Pathological functions of interleukin-22 in chronic liver inflammation and fibrosis with hepatitis B virus infection by promoting T helper 17 cell recruitment

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 criteria30, 31 and had not taken antiviral therapies or immunosuppressive drugs within 6 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.

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 messenger RNA (mRNA) expression profiling analysis or were homogenized for the isolation of LILs and/or embedded in Tissue Tek for in situ immunohistochemical (IHC) staining.33

Animal Experiments

HBV 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, mice were treated with anti-mouse CD137 agonist monoclonal antibody (Ab; clone 2A, 200 μg/dose) five times at 7-day intervals, according to our previous protocols,35 and administered with anti-IL-22 (100 μg/dose; 8E11.9; Genentech, South San Francisco, CA) or isotype control 3 days before each injection of anti-CD137. On day 5 after the final anti-CD137 injection, mice were sacrificed and 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 (Beijing, China). 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). Multiple comparisons were made among the different groups using Kruskal-Wallis' H nonparametric test. Comparisons between various individuals were performed using Mann-Whitney's U test, whereas comparisons between the same individual were performed using Wilcoxon's matched-pairs t test. Correlations between variables were evaluated using Spearman's rank-correlation test. For all tests, a two-sided value of P < 0.05 was considered significant.

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

Expression of IL-22 Signaling Pathway-Associated Genes Was Up-Regulated in Liver Tissues From CHB Patients

Liver tissues from 3 HCs and 4 CHB patients were initially used for screening differentially expressing genes using human Genome U133 Plus 2.0 Array (22,668 genes; Affymetrix, Santa Clara, CA). 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; Supporting Table 1). Four hundred and eighty-four genes were up-regulated and 631 were down-regulated in liver tissues from CHB patients, compared to 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 up-regulated in CHB patients. We initially chose liver tissues from these 4 CHB patients for mRNA screening and identified that the IL-22 pathway was significantly up-regulated in these patients. Subsequently, many other CHB and LC patients were enrolled for further studies described below.

IL-22⁺ Cells Were Accumulated in Liver Tissues From CHB and LC Patients and Were Correlated With Liver Injury and Fibrosis Severity

IHC 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-E). Few IL-22⁺ cells were observed in livers of healthy donors. By contrast, a large number of IL-22⁺ cells infiltrated livers of HBV-infected subjects, including the inflamed portal area and the lobular sinusoids (Fig. 1C,D). In addition, CHB patients with higher G and S scores had more IL-22⁺ cells in their 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 determined which types of cells produce IL-22. IHC double staining showed that IL-22 was coexpressed by CD4⁺ and CD8⁺ T cells, CD68⁺ macrophages, CD56⁺ NK/NKT cells, and gamma-delta T-cell receptor (γδTCR)⁺ T cells as well as NKp46⁺ NK cells (Fig. 2A). This observation was further confirmed using multicolor flow cytometric (FCM) analyses. In peripheral lymphocytes, IL-22 was produced mainly by CD4⁺ T cells, whereas among the LILs, IL-22 was produced not only by CD4⁺ and CD8⁺ T cells, but also by CD3⁻CD56⁺ NK, CD3⁺CD56⁺ NKT, and CD3⁺γδTCR⁺ T cells under stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro (Fig. 2B and Supporting Fig. 1A). These liver-infiltrating IL-22-expressing cell profiles were similarly distributed in the three groups of subjects, except CD3⁻CD8⁺ and CD3⁻CD56⁺ NK cells, which accounted for the greater proportion of hepatic IL-22⁺ cells in LC patients, compared to HC subjects and CHB patients (Supporting Fig. 1B).

We further found that peripheral IL-22 production by CD3 and CD4, but not CD8, T cells was increased in LC patients, compared to 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 to 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 to 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 Therefore, we examined the production of IL-22 by CD4⁺ Th1, Th22, and Th17 cells15, 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 forkhead box protein (FoxP)3-expressing regulatory T (Treg) cells or IL-4-expressing Th2 cells produced IL-22 (Fig. 3A and Supporting Fig. 2). We also confirmed that IL-17A-expressing Th17 cells were mainly characterized by high levels of CD26, CD161, CC chemokine receptor (CCR) 6, and retinoic acid-related orphan receptor-γt expression,36 which proved that IL-22 was secreted by Th17 cells (Supporting Fig. 3).

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

Blockade of IL-22 Attenuates Th17 Cell Infiltration and Liver Fibrosis in an HBV Tg Mouse Model

To further explore the function of IL-22 in chronic liver inflammation and fibrosis, we used an HBV Tg mouse model with repeated anti-CD137 Ab injection.35 HBV Tg mice were repeatedly administered anti-CD137 Ab to activate T cells and induce liver inflammation and fibrosis (Fig. 4A). In consistent with our previous study,35 repeated injection of anti-CD137 Ab induced significant liver fibrosis and inflammation (indicated by increased alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) in HBV Tg mice, whereas blockade of IL-22 reduced liver injury (Supporting Fig. 4), inflammatory cell infiltration, and Sirius Red and α-SMA staining, compared to the immunoglobulin (Ig) control group (Fig. 4B,C). This was also further confirmed by quantifying liver necroinflammation and fibrotic scores. Blockade of IL-22 ameliorated Ishak staging and grading scores (Fig. 4C).

We then determined which lymphocyte population was preferentially reduced after IL-22 blockade in the anti-CD137-treated HBV Tg 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, NK, and dendritic cells (DCs) in the mouse model (Supporting Fig. 5). Similar observation 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. Anti-CD137 treatment significantly increased hepatic and splenic CD4- and CD8-derived IL-17A and IFN-γ production (Fig. 4D,E). Blockade of IL-22 markedly reduced hepatic and splenic IL-17A⁺ Th17 cells, compared to Ig controls. These findings were also confirmed by IHC staining (Fig. 4F), showing that the number of IL-17A⁺ cells was lower in the anti-IL-22-treated group than in the 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,E).

IL-22 Preferentially Promotes Th17 Cell Migration by Stimulating HSCs to Secrete Chemokine (C-X-C Motif) Ligand 10 and Chemokine (C-C Motif) Ligand 20

To gain insight into the mechanism through which blockade of IL-22 reduced Th17 cell migration into liver of the HBV Tg model, we examined the expression of hepatic chemokines, including chemokine (C-X-C motif) ligand (CXCL) 9, 10, and 11 and chemokine (C-C motif) ligand (CCL) 2, 3, 4, 5, 19, 20, and 21, which are known to promote hepatic lymphocyte migration.37 Anti-CD137 treatment increased hepatic expression of CXCL9 and 10 and CCL2, 5, 19, and 20 (Fig. 5). Blockade of IL-22 significantly decreased expression of CXCL10 and CCL20, which are the ligands for C-X-C chemokine receptor (CXCR) 3 and CCR6, respectively, that are expressed by Th17 cells.38

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 (Supporting 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 (Supporting 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, whereas 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; whereas 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.