Interleukin-17–producing CD4+ T cells increase with severity of liver damage in patients with chronic hepatitis B

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Interleukin-17 (IL-17)-producing CD4+ T cells (Th17)-mediated immune response has been demonstrated to play a critical role in inflammation-associated disease; however, its role in chronic hepatitis B virus (HBV) infection remains unknown. Here we characterized peripheral and intrahepatic Th17 cells and analyzed their association with liver injury in a cohort of HBV-infected patients including 66 with chronic hepatitis B (CHB), 23 with HBV-associated acute-on-chronic liver failure (ACLF), and 30 healthy subjects as controls. The frequency of circulating Th17 cells increased with disease progression from CHB (mean, 4.34%) to ACLF (mean, 5.62%) patients versus healthy controls (mean, 2.42%). Th17 cells were also found to be largely accumulated in the livers of CHB patients. The increases in circulating and intrahepatic Th17 cells positively correlated with plasma viral load, serum alanine aminotransferase levels, and histological activity index. In vitro, IL-17 can promote the activation of myeloid dendritic cells and monocytes and enhance the capacity to produce proinflammatory cytokines IL-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-23 in both CHB patients and healthy subjects. In addition, the concentration of serum Th17-associated cytokines was also increased in CHB and ACLF patients. Conclusion: Th17 cells are highly enriched in both peripheral blood and liver of CHB patients, and exhibit a potential to exacerbate liver damage during chronic HBV infection. (HEPATOLOGY 2009.)

More than 350 million people worldwide suffer from persistent infection with hepatitis B virus (HBV) and are at risk for developing liver cirrhosis and hepatocellular carcinoma.1 HBV itself is noncytopathic, but immune-mediated liver damage often occurs in patients with both acute and chronic HBV infection. Such damage has conventionally been attributed to killing of infected hepatocytes by virus-specific cytotoxic CD8+ T cells.2–4 Increasing evidence, however, suggests that non-HBV-specific inflammatory infiltration into the liver is likely responsible for hepatic pathology in patients with chronic hepatitis B (CHB).5, 6 For example, in HBV infection activated HBV-specific CD8+ T cells are often present at high levels in the livers of patients without evident liver inflammation; by contrast, nonantigen-specific lymphocytes were found to be massively infiltrated into the livers of patients with hepatic inflammation.7 An HBV transgenic mouse model further reinforced the concept that liver inflammation initiated by virus-specific CD8+ T cells is amplified by other lymphocytes.4, 8 Indeed, a large number of immune cells, including myeloid dendritic cells (mDCs), plasmacytoid dendritic cells, and FoxP3-positive regulatory T cells can be observed in the livers of mildly and severely affected CHB patients.9–12 These findings, therefore, suggest that multiple types of immune cells may actively participate in HBV-associated liver pathogenesis. Understanding which types of immune cells contribute to liver damage during chronic HBV infection is a prerequisite for discovering effective treatment strategies.

Human interleukin-17 (IL-17)-producing CD4+ T cells (Th17) comprise a newly identified proinflammatory T-cell subset. Several studies have demonstrated that several key cytokines, including IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), and IL-23 create a cytokine milieu that regulates the differentiation and expansion of human Th17 cells.13 Th17 cells can also produce a cocktail of cytokines such as IL-17A, IL-17F, IL-21, IL-22, IL-6, and TNF-α, of which IL-17A is characterized as a major effector cytokine. IL-17A can mobilize, recruit, and activate neutrophils, leading to massive tissue inflammation, and promote the progression of autoimmune disease.14 In alcoholic liver disease, activated liver-infiltrating Th17 cells are also responsible for neutrophil recruitment into the liver.15 Furthermore, serum IL-17 levels are increased and serve as a marker of the severity of acute hepatic injury.16 These studies all provide evidence linking Th17 cells with immune-mediated liver injury.

Th17 cells also play a protective role in the host's defense against some bacterial and fungal infections in mice.14 The Th17 response can be induced by virus antigens,17–23 and the virus-induced Th17 cells may regulate local antiviral immune responses by secreting inflammatory cytokines, which may in turn mediate the tissue damage in humans.22, 24 A recent study indicated that Th17 cells up-regulated antiapoptotic molecules and thus increased persistent infection by enhancing the survival of virus-infected cells, suggesting a novel pathogenic role of Th17 cells during persistent viral infection.25 These studies suggest that Th17 cells may contribute to the immunopathogenesis induced by persistent viral infection; however, the role of Th17 cells in liver damage of CHB patients remains unknown.

The present study characterized Th17 cells in CHB patients and found that the peripheral and intrahepatic Th17 population was selectively enriched and subsequently exacerbated liver damage. These findings may allow the development of rational immunotherapy for enhancing viral control, while limiting or blocking liver inflammation.

Abbreviations

ACLF, acute on chronic liver failure; ALT, alanine aminotransferase; CBA, cytometric bead array; CHB, chronic hepatitis B; HAI, histological activity index; HBcAg, hepatitis B core antigen; HBV, hepatitis B virus; HC, healthy control; IFN, interferon; IL, interleukin; mDC, myeloid dendritic cell; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; Th17, interleukin-17–producing CD4 T cells; TNF-α, tumor necrosis factor alpha.

Patients and Methods

Patients.

Blood samples were collected from 66 CHB patients and 23 HBV-associated acute-on-chronic liver failure (ACLF) who were diagnosed according to the described criteria.10–12, 15, 26 CHB patients were adults with no evidence of liver cirrhosis based on liver biopsy (n = 21), unequivocal clinical and biochemical data (n = 36), or compatible findings on imaging techniques (n = 32). Individuals with concurrent HCV, hepatitis G virus, and human immunodeficiency virus (HIV)-1 infections and autoimmune liver diseases and who met clinical or biological criteria of bacterial or fungal infection were excluded. Thirty age- and sex-matched healthy individuals were enrolled as controls. 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 these subjects are listed in Table 1.

Table 1. Clinical Characteristics of the Populations Enrolled in the Study
GroupCHBACLFHealthy Control
  1. Data are shown as median and range. ND, not determined.

Case662330
Sex (male/female)46/2017/618/12
Age (years)31 (16–48)32 (21–46)29 (16–45)
ALT (U/L)258 (44–1561)614 (120–1,700)21 (8–37)
TBIL (μmol/L)21.3 (7.1–119.2)313 (186–595)8 (4–16)
PTA90.9% (65.4%–129.2%)29.1% (20.3%–39.6%)ND
HBV DNA (copies/mL)23,000,000 (4540–74,0000,000)610,000 (1,358–43,220,000)ND
HBsAg positive66230
HBeAg positive66230
HBeAb positive000
HBcAb positive66230
HBcAb IgM positive000

Liver biopsies from 47 CHB patients undergoing diagnosis and 12 healthy liver transplant donors were collected for immunohistochemical analysis. The degree of hepatic inflammation was graded using the modified histological activity index (HAI) described by Scheuer.27

Flow Cytometric Analysis.

All antibodies were purchased from BD Biosciences (San Jose, CA) except for phycoerythrin (PE)-conjugated anti-IL-17A and fluorescein isothiocyanate (FITC)-conjugated anti-FoxP3 from eBioscience (San Diego, CA). For intracellular IL-17 staining, fresh heparinized peripheral blood (200 μL) was incubated with phorbol 12-myristate 13-acetate (PMA, 300 ng/mL, Sigma, St. Louis, MO) and ionomycin (1 μg/mL, Sigma-Aldrich) in 800 μL RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) for 6 hours. Monensin (0.4 μM, BD PharMingen) was added during the first hour of incubation. The blood was then lysed with fluorescence-activated cell sorting (FACS) lysing solution (BD PharMingen) and further permeabilized, stained with the corresponding intracellular antibody, fixed, and analyzed using FACSCalibur and FlowJo software (Tristar, San Carlos, CA) as previously described.28–30

Cell Isolation.

Peripheral blood mononuclear cells (PBMCs) were isolated and CD4+ T cells, CD11c+ DCs, and monocytes were purified by positive or negative selection using microbeads according to the manufacturer's instructions (Miltenyi Biotech, Bergisch-Gladbach, Germany). The isolated CD4+ T cells were further labeled with PE-conjugated anti-CD45RO, allophycocyanin-conjugated CD45RA, or FITC-conjugated anti-CCR7 antibodies. CD45RAhighCCR7posCD45ROneg (naive) and CD45RAnegCCR7pos/negCD45ROpos (memory) cells were sorted using FACSAria (Becton Dickinson, San Jose, CA). The purity of the mDCs, CD4+ T-cell subsets, and monocytes were each >95%. Unless otherwise stated, freshly isolated cells were incubated in complete RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 20 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 × 10−5 M 2-mercaptoethanol.

Cell Stimulation.

Isolated CD14+ monocytes and mDCs were incubated with medium in a 96-well plate with or without IL-17 (1 ng/mL or 3 ng/mL; PeproTech, Rocky Hill, NJ) for 24 hours. Then the cells were harvested for evaluating the expression of B7-H1, B7-DC, CD86, and CD83. The supernatants were collected to detect cytokine production. For measurement of antigen-specific cytokine production, PBMCs and CD4-deleted PBMCs were cultured with medium alone or with hepatitis B core antigen (HBcAg; 2 μg/mL) in 96-well plates in duplicate for 3 days. Then the cells were collected for messenger RNA (mRNA) quantification and the supernatants were collected for IL-17A detection.

RNA Extraction and Real-Time Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR).

Total RNA was extracted from sorted CD4+ T cells and HBcAg-stimulated cells using the RNeasy Mini Kit (Qiagen, Santa Clarita, CA) according to the manufacturer's instructions. The RNA was reverse-transcribed to complementary DNA (cDNA) using oligo (dT) primers at 42°C for 30 minutes and at 95°C for 5 minutes. Quantitative expressions of the RORγt and IL-17A transcripts were determined by staining with the fluorogenic dye SYBR Green using the reported primers and methods.15 GAPDH was used to normalize the samples in each PCR reaction.12 The absence of nonspecific primer-dimer products was verified by melting-curve and gel-migration analyses. Results are expressed in terms of relative mRNA quantification calculated by using the arithmetic formula 2−ΔCt.

Multiplex Cytometric Bead Assay.

A cytometric bead assay (Bender Medsystems, Copenhagen, Denmark) was employed to measure levels of IL-17, IL-23 p19, IL-1β, IL-6, IL-12 p35, interferon (IFN)-γ, IL-22, IL-8, and GRO-α of plasma and supernatants according to described protocols.30

Immunohistochemical Staining.

Paraffin-embedded, formalin-fixed liver tissue (5 μm) was incubated with anti-IL-17 (AF-317-NA, R&D Systems, Minneapolis, MN) antibody overnight at 4°C after blocking endogenous peroxidase activity with 0.3% H2O2. 3-Amino-9-ethyl-carbazole (red color) was used as the substrate followed by counterstaining with hematoxylin for single staining. Double staining was performed by using the avidin-biotin-peroxidase system with two different substrates: vector blue (blue color) for IL-17, and 3-amino-9-ethyl-carbazole for CD4. Positively stained cells were counted at high-power field (hpf, ×400) according to described protocols.10–12

Virological Assessment.

The virological assay was performed according to our described protocols.10–12 The limit of detection of the assay was 500 copies/mL.

Statistical Analysis.

All data were analyzed using SPSS software (Chicago, IL) and are summarized as means and standard deviations. Comparison between various individuals was performed using the Mann-Whitney U test. Comparison between the same individual was performed using the Wilcoxon matched-pairs T test. Correlation analysis was evaluated by the Spearman rank correlation test. For all tests, two-sided P < 0.05 was considered statistically significant.

Results

Identification of Th17 Cells in CHB Patients.

We first identified peripheral IL-17–producing cells in vitro by way of PMA/ionomycin stimulation. IL-17–producing cells were mainly comprised of CD4+ T cells; in contrast, CD8+ T cells, monocytes, natural killer (NK) cells, B cells, mDCs, and γδ T cells expressed low levels of IL-17 (Fig. 1A). Phenotypic analysis indicated that IL-17+CD4+ T cells expressed high levels of the memory marker CD45RO, but low levels of CD45RA, CD57 (a senescence marker), and Ki67 (a proliferation marker) (Fig. 1B). The expression levels of these markers by Th17 cells were seen at approximately the same levels in all enrolled subjects (data not shown).

Figure 1.

Identification of Th17 cells in CHB patients. (A) Screening IL-17–producing cells from peripheral blood in response to PMA/ionomycin stimulation in CHB patients (n = 3). The values in dotplots represent the percentage of IL-17+ cells. (B) Flow cytometry analysis of phenotypes expressed by IL-17–producing CD4+ T cells from CHB patients (n = 4). The values in the quadrants indicate the percentage of each subset among total CD4+ T cells. (C) The relative mRNA expression of IL-17 and RORγt by isolated total CD4+ T cells, CD45RA+CCR7+CD45RO naive CD4+ T cells and CD45RACCR7+/−CD45RO+ memory CD4+ T cells from CHB patients (n = 3).

RORγt is a unique marker that is restricted primarily to Th17 cells.31 We therefore measured RORγt and IL-17 mRNA expression in various subsets of memory CD4+ T cells in CHB patients and found that RORγt and IL-17 mRNA expression levels were 8-fold higher in memory CD4+ T cells than that in naive CD4+ T cells (Fig. 1C). These data further suggest that IL-17–producing CD4+ T cells can be considered Th17 cells that display memory properties.

Th17 Cells Are Enriched in the Peripheral Blood of CHB Patients.

We then determined the frequencies of Th17 cells, IFN-γ–producing CD4+ T cells (Th1), IL-4–producing CD4+ T cells (Th2), and FoxP3-positive CD4+ T cells (Tregs) in peripheral blood from healthy controls (HCs), CHB, and ACLF patients. All subjects clearly displayed all four of the CD4+ T-cell subsets (Fig. 2A). Notably, the distribution of these subsets in HBV-infected subjects differed from that of HC subjects. We found that the percentage of Th17 cells was significantly increased in CHB patients as compared to HC individuals (P < 0.01; Fig. 2B). Particularly in ACLF patients, the Th17 frequency was further increased over that in CHB patients (P < 0.01). In contrast, there was no significant difference in the frequency of Th1 or Treg between CHB patients and HC subjects, but there was a slight increase in the frequency of the Th2 subset in CHB patients versus HCs (P < 0.05; Supporting Fig. 1A). In ACLF patients the Treg frequency was increased relative to that in CHB patients or HC subjects (both P < 0.05), and no significant alteration was observed in the frequency of Th1 or Th2 cells between ACLF and CHB patients or HC subjects. In addition, we further investigated the activity of Th17 cells through measurement of IL-17 production from purified CD4+ T cells in response to plate-coated anti-CD3 and soluble anti-CD28. CD4+ T cells from CHB patients produced more IL-17 than those of HC subjects under anti-CD3 and anti-CD28 stimulation (Fig. 2C). Thus, these data indicate that Th17 cells were preferentially increased in the peripheral blood of CHB patients and simultaneously displayed increased activity.

Figure 2.

Th17 cells are preferentially increased in peripheral blood of CHB patients. (A) Representative dotplots of IL-17 and IFN-γ, IL-4 or FoxP3 coexpression in peripheral CD4+ T cells of HC subjects, CHB, and ACLF patients. The values in the quadrants indicate the percentage of each CD4+ T-cell subset. (B) Pooled data indicate the percentages of Th17 cells in HC, CHB, and ACLF groups. (C) IL-17A production by purified CD4+ T cells in response to anti-CD3 and anti-CD28 antibody stimulation in HC subjects (n = 10) and in CHB patients (n = 11). **P < 0.01. Horizontal bars represent the median values of indicated index. (D,E) The relative mRNA expression of IL-17 and RORγt, and IL-17A production by PBMCs and CD4-deleted PBMCs stimulated with medium alone or with HBcAg in CHB patients (n = 16). **P < 0.01. The bars represent means with standard deviations.

Interestingly, we found that a minority of Th17 cells secreted IFN-γ or IL-4, or simultaneously expressed FoxP3, regardless of disease status (Supporting Fig. 1B). The frequencies of these double-positive (IL-17+IL-4+, IL-17+IFN-γ+, or IL-17+FoxP3+) CD4 T subsets were also significantly increased in CHB and ACLF patients compared with HC subjects, whereas their frequencies were similar in CHB patients and ACLF patients. These data indicate that in HBV-infected patients some Th17 cells may have properties of Th1, Th2, or Treg cells. We also detected the frequency of IL-22–producing Th17 cells, which have been shown to protect against T-cell–induced hepatitis.32, 33 Surprisingly, only a few Th17 cells have the capacity to produce IL-22 in response to PMA/ionomycin stimulation in HC subjects, CHB, and ACLF patients (Supporting Fig. 2A). There were no significant differences observed in the frequencies of IL-22–producing CD4+ T cells and IL-22–producing Th17 cells among these three groups of subjects (Supporting Fig. 2B,C).

Antigen-specific Th17 cells have been described in HCV infection,23 but it is unknown whether HBV-specific Th17 cells will be present in patients with CHB. Our data indicated that PBMCs from patients with CHB expressed high levels of RORγt and IL-17 mRNA in response to HBcAg (Fig. 2D). Simultaneously, these PBMCs could also produce median amounts of IL-17A after HBcAg stimulation (Fig. 2E). These capacities of PBMCs to express RORγt and IL-17 mRNA and produce IL-17A in response to HBcAg were largely reduced after deletion of CD4+ T cells from PBMCs in patients with CHB (Fig. 2D,E). These data clearly indicated that in CHB patients there are some HBV-specific Th17 cells displaying responsiveness to HBcAg.

Increased Th17 Population Is Positively Correlated with Liver Injury in CHB Subjects.

We analyzed the correlation between Th17 frequency and plasma HBV DNA load or serum alanine aminotransferase (ALT) levels in these CHB and ACLF patients. There were some significant positive correlations between Th17 frequency and both plasma HBV DNA load (r = 0.212, P = 0.024; Fig. 3A) and serum ALT levels (r = 0.390, P < 0.001; Fig. 3B) in these HBV-infected subjects. Further analysis indicated that these positive associations occurred only in patients with CHB (Fig. 3A,B) but not in patients with ACLF. In addition, we also found that CHB patients with high HAI scores (G2-G3) (n = 12) had a greater proportion of Th17 cells than did CHB patients with low HAI scores (G0-G1) (n = 9) (Fig. 3C). These data suggest that peripheral Th17 cell frequency is closely associated with liver injury, indicated by serum ALT levels and liver HAI scores in CHB patients.

Figure 3.

Peripheral Th17 frequency is significantly correlated with liver injury. Significant correlations were found between the Th17 frequency and plasma HBV loads (A) and serum ALT levels (B) in CHB and ACLF patients. Solid line, linear growth trend; r, correlation coefficient. P-values are shown. (C) CHB patients with higher HAI scores (G2-G3, n = 9) had a higher percentage of Th17 cells in their peripheral blood compared with patients with lower HAI scores (G0-G1, n = 12). *P < 0.05. Horizontal bars represent the median percentages of Th17 frequency.

IL-17–Positive Cells Accumulate in the Livers of CHB Patients.

We also examined the distribution of IL-17+ cells in the livers of CHB patients. As shown in Fig. 4A, tonsil tissue from a healthy individual, which served as a positive control, showed obvious IL-17 staining, whereas the liver tissue from a healthy donor had few IL-17+ cells. Interestingly, more IL-17+ cells were found accumulated in the lobular and portal areas of livers in CHB patients (Fig. 4B). The liver-infiltrating IL-17+ cells were differentially distributed in CHB patients with varying G scores: more IL-17+ cells were found to be infiltrated in the livers of patients with a G4 score than those of patients with G2 and G1 scores (Fig. 4B). Using double immunostaining we confirmed that intrahepatic IL-17+ cells were primarily expressed on CD4+ T cells (Fig. 4C). Quantitative analysis of intrahepatic IL-17+ cells documented that livers from CHB patients exhibited more IL-17+ cell infiltration than did livers from HC subjects. In addition, in the lobular area of patients with a G4 score the number of IL-17+ cells per hpf was significantly more than in patients with G2-G3 scores and in HC subjects (Fig. 4D; all P < 0.01). In the portal area the number of IL-17+ cells per hpf was progressively increased in patients with various G phases (Fig. 4E; all P < 0.01 for any two G phases). These data indicate that IL-17+ cells were markedly accumulated in livers of CHB patients, and this infiltration was closely associated with inflammatory injury.

Figure 4.

In situ liver infiltration of IL-17–producing cells is correlated with liver injury in CHB patients. (A) Immunohistochemical staining for IL-17 in tonsil (positive controls; 400×) and in situ liver of healthy controls (400×). (B) Immunohistochemical staining for IL-17 in lobular area and portal area in CHB patients with various degrees of liver injury (400×). (C) Colocalization of CD4 (red, on cell membrane) and IL-17 (blue, in cell plasma) in liver of a representative CHB patient was shown with double labeling (400×). (D,E) Numbers of IL-17–positive cells in liver portal (D) and lobular (E) areas are shown in HC subjects and CHB patients with various degree of liver injury. Each dot represents one individual. *P < 0.05 and **P < 0.01. Horizontal bars represent the median Th17 numbers.

IL-17 Activates Monocytes and mDCs of CHB Patients to Produce Proinflammatory Cytokines In Vitro.

The immune consequence of the increase in peripheral and intrahepatic Th17 cells remains unknown in CHB patients. Previous studies indicate that CHB patients generally display dysfunctional innate immune responses, such as increased release of monocyte-derived proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and mDC-derived cytokines (IL-12 and IL-23).6, 8 To address whether the increase of Th17 cells is associated with these dysfunctional responses in CHB patients, we examined the expression of IL-17R (subunit A) in various cell populations. IL-17R was constitutively expressed by monocytes and mDCs in peripheral blood, but could not be observed in CD4+ T cells, CD8+ T cells, B cells, and NK cells (Fig. 5A). Further analysis indicates that mean fluorescence intensity (MFI) of IL-17R on both mDCs and monocytes was slightly down-regulated in CHB patients compared with that in healthy subjects (Fig. 5B). These data indicate that mDCs and monocytes are uniquely expressed IL-17R, but the overall expression levels seem to be decreased in CHB patients.

Figure 5.

IL-17R expression in monocytes and mDCs. (A) Screening IL-17 receptor (IL-17R)-expressing cells from PBMCs in CHB patients (n = 6). The values in dot plots represent the mean percentage of IL-17R expression. (B) Pooled data indicate the MFI values of IL-17R on monocytes (left) and mDCs (right) from both CHB patients and HC subjects. The bars represent means with standard deviations. **P < 0.01.

Next we detected the responsiveness of mDCs and monocytes to IL-17 in vitro. IL-17 could significantly up-regulate B7-H1, B7-DC, CD86, and CD83 expression on monocytes and mDCs of CHB patients in vitro (Fig. 6A). Increasing IL-17 doses (up to 3 ng/mL) significantly enhanced the expression of these markers, indicating that the effect of IL-17 was dose-dependent. Surprisingly, we found that the MFI levels of these markers were significantly decreased in CHB patients compared with HC subjects in response to IL-17 stimulation in vitro (Fig. 6B). These data indicated that IL-17 can activate both mDCs and monocytes in vitro, and this promotion seemed poorer in CHB patients than HC subjects.

Figure 6.

IL-17 in vitro induces the activation of monocytes and mDCs. (A) IL-17 in vitro up-regulated the expression of the activation markers on monocytes and mDCs. Representative histograms indicate the expression of B7-H1, B7-DC, CD86, and CD83 on isolated monocytes and mDCs from a CHB patient. The red histograms represent the staining of activation markers and the green histograms represent the isotype controls. The data represent the MFI of maturation markers. (B) Pooled data indicate the expression levels of B7-H1, B7-DC, CD86, and CD83 on monocytes and mDCs from both HC subjects and CHB patients in response to IL-17 in vitro. (C) IL-17 in vitro induced monocytes and mDCs to produce proinflammatory cytokine including IL-6, IL-1β, TNF-α, IL-23p19, and IL-12p35. The bars represent means with standard deviations. *P < 0.05 and **P < 0.01.

IL-17 can also significantly stimulate monocytes and mDCs to produce more inflammation-associated cytokines, including IL-1β, TNF-α, IL-6, IL-23p19, and IL-12p35 in a dose-dependent manner; by contrast, unstimulated monocytes and mDCs produced lower levels of these cytokines (Fig. 6C). Similar to maturation markers, IL-17 has a relatively poor capacity to stimulate mDCs and monocytes to produce these cytokines in CHB patients than that of HC subjects. These data indicate that IL-17 can activate monocytes and mDCs and induced them to produce proinflammatory cytokines, a process that is likely involved in the inflammation-mediated liver injury seen in CHB patients.

Alteration of Serum Th17-Associated Cytokines in CHB Patients.

We also detected the serum concentrations of Th17-associated cytokines such as IL-17, IL-23p19, IL-1β, IL-6, IFN-γ, IL-12p35, IL-22, IL-8, and GRO-α (Fig. 7). It was found that the serum IL-17, IL-23p19, and IL-1β concentrations were significantly higher in CHB compared to those of HC subjects, whereas other cytokine levels of CHB patients were similar to those of HC subjects. In ACLF patients nearly all of these cytokines, except IL-22 and GRO-α, were increased in the serum compared with those of HC subjects; among these cytokines, IL-17, IL-6, IFN-γ, and IL-12p35 concentrations were even higher than that seen in CHB patients. These results suggest that CHB patients had significantly altered Th17-associated cytokine profiles.

Figure 7.

The concentration of serum Th17-associated cytokines is increased in CHB patients. Cytometric bead assays were performed to quantify serum IL-17, IL-23p19, IL-1β, IL-6, IFN-γ, IL-12p35, IL-22, IL-8, and GRO-α production in HC subjects (n = 20) and CHB (n = 66) and ACLF patients (n = 13). The bars represent means and standard deviations. *P < 0.05 and **P < 0.01.

Discussion

Increasing evidence suggests that non-HBV-specific inflammatory infiltration into liver is likely responsible for the liver pathology during chronic HBV infection in humans.2–4 However, little is known about how Th17 cells operate in CHB patients. Here, we characterize Th17 cells in CHB patients, and document a significant increase in peripheral and intrahepatic Th17 cells. The increased Th17 cells may further activate mDCs and monocytes to release inflammatory cytokines, a process likely to be involved in liver injury during chronic HBV infection. These properties of Th17 cells may represent an unknown mechanism leading to the pathogenesis of HBV-induced liver disease.

We first characterized Th17 cells in a cohort of CHB patients and found that Th17 cells were mainly enriched in CD4+ T cells and displayed memory phenotypes. This finding was further supported by the observation of higher levels of IL-17 and RORγt mRNA occurring in memory CD4+ T cells relative to naive CD4+ T cells in these CHB patients. We also confirmed that both peripheral and intrahepatic Th17 cell number was relatively preferentially increased in CHB patients compared with other CD4+ T-cell subsets (including IFN-γ–producing Th1 cells and FoxP3-positive Treg cells), suggesting Th17 cells might actively participate in immune-pathogenesis of patients with CHB.

Recent studies have demonstrated that IL-17 from Th17 cells may contribute to T-cell-mediated hepatitis,34, 35 whereas another report indicated that IL-17 did not lead to T-cell hepatitis.33 The present study indicates that the preferential skew of the Th17 subset is associated with liver injury in CHB patients. There are three aspects of evidence to support this notion. First, the peripheral Th17 frequency in these patients with CHB was positively correlated with serum ALT levels, which often serves as a marker of liver injury.1 In addition, according to the liver biopsy diagnosis, patients with higher HAI scores have more Th17 subsets not only in peripheral CD4+ T cells but also in liver in situ than do patients with lower HAI scores. Third, ACLF patients also exhibited a considerably greater increase in peripheral Th17 cells than did CHB patients. This cohort of ACLF patients often presented clinically exacerbated episodes following certain precipitating events and provide a compatible control for CHB patients with mild liver damage.26 Notably, Th17 cells are significantly increased in patients with alcoholic liver disease without HBV or HCV infections.15 Our data also indicated that peripheral Th17 frequency and serum concentrations of Th17-associated cytokines such as IL-17, IL-6, and IL-1β were both significantly increased in patients with acute alcoholic liver disease (data not shown). Thus, the observed increase of Th17 cells in our CHB patients may represent an HBV nonspecific phenomenon. Taken together, these results indicate that Th17 cells are closely associated with the superimposed liver damage induced by HBV infection.

The precise mechanism of Th17 cells inducing liver damage in CHB patients remains unknown. The present study found that IL-17R was uniquely expressed on peripheral monocytes and mDCs in CHB patients. In addition, IL-17 in vitro can activate mDCs and monocytes and enhance their capacity to produce proinflammatory cytokines in a dose-dependent pattern. These proinflammatory cytokines are critical for liver damage during hepatitis B progression.2 Indeed, our previous studies indicate that multiple immune cells, including mDCs, plasmacytoid DCs, and FoxP3-positive regulatory T cells, can infiltrate the liver and actively participate in the immune-pathogenesis in mild and severe CHB patients.10–12 Thus, IL-17 can directly function on these IL-17R–expressing cells and exacerbate the inflammatory microenvironment of the liver. Notably, both mDCs and monocytes from CHB patients displayed lower levels of IL-17R expression and poorer responsiveness to IL-17 in vitro relative to those of HC subjects. This phenomenon can be explained by the negative feedback effects of high levels of IL-17 on the IL-17R–expressing cells because IL-17 can significantly down-regulate IL-17R expression on these mDCs and monocytes (Supporting Fig. 3). Future studies should investigate the factors underlying the low responsiveness of mDCs and monocytes to IL-17 stimulation in vitro in CHB patients.

Notably, a recent study indicated that hepatic stellate cells can also express IL-17R; following IL-17 stimulation in vitro they can secret IL-8 and GRO-α and subsequently recruit neutrophils into the livers of patients with alcoholic liver disease.15 Therefore, it is necessary to understand whether IL-17 protein secreted by liver-infiltrating Th17 cells of CHB patients also enhances this chemokine production by liver parenchymal cells, which further recruit immune cells into the livers of CHB patients. Furthermore, we found that peripheral Th17 cells from CHB patients have little capacity to produce IL-22, a cytokine which has been shown to protect against T-cell hepatitis.32, 33 This loss of Th17-producing IL-22 might also exacerbate liver injury in CHB patients. Future studies should investigate whether these Th17 cells are inherently defective, or whether the CHB patients are simply lacking a cofactor for IL-22 production. Taken together, these data emphasize that liver Th17 cells may reinforce the pathogenic inflammatory microenvironment in the livers of CHB patients.

Interestingly, we found in this study that HBcAg can induce IL-17 production, which is similar to the observation with HCV infection in which HCV antigens have an ability to induce virus-specific Th17 expansion.23 These preliminary data indicated that persistent virus replication such as HBV and HCV, at least in part, may be a contributing factor to Th17 expansion in these patients. In addition, a fraction of Th17 cells coexpressing IFN-γ, IL-4, and FoxP3 was increased in CHB patients as compared to healthy controls. Although few data define the role of these double-positive cells at present, it is likely that they represent a subset of interim cells differentiating from Th1, Th2, or Tregs. Indeed, recent studies have confirmed that Th1, Th2, and Treg cells have the potential to differentiate into Th17 cells under certain conditions.36 In CHB patients, the increased Th17-related cytokines such as IL-1β and IL-6 as well as IL-23 may facilitate Th17 differentiation and expansion. It will be of interest to elucidate the factors that selectively facilitate Th17 differentiation and expansion in CHB patients in the future.

In summary, our findings demonstrate, for the first time, that peripheral and intrahepatic Th17 cells are preferentially increased in CHB patients, which might activate mDCs and monocytes to release inflammatory cytokines during chronic HBV infection. Thus, Th17 cells may participate in the immunopathogenesis of chronic HBV infection.

Acknowledgements

We thank all HBV-infected individuals and healthy participants in this study.

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