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  • The Transforming Growth Factor �1/Interleukin-31 Pathway Is Upregulated in Patients with Hepatitis B Virus-Related Acute-on- Chronic Liver Failure and Is Associated with Disease Severity and Survival

    Xueping Yu,a Ruyi Guo,a Desong Ming,b Yong Deng,c Milong Su,b Chengzu Lin,a Julan Li,a Zhenzhong Lin,b Zhijun Sua

    Department of Infectious Diseases, The First Hospital of Quanzhou Affiliated to Fujian Medical University, Quanzhou, Chinaa; Department of Clinical Laboratory, The First Hospital of Quanzhou Affiliated to Fujian Medical University, Quanzhou, Chinab; Department of Infectious Diseases, The Second People’s Hospital of Pingxiang, Pingxiang, Chinac

    The transforming growth factor �1/interleukin-31 (TGF-�1/IL-31) pathway plays an important role in the process of cell injury and inflammation. The purpose of this work was to explore the role of the TGF-�1/IL-31 pathway in the cytopathic process of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF). The quantitative serum levels of TGF-�1, IL-9, IL-10, IL- 17, IL-22, IL-23, IL-31, IL-33, and IL-35 were analyzed among chronic hepatitis B (CHB) patients (n � 17), ACLF patients (n � 18), and normal control (NC) subjects (n � 18). Disease severity in patients with ACLF was assessed using the model for end- stage liver disease (MELD) and Child-Pugh scores. Serum TGF-�1 levels were strongly positively correlated with IL-31 in all sub- jects, and both of them were positively correlated with IL-17, IL-22, and IL-33. In CHB and ACLF patients, serum levels of TGF-�1 and IL-31 were both increased significantly compared with those in NC subjects and positively correlated with total bili- rubin (TBil) and alpha-fetoprotein (AFP) levels. ACLF patients showed the highest levels of TGF-�1 and IL-31, which were posi- tively correlated with Child-Pugh scores. Furthermore, the recovery from the liver injury in CHB was accompanied by decreased TGF-�1 and IL-31 levels. More importantly, serum levels of TGF-�1 and IL-31 were markedly upregulated in ACLF nonsurvi- vors, and IL-31 displayed the highest sensitivity and specificity (85.7% and 100.0%, respectively) in predicting nonsurvival of ACLF patients. Increasing activity of the TGF-�1/IL-31 pathway is well correlated with the extent of liver injury, disease severity, and nonsurvival of ACLF patients, while reducing activity is detected along the recovery from liver injury in CHB, suggesting its potential role in the pathogenesis of liver injury during chronic HBV infection.

    Hepatitis B virus (HBV)-related acute-on-chronic liver failure(ACLF) is triggered mainly by severe extensive liver injury, and the exact mechanisms of massive destruction of HBV-in- fected hepatocytes remain unclear. However, one of the current assumptions is that the imbalance of the cytokine network, the so-called cytokine storm theory (1), points to potential involve- ment of inflammatory cytokines in destroying the HBV-infected cells, which may provide an explanation for the aggravation of liver injury.

    Transforming growth factor-�1 (TGF-�1) is a 25-kDa ho- modimeric protein composed of two subunits linked by a disul- fide bond and is a powerful inhibitor of DNA synthesis and cellu- lar proliferation (2). It also mediates formation of extracellular matrix and facilitates cell differentiation (3). Previous studies have shown that TGF-�1 plays a role in developing liver failure (LF). Miwa et al. found that the mRNA and protein expression of TGF-�1 were significantly upregulated in both the plasma and liver tissue in patients with fulminant liver failure (FLF) (4). Yo- shimoto et al. found that the overexpression of TGF-�1 delayed liver regeneration and promoted perisinusoidal fibrosis and hepa- tocyte apoptosis in the rat model of FLF (5).

    Interleukin-31 (IL-31), is a newly discovered proinflammatory cytokine and is produced mainly by CD4� T cells, especially when cells are skewed toward a Th2 phenotype (6). It acts through the oncostatin receptor (OSMR) and heterodimeric receptors of IL-31 (IL-31R), a complex that stimulates the JAK-STAT, the phosphoinositol 3-kinase (PI3K)/AKT, and the RAS/extracellular

    signal-regulated kinase (ERK) signal pathways (7, 8). There is emerging evidence showing that the IL-31/IL-31R signaling path- way plays an important role in the pathogenesis of atopic and allergic diseases and inflammatory diseases such as allergic contact dermatitis (9, 10), nonatopic eczema (11), spontaneous urticaria (12), nasal polyps (13), asthma (14), and familial primary cutane- ous amyloidosis (15). Nevertheless, there is a paucity of data ex- ploring the potential role of IL-31 in the pathogenesis of ACLF.

    Biological functions of TGF-�1 depend on the signal transduc- tion and regulation of Smad proteins. Smad2/3 are the key ele- ments in mediating TGF-�1-induced inflammatory diseases (16). Ge et al. (17) found that TGF-�1 induced Smad2 phosphorylation and blockade of Smad2/3 prevented TGF-�1-modulated IL-6 in-

    Received 7 October 2014 Returned for modification 10 November 2014 Accepted 18 February 2015

    Accepted manuscript posted online 25 February 2015

    Citation Yu X, Guo R, Ming D, Deng Y, Su M, Lin C, Li J, Lin Z, Su Z. 2015. The transforming growth factor �1/interleukin-31 pathway is upregulated in patients with hepatitis B virus-related acute-on-chronic liver failure and is associated with disease severity and survival. Clin Vaccine Immunol 22:484 –492. doi:10.1128/CVI.00649-14.

    Editor: R. L. Hodinka

    Address correspondence to Zhijun Su, [email protected]

    Copyright © 2015, American Society for Microbiology. All Rights Reserved.


    484 May 2015 Volume 22 Number 5Clinical and Vaccine Immunology

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  • crease. Activated Smad2 can bind to the IL-6 promoter region, including IL-31, a new member of the IL-6 family (17). Shi et al. also found that TGF-�1 induced Smad2 phosphorylation and then activated the binding of Smad3 to IL-31 promoters before finally stimulating the IL-31-JAK-STAT signal pathway (18). Therefore, IL-31, which increased with elevated TGF-�1 expres- sion, was considered a downstream molecule of the TGF-�1– Smad2/3 pathway (18). Recently studies have shown that the TGF-�1–Smad2/3/IL-23 pathway plays an important role in the progression of bleomycin-induced pulmonary fibrosis in mice (18, 19), suggesting that the TGF-�1–Smad2/3/IL-23 pathway is one of the key players in inducing cell injury, a critical part of the pathological process of many human diseases. We decided to in- vestigate the TGF-�1–Smad2/3/IL-23 pathway in ACLF because ACLF is always preceded by acute, severe, and extensive liver in- jury in chronically HBV-infected patients. More importantly, analysis of the TGF-�1–Smad2/3/IL-23 pathway could shed new light on the pathogenesis of massive liver injury and lead to new treatment strategies.

    In this study, we analyzed the serum levels of TGF-�1, IL-9, IL-10, IL-17, IL-22, IL-23, IL-31, IL-33, and IL-35, and investi- gated their relationships with the disease severity and survival in patients with ACLF and chronic hepatitis B (CHB) to address the potential role of the TGF-�1/IL-31 pathway in liver injury of ACLF and to build a foundation for identifying new therapeutic targets.

    MATERIALS AND METHODS Subjects. Blood samples were collected on the next morning after admis- sion from 17 CHB patients and 18 ACLF patients who were hospitalized or followed up from July 2012 to November 2013 in the Department of Infectious Diseases of The First Hospital of Quanzhou Affiliated to Fujian Medical University. The criteria for diagnoses of CHB and ACLF have been described in a previous study (20–25). All CHB patients who had positive hepatitis B surface antigen detection (HBsAg) and hepatitis-re- lated clinical manifestations or a histological confirmation of hepatitis and abnormal alanine aminotransferase (ALT) levels (�40 U/liter) for more than 6 months were included. The diagnostic criteria for ACLF include mainly a history of CHB or liver cirrhosis, serum total bilirubin (TBil) 10 times or more greater than the normal level (�171 �mol/liter), and prothrombin time activity (PTA) of �40%. All participants were enrolled following the order of patient hospital admission, and there was

    no exclusion based on gender. The predominance in male patients most likely reflects the demographic features, where the majority of patients with advanced or end-stage liver disease are males.

    No patients had received antiviral therapy or immunomodulating agents, such as glucocorticoid hormones and thymosin, before enroll- ment. Patients with hepatitis A, hepatitis C, hepatitis D, or human immu- nodeficiency virus (HIV) infection and those with alcohol-induced hep- atitis or drug-induced autoimmune liver diseases and hepatic carcinoma (HCC) were excluded. Fresh blood samples from 18 healthy individuals without noticeable or detectable cell injury were designated normal con- trols (NC). Clinical characteristics of the enrolled cohorts are listed in Table 1.

    Eighteen ACLF patients were divided into two groups by their final clinical outcome. The survival group (n � 11) included patients whose liver function and blood coagulation were recovered gradually within 6 months after blood sampling and were still alive by the completion of this study. The nonsurvival group (n � 7) included patients whose liver func- tion deteriorated progressively and who died within 6 months after blood sampling. The d