- Research Article
- Open Access
Interleukin-17F expression is elevated in hepatitis C patients with fibrosis and hepatocellular carcinoma
- Ming-Sian Wu†1,
- Chun-Hsiang Wang†2,
- Fan-Chen Tseng1,
- Hsuan-Ju Yang2,
- Yin-Chiu Lo1,
- Yi-Ping Kuo1,
- De-Jiun Tsai1,
- Wan-Ting Tsai1 and
- Guann-Yi Yu1, 3Email author
© The Author(s). 2017
Received: 3 May 2017
Accepted: 19 July 2017
Published: 26 July 2017
The role of interleukin (IL) 17A in chronic liver diseases had been extensively studied, but the function of IL-17F, which shares a high degree of homology with IL-17A, in the progression of chronic hepatic diseases is poorly understood. The aim of the study was to evaluate the association between IL-17F and liver diseases including, fibrosis and hepatocellular carcinoma (HCC).
Hepatic tumor samples from both hepatitis C virus (HCV) positive and negative patients (without HBV and HCV, NBNC) were examined with quantitative PCR and immunohistochemistry staining for inflammatory cytokine genes expression. In addition, 250 HCV patients naïve for interferon treatment were also subjected to enzyme-linked immunosorbent Assay (ELISA) for their serum cytokine concentrations.
Serum IL-17F concentrations were significantly elevated in HCV patients with severe fibrosis stages. In accordance with serum data, IL-17F expression was also found higher in HCV-associated HCC tissues compared with NBNC HCC tissues at both the mRNA and protein levels.
Our data suggest that IL-17F might be used as a valuable biological marker than IL-17A during chronic fibrosis progression and HCC development in HCV patients.
Approximately 80 million people worldwide have viremic hepatitis C virus (HCV) infection, which causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) . The full progression of end-stage liver diseases in HCV-infected patients takes about two to three decades, which provides a window for intervention. Liver fibrosis is a protective response to chronic hepatic injury that leads to accumulation of extracellular matrix proteins . Extensive fibrosis may result in cirrhosis and, in severe cases, lead to liver failure requiring liver transplantation. Severe fibrosis and cirrhosis are the outcome of continuous liver injuries, which can be caused by chronic virus infection or long-term alcohol consumption. Liver fibrosis is one of the known risk factors for HCC . Chronic inflammation caused by innate and adaptive immune responses to HCV infection is involved in the progression of HCV-associated diseases, such as liver cirrhosis and HCC [4, 5]. Identification of factors involved in HCV pathogenesis or biomarkers associated with liver diseases may provide new intervention and treatment approaches for HCV-related diseases.
T helper (Th) 17 cells are a subset of CD4+ T cells that mediate a protective role against bacterial and fungal infections [6, 7] as well as a pathological role in inflammation-associated diseases, such as autoimmune diseases and cancer [8–10]. Transforming growth factor (TGF)-β, interleukin (IL)-6, IL-21, and IL-23 are the key cytokines for Th17 cell maturation and production of IL-17 family cytokines, the secretion of which is a quintessential defining feature for Th17 cells [11–13]. The IL-17 family has six members, including IL-17A–F. IL-17A and IL-17F have high sequence homology and are expressed as homodimers or as an IL-17A + F heterodimer to induce expression proinflammatory cytokines, chemokines, antimicrobial peptides, and matrix metalloproteinases in IL-17 receptor-bearing cells .
Th17 cells play an important role in many liver diseases, such as liver fibrosis, alcoholic liver disease, chronic hepatitis B, and autoimmune liver disease [15–18]. The Th17 cell population is increased in chronic hepatitis C patients and Th17 cell abundance correlates positively with liver injury but inversely with HCV RNA load . Although IL-17A had been extensively studied in chronic liver diseases, the function of IL-17F, which shares a high degree of homology with IL-17A, in the progression of chronic hepatic diseases is poorly understood. IL-17F can be secreted by CD8+ T cells, γδ T cells, NKT cells, LTi-like cells, and epithelial cells in addition to being secreted by Th17 cells (CD4+ T cells) [8, 20]. IL-17F has a weaker receptor binding affinity than IL-17A and therefore induces less expression of proinflammatory cytokines than IL-17A . The aim of this study was to evaluate the association between the expression of IL-17F and HCV-associated diseases by the evaluation of Th17-related cytokine expression in HCC tissue and serum from HCV patients.
RNA samples derived from cancerous and non-cancerous parts of HCC tissue from patients infected with HCV and controls negative for both HBV and HCV (“non-B, non-C”, NBNC) were obtained from the Taiwan Liver Cancer Network (TLCN) .
Treatment naïve HCV cohort
A total of 250 patients who had serologically demonstrated HCV infection and were treated at Tainan Municipal Hospital (TMH), Tainan, Taiwan from 2003 to 2008 were included in our serum cytokine analysis. Baseline sera were collected from the patients within two months before they underwent HCV antiviral therapy. Patients infected with HBV or HIV were excluded from the study. The diagnoses of hepatic steatosis, fibrosis, and HCC of the HCV cohort were independently confirmed by two pathologists at TMH. Based on the METAVIR grading system, liver fibrosis status was classified as: no fibrosis (F0), portal fibrosis without septa (F1), with few septa (F2), with numerous septa but without cirrhosis (F3), and with cirrhosis (F4).
Serum cytokine detection
IL-6, IL-17A, IL-17F, and IL-21 concentrations in serum samples were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Human IL-6, IL-17A, and IL-17F ELISA kits were purchased from R&D Systems (MN, USA), and the IL-21 immunoassay kit was obtained from eBioscience (CA, USA). The kits’ analytic sensitivities were 9.375 pg/ mL for IL-6, 15.625 pg/mL for IL-17A, 312.5 pg/mL for IL-17F, and 8 pg/mL for IL-21.
Cytokine mRNA quantification
Primer sequences for quantitative RT-PCR
5′ CCTGGTGCTCCGTCTTAGAG 3’
5′ TTTCCCAGCAAAGATCAACC 3’
5′ AATCTCCACCGCAATGAGGA 3’
5′ ACGTTCCCATCAGCGTTGA 3’
5′ GAAGCTTGACATTGGCATCA 3’
5′ GATGCAGCCCAAGTTCCTAC 3’
Paraffin-embedded sections of the HCC cohort were subjected to immunohistochemistry (IHC) by following the protocol described previously with minor modification . In brief, paraffin-embedded sections were deparaffinized and incubated in citrate buffer (pH 6) with 0.05% Tween 20 at 95–98 °C for 20 min. After 3% H2O2 and 1% bovine serum albumin blocking, slides were incubated with the anti-IL-17F antibody (ab168194, Abcam) overnight at 4 °C, followed by incubation with a secondary antibody (EnVision+ system-HRP-labeled polymer, DakoCytomation) at room temperature for 1 h. The sections were then stained with 3,3′-diaminobenzidine substrate and counterstained with hematoxylin. Samples were considered IL-17F-immunopositive if at least 10% of randomly selected fields contained positive staining signals.
Statistical analyses were performed with SPSS software (IBM, NY, USA). Serum cytokine and mRNA expression were across-analyzed with demographic and clinical groups using the Mann-Whitney U test and Kruskal-Wallis tests. The relative distribution of IL-17A and IL-17F mRNA expression in cancerous and non-cancerous tissue was analyzed by the Fisher exact test and Mann-Whitney U test. In all cases, p < 0.05 was considered statistically significant.
IL-17F expression was elevated in HCV-associated HCC
Demographic Summary of TLCN HCC cohort
HCV (n = 40)
NBNC (n = 32)
66.3 ± 8.4 (59.3–72.0)
66.0 ± 14.9 (61.0–76.0)
Presence of cirrhosis
Tumor size (cm)
4.5 ± 2.3 (3.0–5.9)
8.0 ± 5.1 (3.5–11.8)
Association of serum IL-17F level with liver fibrosis progression in treatment naïve HCV patient cohort
Variables of HCV infected patients from TMH
56.6 ± 10.5 (51.5–64.8)
1246.7 pg/mL (0–877.7)
4.3 pg/mL (0–0)
73.2 pg/mL (0–84.7)
50.6 pg/mL (0–0)
Correlations of serum IL-17F level with the HCV infection-associated liver diseases and other proinflammatory cytokines
Correlation Coefficient (r)
The present study showed that a higher percentage of HCV-associated HCC tissues than of NBNC-HCC tissues were found to have IL-17F mRNA expression. IL-17F mRNA expression was higher in tumor tissue than in non-tumor counterparts. Furthermore, the serum IL-17F levels were elevated in HCV-infected patients with severe liver fibrosis than in patients with mild liver fibrosis.
Chronic HBV, HCV infection and steatohepatitis are common causes of liver fibrosis [26–28]. The relevance of Th17 and IL-17 in liver fibrosis has been studied in human patients and mouse models [29, 30]. Chang et al. showed that circulating and liver-infiltrating Th17 cell levels correlate with severity of liver inflammation . However, serum IL-17A levels are low in HCV patients and appear not to correlate with HCV-related fibrosis . In our HCV cohort study, the mean serum IL-17F level was much higher than that of IL-17A. The severity of liver fibrosis in HCV patients was associated only with IL-17F, but not with IL-17A, suggesting that IL-17F might be a better biomarker than IL-17A for HCV-associated fibrosis progression. The serum IL-17F level did not have a significant correlation with serum alanine aminotransferase activity in the HCV cohort, suggesting that IL-17F elevation was not associated with acute liver inflammation.
IL-17A and IL-17F have highly homologous amino acid sequences, bind the same receptor, and activate similar proinflammatory responses, but these two cytokines differ in tissue distribution and receptor binding affinity . In the present HCV cohort, more serum samples were positive for IL-17F than with IL-17A, and the mean IL-17F level was much higher than the mean IL-17A level. High levels of IL-17F can be due to a high transcription activity, a long protein half-life, or production by different cell types. Given that the expression of serum IL-17A and IL-21, two key cytokines produced by Th17 cells, was associated with IL-17F expression, it is likely that Th17 cells contribute to IL-17F expression. However, in our IL-17F IHC analysis of HCC tissue sections, IL-17F expression was found mainly in hepatocytes. Serum IL-17F in HCV-infected patients may be produced partially by hepatocytes. The stimuli capable of triggering IL-17F expression in hepatocytes and other cell types and the responses activated by IL-17F-dependent signaling in the liver will need to be addressed further.
A pathological role of IL-17 and Th17 in HCC development has been proposed based on recent studies [29, 33–35]. Proinflammatory, anti-apoptosis, and pro-angiogenesis signals may contribute to the tumor promotion role of Th17 cells and IL-17 in HCC development . The role of IL-17F in HCC remains undetermined. In the present study, we found that IL-17F was elevated in patients with HCV and advanced fibrosis and that IL-17F mRNA was also elevated in HCV-associated tumor tissue. Notably, the IL-17F protein was found mainly in hepatocytes. The exact function of IL-17F in HCC development needs to be clarified with long-term follow-up cohort study.
The importance of IL-17F in chronic hepatic diseases was poorly investigated; however, we had found Th-17 associated inflammatory cytokine, IL-17F, which took a major part in severe liver fibrosis and HCC symptoms in HCV patients than other inflammatory cytokines, IL-6, and IL-17A. It appears that IL-17F can be a precise biomarker for the diagnosis of liver fibrosis and HCC progression in the future.
We thank Tainan Municipal Hospital and Taiwan Liver Cancer Network for providing patient serum, RNA and tissue sections.
This work was supported by National Health Research Institutes (Taiwan), grant number IV-104-pp.-21.
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
MSW and CHW contributed equally to this work. MSW, CHW, and GYY were in involved in experimental design, data analysis, and manuscript preparation. FCT performed statistical analyses. HJY, YCL, YPK, DJT, and WTT performed related experiments. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The studies were approved by Institutional Review Board committees of Tainan Municipal Hospital (IRB1011205) and National Health Research Institutes (EC1000801).
Consent for publication
The authors declare that they have no competing interests with the contents of this article.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Gower E, et al. Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol. 2014;61(1 Suppl):S45–57.View ArticlePubMedGoogle Scholar
- Friedman SL. Liver fibrosis -- from bench to bedside. J Hepatol. 2003;38(Suppl 1):S38–53.View ArticlePubMedGoogle Scholar
- Fattovich G, et al. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology. 2004;127(5 Suppl 1):S35–50.View ArticlePubMedGoogle Scholar
- Donato F, et al. Alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women. Am J Epidemiol. 2002;155(4):323–31.View ArticlePubMedGoogle Scholar
- El-Serag HB. Hepatocellular carcinoma and hepatitis C in the United States. Hepatology. 2002;36(5 Suppl 1):S74–83.View ArticlePubMedGoogle Scholar
- Tesmer LA, et al. Th17 cells in human disease. Immunol Rev. 2008;223:87–113.View ArticlePubMedPubMed CentralGoogle Scholar
- Hohl TM, Rivera A, Pamer EG. Immunity to fungi. Curr Opin Immunol. 2006;18(4):465–72.View ArticlePubMedGoogle Scholar
- Komiyama Y, et al. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J Immunol. 2006;177(1):566–73.View ArticlePubMedGoogle Scholar
- Miossec P. IL-17 and Th17 cells in human inflammatory diseases. Microbes Infect. 2009;11(5):625–30.View ArticlePubMedGoogle Scholar
- Dardalhon V, et al. Role of Th1 and Th17 cells in organ-specific autoimmunity. J Autoimmun. 2008;31(3):252–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Wei L, et al. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J Biol Chem. 2007;282(48):34605–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Bettelli E, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441(7090):235–8.View ArticlePubMedGoogle Scholar
- Veldhoen M, et al. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24(2):179–89.View ArticlePubMedGoogle Scholar
- Iwakura Y, et al. Functional specialization of interleukin-17 family members. Immunity. 2011;34(2):149–62.View ArticlePubMedGoogle Scholar
- Lemmers A, et al. The interleukin-17 pathway is involved in human alcoholic liver disease. Hepatology. 2009;49(2):646–57.View ArticlePubMedGoogle Scholar
- Lan RY, et al. Hepatic IL-17 responses in human and murine primary biliary cirrhosis. J Autoimmun. 2009;32(1):43–51.View ArticlePubMedGoogle Scholar
- Yasumi Y, et al. Interleukin-17 as a new marker of severity of acute hepatic injury. Hepatol Res. 2007;37(4):248–54.View ArticlePubMedGoogle Scholar
- Tan Z, et al. IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation. J Immunol. 2013;191(4):1835–44.View ArticlePubMedGoogle Scholar
- Chang Q, et al. Th17 cells are increased with severity of liver inflammation in patients with chronic hepatitis C. J Gastroenterol Hepatol. 2012;27(2):273–8.View ArticlePubMedGoogle Scholar
- Takatori H, et al. Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22. J Exp Med. 2009;206(1):35–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Ishigame H, et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity. 2009;30(1):108–19.View ArticlePubMedGoogle Scholar
- Chang IC, et al. The hepatitis viral status in patients with hepatocellular carcinoma: a study of 3843 patients from Taiwan liver cancer network. Medicine (Baltimore). 2016;95(15):e3284.View ArticleGoogle Scholar
- Kim S, Kim T. Selection of optimal internal controls for gene expression profiling of liver disease. BioTechniques. 2003;35(3):456–8. 460PubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.View ArticlePubMedGoogle Scholar
- Grivennikov S, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell. 2009;15(2):103–13.View ArticlePubMedPubMed CentralGoogle Scholar
- Yilmaz B, et al. Chronic hepatitis B associated with hepatic steatosis, insulin resistance, necroinflammation and fibrosis. Afr Health Sci. 2015;15(3):714–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Sebastiani G, Gkouvatsos K, Pantopoulos K. Chronic hepatitis C and liver fibrosis. World J Gastroenterol. 2014;20(32):11033–53.View ArticlePubMedPubMed CentralGoogle Scholar
- Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115(2):209–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Hammerich L, Heymann F, Tacke F. Role of IL-17 and Th17 cells in liver diseases. Clin Dev Immunol. 2011;2011:345803.View ArticlePubMedGoogle Scholar
- Meng F, et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology. 2012;143(3):765–76. e1–3View ArticlePubMedPubMed CentralGoogle Scholar
- Foster RG, et al. Interleukin (IL)-17/IL-22-producing T cells enriched within the liver of patients with chronic hepatitis C viral (HCV) infection. Dig Dis Sci. 2012;57(2):381–9.View ArticlePubMedGoogle Scholar
- Yang XO, et al. Regulation of inflammatory responses by IL-17F. J Exp Med. 2008;205(5):1063–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang JP, et al. Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients. J Hepatol. 2009;50(5):980–9.View ArticlePubMedGoogle Scholar
- Li J, et al. Interleukin 17A promotes hepatocellular carcinoma metastasis via NF-kB induced matrix metalloproteinases 2 and 9 expression. PLoS One. 2011;6(7):e21816.View ArticlePubMedPubMed CentralGoogle Scholar
- Kuang DM, et al. Activated monocytes in peritumoral stroma of hepatocellular carcinoma promote expansion of memory T helper 17 cells. Hepatology. 2010;51(1):154–64.View ArticlePubMedGoogle Scholar