22.

Patients and Methods

Study subjects

Seventy-four chronic hepatitis B (CHB) patients and 36 LC patients were recruited for this study. All patients were diagnosed according to our previously described criteria^30,31 and had not taken antiviral therapies or immunosuppressive drugs within six months before sampling. Individuals with concurrent HCV, hepatitis D virus or human immunodeficiency virus infection, autoimmune liver disease or alcoholic liver disease were excluded. Forty-eight age- and sex-matched individuals were enrolled as healthy controls (HCs). The study protocol was approved by the ethics committee of our unit and written informed consent was obtained from each subject. The basic characteristics of the enrolled subjects are listed in Table 1.

Table 1.

Clinical characteristics of enrolled subjects

Category	Healthy control (HC)	Chronic hepatitis B (CHB)	Liver cirrhosis (LC)
Cases	48	74	36
Age (years)	29 (21–48)	31 (19–52)	48 (25–68)
Gender (M/F)	38/10	56/18	28/8
Alanine aminotransferase (IU/L)	ND	72 (6–627)	35 (8–218)
AST (IU/L)	ND	43 (14–481)	47 (15–278)
Serum albumin (g/L)	ND	39 (32–50)	33 (23–44)
Total bilirubin (μmol/L)	ND	14.4 (0.82–414)	26.7 (7.1–181)
Direct bilirubin (μmol/L)	ND	5.4 (1.6–236)	12.9 (4.0–301)
Prothrombin activity (%)	ND	92 (68–190)	66.4 (22–101)
HBeAg positive/negative	ND	51/23	18/18
Serum HBV levels (IU/ml)	ND	3.5×107 (100-4.6×109)	2.8×105 (100-1.1×108)

Liver biopsies were collected from 30 CHB and 16 LC patients, and 10 healthy liver tissue samples were obtained from donors whose livers were used for transplantation. The degree of hepatic inflammation and fibrosis was graded using the Modified histology activity index (HAI) described by Scheuer.³² In addition to the tissues used for pathological evaluation, liver biopsy specimens were employed for extraction of total RNA for hepatic mRNA expression profiling analysis, or were homogenized for the isolation of LILs and/or embedded in Tissue Tek for in situ immunohistochemical staining.³³

Animal experiments

HBV transgenic (Tg) mice C57BL/6J-TgN (Alb1 HBV) 44Bri, which express S, pre-S, and X genes under the mouse albumin promoter, were used in this study as previously reported.³⁴ To induce chronic liver inflammation and fibrosis, the mice were treated with anti-mouse CD137 agonist monoclonal antibody (clone 2A, 200 μg/dose) five times at 7-day intervals according to our previous protocols,³⁵ and administered with anti-IL-22 (8E11.9; Genentech; 100 μg/dose) or isotype control 3 days before each injection of anti-CD137. On day 5 after the final anti-CD137 injection, the mice were sacrificed and their livers were collected for pathological and immunological evaluation. All mice were maintained in the animal facility at the Institute of Biophysics, Chinese Academy of Sciences. All studies involving animals were approved by the Institutional Laboratory Animal Care and Use Committee.

Statistical analysis

All data were analyzed using SPSS 13.0 for Windows software (SPSS Inc., Chicago, IL, USA). Multiple comparisons were made among the different groups using the Kruskal–Wallis H non-parametric test. Comparisons between various individuals were performed using the Mann–Whitney U-test, while comparisons between the same individual were performed using the Wilcoxon matched pairs t-test. Correlations between variables were evaluated using the Spearman rank correlation test. For all tests, two-sided P < 0.05 was considered significant.

All other materials and methods are given in the Supplemental Information.

Results

Expression of IL-22 signaling pathway-associated genes was upregulated in liver tissues from CHB patients

Liver tissues from three healthy controls (HCs) and four CHB patients were initially used for screening differentially-expressing genes using human Genome U133 Plus 2.0 Array (22,668 genes, Affymetrix). All CHB patients had similar disease background and inflammatory grading (G) scores except for fibrotic staging (S): CHB2 and CHB3 had low S scores (G2S1 and G3S2) and CHB1 and CHB4 had high S scores (both G3S4) (Supplemental Table 1). Four hundred and eighty-four genes were upregulated and 631 genes were downregulated in liver tissues from the CHB patients compared with the HC subjects (the screening standard for differentially-expressed genes was defined as: P < 0.05, fold change > 2). Hierarchical clustering indicated that these differentially-expressed genes segregated into two main groups (HC and CHB, Fig. 1A). We then searched the GeneGo database to identify potentially relevant pathways in these differentially-expressed genes. The IL-22 signaling pathway was the second highest scoring pathway among the top 10 maps (Fig. 1B), suggesting that the IL-22 pathway was significantly upregulated in the CHB patients. We initially choose the liver tissues from these four CHB patients for mRNA screening and identified that IL-22 pathway was significantly up-regulated in these patients. Subsequently, many other CHB and LC patients were enrolled for further studies described below.

Figure 1. Upregulation of IL-22 signaling pathway was identified in HBV-associated CHB and LC patients.

(A) Heat map analysis of 1115 linkage hierarchically clustered (with self-organized mapping) gene expression data from liver tissues of CHB patients (n = 4) compared with HC subjects (n = 3). TreeView visualization demonstrated that the primary data set clustered into two main groups: HC and CHB. (B) Pathway maps represent a set of signaling and metabolic maps. Sorting was performed for statistically significant maps. Experimental data are represented as blue (downregulation) and red (upregulation) histograms. The height of the histogram corresponds to the relative expression of a particular gene/protein. (C) Representative immunohistochemical staining of IL-22 in liver from a HC subject, and CHB and LC patients. Positive cells are red (400×). (D) Quantification of IL-22⁺ cells showed that they were largely accumulated in the liver of HBV infected patients, especially LC patients. Each circle represents one individual; horizontal bars represent median values. (E) Correlation analysis of intrahepatic IL-22⁺ cell counts and liver inflammation grading (G) and fibrosis staging (S) in CHB patients with an available liver biopsy. Solid line, linear growth trend; r, correlation coefficient. P values are shown. HC, healthy control; CHB, chronic hepatitis B; LC, liver cirrhosis; hpf, high power field.

IL-22⁺ cells were accumulated in liver tissues from CHB and LC patients, and were correlated with liver injury and fibrosis severity

Immunohistochemical staining was subsequently used to investigate IL-22 protein expression in liver tissues from a larger cohort including 8 HC subjects, 30 CHB patients and 9 LC patients (Fig. 1C–1E). Few IL-22⁺ cells were observed in the liver of healthy donors. By contrast, a large number of IL-22⁺ cells infiltrated the livers of HBV-infected subjects, including the inflamed portal area and the lobular sinusoids (Fig. 1C–1D). In addition, CHB patients with higher G and S scores had more IL-22⁺ cells in the livers compared to those with lower G and S scores (Fig. 1E). Further analysis indicated that both lobular and portal hepatic IL-22⁺ cell numbers were positively associated with S score, and showed a trend of positive association with G score in these patients (Fig. 1E).

IL-22 is produced by multiple LILs

Next we determine which types of cells produce IL-22. Immunohistochemical double staining showed that IL-22 was coexpressed by CD4⁺ and CD8⁺ T cells, CD68⁺ macrophages, CD56⁺ NK/NKT cells and γδTCR⁺ T cells as well as NKp46⁺ NK cells (Fig. 2A). This observation was further confirmed using multicolor flow cytometric analyses. In peripheral lymphocytes, IL-22 was produced mainly by CD4⁺ T cells; while among the LILs, IL-22 was produced not only by CD4⁺ and CD8⁺ T cells but also by CD3⁻CD56⁺ NK cells, CD3⁺CD56⁺ NKT cells and CD3⁺γδTCR⁺ T cells under stimulation with PMA/ionomycin in vitro (Fig. 2B and Supplemental Fig. 1A). These liver-infiltrating IL-22-expressing cell profiles were similarly distributed in the three groups of subjects except CD3⁻CD8⁺ cells and CD3⁻CD56⁺ NK cells, which accounted for the greater proportion of hepatic IL-22⁺ cells in LC patients compared with HC subjects and CHB patients (Supplemental Fig. 1B).

Figure 2. Intrahepatic multiple immune cells produce IL-22.

(A) Representative immunohistochemical double staining identifies the cellular source of IL-22 in the liver of CHB patients. Arrows indicate double positive cells (400×). (B) Dot plots indicate that IL-22 is mainly produced by T cells in peripheral blood, but by multiple immune cells in the liver. Values represent percentages of indicated cell subsets expressing IL-22. (C and D). Statistical analysis of percentages of peripheral CD3, CD4 and CD8 T cells expressing IL-22 in HC, CHB and LC groups (C) and LC patients with various Child–Pugh scores (D). (E) Statistical analysis of percentages of intrahepatic CD3, CD4, CD8, NK, NKT and γδ T cells expressing IL-22 in HC, CHB and LC groups. (C, D and E) Data are shown as the mean ± standard deviation. *P < 0.05, compared with HC group; ^#P < 0.05, compared with CHB group.

We further found that peripheral IL-22 production by CD3 and CD4 but not CD8 T cells was increased in LC patients compared with CHB and HC subjects (Fig. 2C). Particularly, IL-22 production by CD4 and CD8 T cells was higher in LC patients with Child–Pugh C score compared with those with Child–Pugh A or B scores (Fig. 2D). By contrast, intrahepatic IL-22 production by γδT cells and NK cells was significantly elevated in HBV-infected patients compared with HC subjects, especially in LC patients who displayed a greater potential for IL-22 production (Fig. 2E).

Th17 cell subsets are responsible for hepatic CD4 Th cell-derived IL-22 production in LC patients

The above data show that CD4 T cell subsets are the major IL-22 producers in peripheral lymphocytes and are one of the main IL-22 producers in intrahepatic lymphocytes, which have been involved in several types of human diseases.^16,17 We therefore examined the production of IL-22 by CD4⁺ Th1, Th22 and Th17 cells,^15,18,19 and found that both peripheral and intrahepatic IFN-γ-expressing Th1 and IL-17A-expressing Th17 cells produced IL-22 in response to PMA/ionomycin stimulation, whereas few FoxP3-expressing regulatory T (Treg) cells or IL-4-expressing Th2 cells produced IL-22 (Fig. 3A and Supplemental Fig. 2). We also confirmed that IL-17A-expressing Th17 cells were mainly characterized by high levels of CD26, CD161, CCR6 and RORγt expression,³⁶ which proved that IL-22 was secreted by Th17 cells (Supplemental Fig. 3).

Figure 3. Hepatic IL-22-producing CD4 Th subsets are polarized toward Th17 cells in LC patients.

(A) Dot plots indicate various Th subset composition among peripheral and hepatic IL-22⁺ CD4 T cells in a HC subject and CHB and LC patients. Values in the quadrants represent the percentages of IL-22⁺CD4 T cells expressing IL-17A and IFN-γ. (B) Statistical analysis of percentages of peripheral and intrahepatic IL-22-producing Th1 cells (IL-22⁺IL-17A⁻IFN-γ⁺), Th17 cells (IL-22⁺IL-17A⁺IFN-γ⁻), Th22 cells (IL-22⁺IL-17A⁻IFN-γ⁻) and Thx (IL-22⁺IL-17A⁺IFN-γ⁺) cells among CD4 T cells in HC, CHB and LC groups. Data are shown as the mean ± standard deviation. (C) Statistical analysis of the composition of both peripheral and intrahepatic Th subsets among IL-22⁺ CD4 T cells in HC, CHB and LC groups. The numbers indicate the mean values of the various Th subsets. (B and C) A total of 28 healthy subjects, 51 CHB patients and 17 LC patients were used for the peripheral blood cytokine analysis, and 5 healthy subjects, 10 CHB patients and 5 LC patients were used for the liver cytokine analysis. *P < 0.05, compared with HC group; ^#P < 0.05, compared with CHB group.

We further categorized IL-22⁺ CD4 T cells into four subsets according to IFN-γ and IL-17A expression: IL-17A⁺IFN-γ⁻ Th17 cells, IL-17A⁻IFN-γ⁺ Th1 cells, IL-17A⁻IFN-γ⁻ Th22 cells and IL-17A⁺IFN-γ⁺ Thx cells (Fig. 3A). It was found that the percentages of these IL-22⁺ Th subsets within the peripheral CD4 T cells were higher in CHB and LC patients than in HC subjects; while among the liver-infiltrating CD4 cells, LC patients showed a significant increase in IL-22⁺Th17 cells and a reduction in Th22 cells compared with HC and CHB individuals (Fig. 3B). Further analysis indicated that the distribution of these Th subsets within peripheral IL-22⁺ CD4 T cells was similar among the three groups. By contrast, within the intrahepatic IL-22⁺ CD4 T cells, Th17 cell proportion was increased whereas Th22 cell proportion was reduced in CHB patients compared with HC subjects. This change was more pronounced in LC patients compared with CHB patients (Fig. 3C). These results signify that the increased hepatic IL-22 expression were mainly derived from Th17 cells in LC patients as compared with that in CHB and HC subjects.

Blockade of IL-22 attenuates Th17 cell infiltration and liver fibrosis in a HBV transgenic mouse model

To further explore the function of IL-22 in chronic liver inflammation and fibrosis, we used a HBV transgenic mouse model with repeated anti-CD137 antibody injection.³⁵ As illustrated in Fig. 4A, HBV transgenic mice were repeatedly administered anti-CD137 antibody to activate T cells and induce liver inflammation and fibrosis. In consistent with our previous study,³⁵ the repeated injection of anti-CD137 antibody induced significant liver fibrosis and inflammation (indicated by increased ALT and AST) in HBV transgenic mice; while blockade of IL-22 reduced liver injury (Supplemental Fig. 4), inflammatory cell infiltration and Sirius red and α-SMA staining compared with the Ig control group (Fig. 4B–4C). This was also further confirmed by quantifying liver necroinflammation and fibrotic scores. As illustrated in Fig. 4C, blockade of IL-22 ameliorated Ishak staging and grading scores.

Figure 4. Blockade of IL-22 reduces liver inflammation and fibrosis, and selectively reduces hepatic Th17 cell recruitment in a HBV transgenic mouse model.

(A) Schematic schedule of anti-CD137 treatments and anti-IL-22 blockade in HBV transgenic mice. (B) Representative images of liver tissue sections stained with hematoxylin and eosin, sirius red (200×) or anti-α-SMA antibody (400×). Sirius red positive and α-SMA positive cells are red. (C) Ishak grading and staging scores were quantified for mice in the Tg (n = 7), Ig control (n = 8) and anti-IL-22 blocking (n = 8) groups. The number of α-SMA positive cells and the percentage of Sirius red areas (400×) were quantified from 10 fields for each sample. (D and E) Pooled data indicate the proportions of hepatic and splenic CD4 (D) and CD8 (E) T cells expressing IL-17A, IFN-γ or both in mice in the Tg (n = 7), Ig control (n = 8) and anti-IL-22 blocking (n = 8) groups. *P < 0.05. (F) Representative images of liver tissue sections stained with anti-IL-17A (400×). IL-17A positive cells are red. Liver tissues from four mice in each group were stained.

We then determined which lymphocyte population was preferentially reduced after IL-22 blockade in the anti-CD137-treated HBV transgenic mouse model. We found that anti-CD137 treatment enhanced CD3 T cell (especially CD8 T cells) and granulocyte recruitment into the liver, but reduced infiltration of B cells, NK cells and dendritic cells (DCs) in the mouse model (Supplemental Fig. 5). Similar observation was also occurred in the spleen. Interestingly, blockade of IL-22 failed to alter the anti-CD137 treatment-induced hepatic and splenic lymphocyte proportion mentioned above.

We also analyzed the liver-infiltrating Th1 and Th17 cell subsets. As illustrated in Fig. 4D–4E, anti-CD137 treatment significantly increased hepatic and splenic CD4- and CD8-derived IL-17A and IFN-γ production. Blockade of IL-22 markedly reduced hepatic and splenic IL-17A⁺ Th17 cells compared to Ig controls. These findings were also confirmed by immunohistochemical staining in Fig. 4F showing that the number of IL-17A⁺ cells was lower in anti-IL-22-treated group than in control Ab-treated group. By contrast, the number of IFN-γ⁺ Th1 and IL-17A⁺IFN-γ⁺ cells was comparable between the three groups (Fig. 4D–4E).

IL-22 preferentially promotes Th17 cell migration by stimulating HSCs to secret CXCL10 and CCL20

To gain insight into the mechanism through which blockade of IL-22 reduced Th17 cell migration into the liver of the HBV transgenic model, we examined the expression of hepatic chemokines including CXCL9, 10 and 11 and CCL2, 3, 4, 5, 19, 20 and 21, which are known to promote hepatic lymphocyte migration.³⁷ As illustrated in Fig. 5A, anti-CD137 treatment increased hepatic expression of CXCL9 and 10 and CCL2, 5, 19 and 20. Blockade of IL-22 significantly decreased the expression of CXCL10 and CCL20, which are the ligands for CXCR3 and CCR6, respectively, that are expressed by Th17 cells.³⁸

Figure 5. IL-22 stimulates HSCs to produce chemokines and subsequently recruits Th17 cells both in vivo and in vitro.

(A) Relative expression of 10 chemokines in liver from mice in the Tg (n = 7), Ig control (n = 8) and anti-IL-22 blocking (n = 8) groups. *P < 0.05, compared with WT; ^#P < 0.05, compared with Ig control group. Data are shown as the mean ± standard deviation. (B) Relative expression of 9 chemokines in HSCs incubated with IL-22 or not. Data are shown as the mean ± standard deviation. Six independent experiments were performed. (C) IL-22 treated HSCs preferentially promote Th17 cell migration in vitro. Representative images indicate that IL-22 treated HSCs have a greater potential to recruit more lymphocytes into the lower chamber in a Transwell assay (upper). FACS dot plots indicate expression of IL-17A and IFN-γ by CD4 T cells under various conditions. Values in each quadrant indicate the percentages of IL-17A- and IFN-γ-expressing CD4 T cells. Four independent experiments were performed. (D) Pooled data indicate the chemotactic index of IL-17A-producing Th17 cells and IFN-γ-producing Th1 cells under various conditions. Chemotactic index is defined as the ratio between the percentages of Th17 cells or Th1 cells in experimental wells and the corresponding Th17 or Th1 cell percentages in medium blank wells. Four independent experiments were performed. Data are shown as the mean ± standard deviation. *P < 0.05.

It was reported that IL-22 stimulates hepatocytes to produce acute phase proteins, thereby promoting liver inflammation.³⁹ Here, we evaluated the potential of IL-22 to stimulate chemokine production by HSCs and HepG2 cells and found that IL-22 alone induced HSCs to secrete high levels of CXCL10 and CCL20 (Fig. 5B) but fail to stimulate HepG2 cells to produce CXCL10 and CCL20 (Supplemental Fig. 6). By contrast, IFN-γ mainly stimulated both HepG2 and LX-2 cells to produce high levels of CXCL9, CXCL10 and CXCL11, and IL-17A just stimulated HepG2 but not LX-2 cells to produce these chemokines (Supplemental Table 4). These data indicated that IL-22-induced CXCL10 and CCL20 by LX-2 cells possibly promote Th17 cell recruitment from blood into the liver and further position near HSCs.⁴⁰

We then performed a transwell chemotactic assay in vitro to investigate lymphocyte migration from the upper chamber into the lower chamber containing IL-22 treated HSCs. IL-22 treated HSCs had greater lymphocyte chemoattractant potential than medium-cultured HSCs, while blockade of CXCR3, CCL20 or both significantly reduced lymphocyte migration into the lower chamber (Fig. 5C, top). We further found that the CD4 T cells from both the upper and lower chambers had the potential to produce IL-17A and IFN-γ on PMA/ionomycin stimulation (Fig. 5C, bottom). Particularly, IL-22 treated HSCs had greater Th17 chemoattractant potential than medium-cultured HSCs; while blockade of CXCR3, CCL20 or both significantly reduced Th17 cell migration into the lower chamber (Fig. 5D). Taken together, these results demonstrate that IL-22 stimulates HSCs to secrete CXCL10 and CCL20, thereby promoting intrahepatic Th17 recruitment.

Discussion

IL-22 has been implicated in the regulation of different set of conditions of liver diseases.^24–29 The current study presents several key points regarding the effects of IL-22 on chronic liver inflammation and fibrosis: (i) multiple intrahepatic cells, especially Th17 cells, are main producers of IL-22 in HBV-infected LC patients; (ii) hepatic IL-22 expression is positively correlated with liver inflammation and fibrosis in LC patients; and (iii) hepatic increased IL-22 exacerbates chronic liver inflammation and fibrosis by promoting Th17 cell recruitment in vitro and in vivo of a HBV transgenic mouse model.

Although upregulation of IL-22 has been found in CHB patients in recent studies,^24,27,28 the increased extent and cellular origin of IL-22 remain obscure in chronic HBV-infected patients. The current study has demonstrated that the IL-22 signaling pathway is the second highest scoring pathway in chronic HBV-infected livers as compared with healthy livers through the human mRNA expression profile screening. Furthermore, this study clearly indicates that multiple intrahepatic immune cells, including CD4 and CD8 T cells, NK/NKT cells, γδT cells and macrophages, have the potential to generate IL-22 independent of disease status. Notably, IL-22 is also produced by Th1 cells, Th17 cells and Th22 cells but not by FoxP3⁺ Treg cells or Th2 subsets in both the liver and peripheral blood, which is consistent with previous reports.^15,18,19 As compared with our previous study,⁴¹ the present study demonstrated a more clear expression of IL-22 in Th17 population and a preferential polarization of intrahepatic IL-22-producing cells toward Th17 cells in LC patients compared with HC and CHB subjects. The different anti-IL-22 Abs and the protocols used for IL-22 intracellular staining within Th17 cells may explain the differences between our previous and current studies. Finally, although the mechanisms underlying the Th17 cell skew in LC patients remain obscure, intrahepatic inflammatory environment may be responsible for facilitating the generation of IL-22-producing cells in LC patients.⁴²

The hepatoprotective effect of IL-22 has been well documented in acute liver injury;^21,22 however, there is also evidence that IL-22 plays a detrimental role in exacerbating chronic liver injury and inflammation.^24,43 These studies indicated the protective or pathogenic role of IL-22 in liver disease remains contradictory, in part related to the models that were used, but also to liver injured conditions being examined (ie. acute vs chronic).⁴³ The clinical studies regarding the correlation between hepatic IL-22 expression and liver fibrosis in patients are also controversial. One clinical study revealed that hepatic IL-22 expression inversely correlates with inflammation grading and fibrosis staging in patients with chronic HBV infection;²⁷ whereas another study reported that elevation of systemic IL-22 levels are predictive for reduced survival in patients with advanced liver cirrhosis.⁴⁴ The current study demonstrated that intrahepatic IL-22-producing cells were positively associated with the severity of liver fibrosis in HBV-infected patients. In mouse models, IL-22 overexpression or treatment with IL-22 ameliorates liver fibrosis induced by acute CCl₄ administration or bile duct ligation,^25,45 suggesting that IL-22 acts as an anti-fibrotic cytokine. Although these two models have been widely used to study liver fibrogensis, they do not mimic the immune-mediated liver fibrotic progression of human HBV infection. We, therefore, developed a T cell-mediated liver disease mouse model in HBV-transgenic mice through repeated injection of anti-CD137 Ab, which potentially induced T-cell immune responses, and subsequently mimics human HBV-associated liver disease progression from chronic liver inflammatoty injury, fibrosis/cirrhosis to HCC.³⁵ By using this immune-mediated liver fibrosis model, we demonstrated that IL-22 appears to be a pro-fibrotic cytokine in the HBV transgenic mouse.³⁵ Thus, it is imperative to determine how IL-22 exerts its effects (either positively or negatively) in various liver diseases in future study.

The obvious question is why IL-22 has anti-fibrotic effects in CCl₄ and bile duct ligation models but pro-fibrotic functions in the HBV transgenic mouse model. HSCs, which are considered to be the primary source of ECM in liver fibrosis, were found to express high levels of IL-22R1 in the present study (Supplemental Fig. 7). Notably, we have previously demonstrated that IL-22 treatment strongly activates pSTAT3 and induces HSC senescence.^25,27 The ultimate effect of IL-22 on liver fibrosis is determined by its effect on hepatocyte protection and liver inflammatory damage. Both CCl₄ and bile duct ligation induce severe hepatocyte damage. IL-22 over-expression or treatment prevents hepatocyte damage, thereby ameliorating injury-driven liver fibrosis in these models. By contrast, repeated injection of anti-CD137 activated immune cells in HBV transgenic mice, resulting in chronic liver inflammation. Thus, endogenous IL-22 promotes liver inflammation and subsequently exacerbates liver fibrosis in anti-CD137 treated HBV transgenic mice. Indeed, IL-22 has been implicated as a pro-inflammatory cytokine in human diseases such as psoriasis, rheumatoid arthritis and Crohn’s disease.^16,17 The balance between HSC senescence and inflammation-promoting fibrosis may determine the outcome of the action of IL-22 in liver fibrosis.

This study also identified a novel mechanism by which IL-22 exacerbates liver fibrosis by enhancing intrahepatic Th17 migration, which was reported to promote liver fibrosis by targeting multiple types of cells.⁴⁵ The present findings supported the notion that blockade of IL-22 in vivo preferentially reduced intrahepatic Th17 cell infiltration and treatment of IL-22 in vitro stimulated HSCs to promote Th17 chemotaxis. Indeed, a study on airway inflammation has indicated that IL-22 has a pro-inflammatory effect in the presence of IL-17, and may be regulated by IL-17; while IL-22 appears to be protective in the absence of IL-17.⁴⁶ Thus, the increased Th17 cells in HBV-infected livers produce more IL-22, forming a positive feedback loop that ultimately promotes liver fibrosis during chronic liver injury and inflammation (Fig. 6). Thus, in combination with our previous findings that CD8 T cell-derived IFN-γ actively recruited hepatic macrophages to produce fibrosis-promoting cytokines and chemokines in the early phase of chronic liver disease,³⁵ CD4 T cell-derived IL-22 likely play important roles in promoting liver inflammation and fibrosis at late stages of chronic liver diseases by activating HSCs. A key point in future study is to understand the differential role of IL-22, IL-17 and IFN-γ in promoting liver inflammation and fibrosis at various stages of chronic liver diseases with HBV infection.

Figure 6. Role of IL-22 in promoting liver fibrosis.

In CHB patients, multiple intrahepatic immune cells, including Th17 cells, have the potential to produce IL-22 (1), which activates IL-22R1-expressing HSCs (2) and stimulates HSCs to produce high levels of chemokines such as CXCL10 and CCL20, which selectively induce Th17 cell migration into the inflammatory liver (3). Subsequently, the increased intrahepatic Th17 cell responses produce more IL-22 and simultaneously recruit more inflammatory cells into the liver. Thus, IL-22, HSCs and Th17 cells form a positive feedback loop and may promote progression of liver fibrosis through both enhancing liver inflammation in HBV infected patients. The ultimate effect of IL-22 in liver fibrosis is determined by the balance between induction of senescence in HSCs, and promotion of liver inflammation.

In summary, our findings suggest that increased hepatic IL-22 may promote liver fibrosis progression in chronically HBV infected patients, possibly through the induction of intrahepatic Th17 migration. Thus, inhibition of IL-22 would be a potential therapeutic option for the treatment of liver fibrosis/cirrhosis in patients with chronic HBV infection. A better understanding of the relative function of various IL-22-producing cells in liver disorders may shed light on the pathogenic mechanisms and help in the development of appropriate immune interventions.

Abbreviations

LC

liver cirrhosis

HSC

hepatic stellate cell

α-SMA

α-smooth muscle actin

HBV

hepatitis B virus

NK

natural killer

LIL

liver-infiltrating lymphocyte

IL

interleukin

STAT3

signal transducers and activators of transcription 3

CCl4

carbon tetrachloride

HCV

hepatitis C virus

CHB

chronic hepatitis B

HC

healthy control

FACS

fluorescence activated cell sorting

G

grading

S

staging

PMA

phorbol 12-myristate 13-acetate

FoxP

forkhead box protein

Treg

regulatory T cell

Tg

Transgenic

WT

wild-type

HAI

histology activity index

DC

dendritic cell

TNF

tumor necrosis factor

IFN

interferon

ALT

alanine aminotransferase

R

receptor

Th

T helper

PBMC

peripheral blood mononuclear cell

Ab

Antibody