Chronic hepatitis B virus infection remains a major global public health problem. Peginterferon-alpha-2a (PEG-IFN) has direct antiviral and immunoregulatory effects, and it has become one of the first choice drugs for the treatment of chronic hepatitis B (CHB). Cytokines play an important role in immunity, and they directly inhibit viral replication and indirectly determine the predominant pattern of the host immune response.

To determine the correlation between cytokine/chemokine expression levels and response to PEG-IFN treatment in patients with CHB.

Forty-six kinds of cytokines were analyzed before PEG-IFN therapy and at 24 wk during therapy in 26 CHB patients.

The monokine induced by INF-γ (CXCL9) and serum interferon-inducible protein 10 ( IP-10) levels at baseline were higher in virological responders than in non-virological responders (NRs) and decreased during treatment, whereas the NRs did not exhibit significant changes. The macrophage inflammatory protein 1d (MIP-1d) levels at baseline and during treatment were significantly higher in the virological responders than in the NRs, while thymus and activation-regulated chemokine (TARC) levels at baseline and during treatment were significantly lower in the virological responders than in the NRs. The CXCL9, IP-10, MIP-1d, and TARC baseline levels exhibited the expected effects for interferon treatment. The area under the receiver operating characteristic curve values of CXCL9, IP-10, MIP-1d, and TARC for predicting virological responses were 0.787, 0.799, 0.787, and 0.77 (P = 0.01, 0.013, 0.01, and 0.021), respectively.

We found that cytokine levels before and during treatment may represent potential biomarkers to select CHB patients who can respond to PEG-IFN. Therefore, cytokines can be used as an indicator of antiviral drug selection before CHB treatment.

Despite the development of a highly effective and safe vaccine over 30 years ago, chronic hepatitis B virus (HBV) infection remains a global major public health problem, and at least 257 million people are chronically infected with HBV worldwide[1]. Approximately 887000 chronic hepatitis B (CHB) patients die annually from complications associated with the disease, including cirrhosis, hepatocellular carcinoma, and liver failure[1-10]. Effective antiviral drugs inhibit replication and reduce the risks of CHB complications[11]. Host and viral factors play key roles in the natural history of CHB, the disease activity, and the effectiveness of antiviral therapy[12]. CHB patients generally have an impaired host immune response, which may be associated with a persistent high viral load and subsequent T cell failure[11,13]. At present, the antiviral strategy for CHB mainly lies in the effective suppression of the virus and the recovery of HBV-specific immune responses[12]. Because peginterferon-alpha-2a (PEG-IFN) has direct antiviral and immunoregulatory effects, it has become one of the first choice drugs for the treatment of CHB[14]. Antiviral treatment with PEG-IFN effectively inhibits HBV replication and may lead to seroconversion of hepatitis B e antigen (HBeAg), clearance of hepatitis B surface antigen (HBsAg), normalization of alanine aminotransferase (ALT) levels, and histologic improvement[11]. If patients achieve an IFN induced virological response, they may achieve long-term therapeutic effects while reducing the risks of cirrhosis and HCC[11,14]. When patients with CHB receive PEG-IFN treatment, there is no guarantee of a sustained virology response, and studies show that only 30% of patients benefit from this treatment[15]. Since PEG-IFN has the properties of immune regulation, the host immune status may affect the efficacy of PEG-IFN in the treatment of CHB[11,12]. Several studies in recent years have shown that cytokines and chemokines may play a potential role in chronic viral hepatitis[12,16,17].

Cytokines play an important role in immunity, and they directly inhibit viral replication and indirectly determine the predominant pattern of the host immune response[18]. Chemokines are a group of small cytokines that induce the migration of circulating white blood cells to sites of inflammation or injury and enhance the phagocytosis abilities of inflammatory cells[19]. Due to the highly complex control of cytokine secretion, the interactions between cytokines, and with their receptors, are widely spread in numerous regulatory networks[19]. Screening multiple biomarkers can clarify the immune mechanism of HBV infection and predict the response to antiviral therapy. Moreover, a study has demonstrated that T cell responses and circulating cytokines are associated with HBV replication and liver function. Interleukin (IL)-10, IL -12, and IL -21 play crucial roles in the immune clearance stage; thus, they are related to the seroconversion of HBeAg[11,16,18,19]. Nevertheless, the relationship between multiple cytokines and chemokines and the responses to PEG-IFN therapy in patients with CHB has not previously been clarified.

In this study, we analyzed the serum levels of cytokines in CHB patients receiving PEG-IFN treatment and responses to the therapy. Forty-six cytokines, including macrophage inflammatory protein 1d (MIP-1d), CXCL9, CXCL6, IP10, and TARC, in serum samples of CHB patients were simultaneously tested. The predictive value of the cytokines for the responses to PEG-IFN was also assessed.

Liver inflammation due to HBV infection is immune mediated. Acquired immunity and innate immunity are considered related to the pathogenesis of CHB[21]. In this study, we obtained simultaneous measurements of 46 kinds of cytokines in CHB patients to assess the roles of cytokines according to the responses to PEG-IFN, which is a widely used therapeutic agent for the treatment of CHB. However, interferon therapy for CHB remains a clinical challenge. Although successful PEG-IFN therapy can induce a long-lasting response and reduce liver-related complications, the efficacy rate of conventional interferon therapy for 6 mo was only 25% to 40%[22,23]. Therefore, individualized treatment strategies according to pretreatment parameters should be established to identify patients with the highest chance of response. However, there is currently no ideal pre-treatment predictor of VR.

In our study, the serum levels of cytokines changed in the CHB patients during PEG-IFN therapy, and the baseline TARC, MIP-1d, CXCL9, CXCL6, and IP10 levels were strongly correlated with the virological response in patients with CHB. Among these cytokines, high baseline levels of MIP-1d, CXCL9, and IP10 had better prediction of the virological response to interferon therapy. CXCL6, CXCL9, and IP10 are all members of the chemokine ELR-CXC subfamily, in which CXCL9 and IP10 can be induced by IFN-γ[12,24]. These three cytokines are secreted by mono-nuclear/macrophage T lymphocytes, B lymphocytes, hepatocytes, fibroblasts, etc. CXCL9 and IP10 bind to their co-receptor CXCR3, activate T cells, NK cells, etc, and exert antiviral effects[12,25-27]. Bièche et al[28] reported that CXCL9 and IP-10 were significantly increased in the serum of patients with CHB[28]. A study found a high correlation between high baseline levels of IP10 and hepatitis C virus (HCV) clearance induced by PegIFNα treatment in HCV-infected patients[29]. The baseline levels of CXCL9 and IP10 were significantly higher in the response group than in the non-response group; however, there was a significant decrease in the response group after 24 wk of treatment, and there was no significant difference in the non-response group before and after treatment. Studies have indicated that CXCL9 and IP10 mRNAs were significantly decreased in peripheral blood mononuclear cells of CHB patients after 12-24 wk of PegIFNα treatment, which was similar to the findings in patients with chronic hepatitis C infection[30-32]. According to our study and reports in the literature, the serum levels of CXCL9 and IP10 and the mRNA levels of CXCL9 and IP10 in peripheral blood mononuclear cells decreased in patients who responded to PEG-IFN treatment, which might reflect a reduced hepatic necro-inflammatory activity after treatment. CXCL9 and IP-10 were reported to correlate with hepatic injury during hepatic flares in CHB, and a strong correlation of serum CXCL9 and IP-10 levels with ALT levels was also noted[33]. Our results also showed positive correlations of CXCL6 and CXCL9 with ALT levels (r = 0.530, P = 0.005; r = 0.588, P = 0.002). However, IP-10 was not correlated with ALT, which was different from that in the previous literature and may be related to the relatively few samples tested and individual differences of the hosts. Mac-2-binding protein glycosylation isomer (M2BPGi), also called WFA⁺-M2BP, is a serum biomarker for assessing liver fibrosis in patients with viral hepatitis[34], and its levels significantly correlated with serum IP-10 levels[35]. A recent study suggested that baseline serum M2BPGi level can be a useful marker in the CHB patients during PEG-IFN therapy[36]. The positive correlations between these parameters may imply that these chemokines have positive impacts on the liver inflammation and HBV control, albeit the relevance was not satisfactory due to the complex interactions between other host and viral factors that contribute to liver inflammation. These findings indicate that CXCL6, CXCL9, and IP10 are associated with hepatic necro-inflammation, and these chemokines are induced and participate in immune responses against HBV infection.

We also found that TARC in patients with an interferon-treated response was lower than that in the NRs at baseline; however, the level in the response group remained lower than that in the non-response group after 24 wk of treatment. TARC is a member of the CC chemokine group that is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes, bronchial epithelial cells, and fibroblasts[37,38]. TARC is a ligand for CCR4, which is predominantly expressed on Th2 lymphocytes, basophils, and natural killer cells[38]. ARC can recruit a large number of Th2 cells, despite the fact that the Th2 cell predominance is to maintain the chronic carrying state of virus infection[39]. Th2 cell activation inhibits both acute and chronic inflammatory reactions[40]. Therefore, the higher expression of TARC in the non-response group treated with interferon is related to the poor prognosis.

MIP-1d/CCL15, a member of the CC chemokine family that is expressed only in the gut and the liver, acts mainly via the CC chemokine receptor CCR1[41,42]. MIP-1d was demonstrated to have a chemoattractant role for monocytes, lymphocytes, neutrophils, eosinophils, and dendritic cells. In addition, this molecule plays an important role in the development of inflammatory diseases[41,43]. Our study found that MIP-1d was higher in the response group than in the non-response group at baseline and after 24 wk of treatment. The higher level in the response group than in the non-response group may be associated with better recruitment of T cells in the response group.

CXCL6/GCP-2 is a member of the ELR-CXC chemokines. CXCL6 is mainly secreted by macrophages and epithelial and stromal cells[44]. The receptors of CXCL6 are CXCR1 and CXCR2, and CXCL6 has the functions of regulating immunity, tumor growth, and metastasis, as well as promoting angiogenesis. Liver tissue can express CXCL6, which can induce Kupffer cell, neutrophil, and lymphocyte infiltration in liver inflammation[44,45]. The expression of CXCL6 was elevated in liver inflammation and fibrosis, and in alcoholic hepatitis, CXCL6 levels are elevated and related to the prognosis of patients[45]. In our study, CXCL6 was significantly higher at both baseline and 24-wk in the response group than in the non-response group and showed a significant correlation with ALT.

In conclusion, our results suggest that MIP-1d, CXCL9, CXCL6, IP-10, and TARC have predictive significance in interferon therapy. Cytokines are fundamental molecules in the complex signaling network of cell-mediated immunity. Analyses of the changes in cytokine expression patterns during treatment can help to understand the pathogenesis of CHB and predict treatment responses. However, there were limited subjects in this study, and unfortunately, we could not collect all samples from each patient at all treatment phases. Therefore, the statistical power was not adequate for an assessment of the usefulness of cytokines in monitoring CHB patients. Further large and long-term follow-up studies are required to determine the predictive value of cytokines in CHB patients receiving PEG-IFN treatment. In addition, the differences in these factors between the response and non-response groups and their specific biological roles in HBV infection require further investigation.