Approximately 290 million people are chronically infected with the hepatitis B virus (HBV) worldwide (1). More than 650,000 people die each year from end-stage liver disease associated with HBV, including liver failure, cirrhosis, and hepatocellular carcinoma (1–3). Achieving the functional cure, meaning both HBV DNA and hepatitis B surface antigen (HBsAg) undetectable (4–6), can significantly improve the disease progression of chronic hepatitis B (CHB) and is considered the ideal endpoint for antiviral treatment (4–7). Nucleos(t)ide analogs (NAs) and interferon (IFN) are the main first-line agents for CHB, which can significantly inhibit HBV replication. However, it is hard to achieve a functional cure with either drug alone. Although many studies have shown that sequential or combination therapy with the two drugs can significantly improve the probability of functional cure in some specific populations, it is still not satisfactory (8–12). Therefore, it is particularly important to explore the mechanisms behind HBsAg loss and to identify predictive markers to expand the population suitable for treatment.

The development of HBV infection is mainly affected by the host’s immune response. In acute infection, 95% of adult patients showed an adequate immune response and eventually cleared the virus. Otherwise, patients can become chronically infected when the host immune response is inadequate or inappropriate (13, 14). As important immune system components, cytokines may introduce immune dysregulation or tolerance and be associated with progression in CHB (15–18). Inflammatory cytokines, such as IFN-α, CXCL8, CXCL9, and CXCL10, can induce inflammatory immune cell recruitment and promote hepatocyte apoptosis in CHB (15, 19–21). For instance, an elevated serum IFN-α and CXCL8 could promote NK-cell-mediated liver cell injury (20, 21), high serum CXCL9 and CXCL10 levels were reported to correlate with the development of hepatitis flares (20–22), IL-2 and IFN-γ were upregulated with high ALT levels (21). The changes of multiple cytokines can more comprehensively and accurately reflect the immune network in the liver to guide clinical treatment better.

It was also demonstrated that in the case of antiviral therapy, changes in serum cytokines were related to the response to antiviral therapy. Li M. et al. reported that an early rise in IFN-α2 levels during pegylated interferon alpha (PEG-IFN-α) treatment was related to functional cure in hepatitis B e antigen (HBeAg) positive CHB patients (23, 24). In addition, CXCL10 has also shown a predictive role in PEG-IFN-α treatment of chronic hepatitis C (25). Still, the detailed relationship among virological and biochemical markers, and cytokines behind different responses to anti-HBV treatment remains unclear.

In this study, we investigated the dynamic changes of serum cytokines levels during PEG-IFN-α add-on therapy in NAs-suppressed CHB patients with HBsAg levels < 1,500 IU/ml, and explored the relationships between the changes of cytokines, the virological response, and the fluctuation of liver inflammation.

CHB is an immune-related disease; immune cells and cytokines play an essential part in disease progression. Several studies have demonstrated that baseline quantitative HBsAg < 1,500 IU/ml can effectively predict HBsAg loss after a finite course of IFN-based therapy (27, 28). However, HBsAg alone as a predictor could not fully predict the efficacy (15, 29) nor reflect the liver’s immune status (30). Cytokines in serum can indirectly reflect intrahepatic immune response and inflammation (10). Dynamic detection of multiple cytokines can reflect the immune changes more thoroughly to reveal the mechanism of antiviral therapy. However, the association of cytokines with the response to antiviral treatment is yet to be entirely clear. In this study, we found that the slight early increase of Th1 cytokine (IL2), Th2 cytokine (IL4), Th17 cytokine (IL17A and IL6), and proinflammatory cytokines (IL1B, CCL3, and CXCL8) was associated with HBsAg loss or decrease >1 log in the Add-on group, and sustained high levels of proinflammatory chemokines (CCL5 and CXCL10) were associated with poor response during combination therapy of NAs and PEG-IFN-α.

The incidence of liver cirrhosis and hepatocellular carcinoma is significantly reduced after HBsAg loss, which is the treatment goal of CHB (4, 31, 32). An expert consensus recommends a 1log drop in HBsAg in early treatment as an indication for continued PEG-IFN-α therapy (27). Multiple studies have confirmed that a 1log drop in HBsAg predicts functional cure (33–37). Therefore, we defined good response and partial response as HBsAg loss and decreasing > 1 log, respectively.

In CHB, Th1/Th2 and Th17/Treg were in immune imbalance (30, 38, 39), and antiviral treatment partially restored immune cell function (30). IL2 (Th1 cytokine) activated CD8+T cells and NK cells and promoted their proliferation and cytosolic activity (40–42). NK cells can indirectly regulate T cells by releasing or consuming cytokines (43, 44). Peg-IFN-α increased IL2 activity in vitro ( 45) and enhanced the inhibition of NK cells on regulatory T cell proliferation and differentiation through IFNG (46). IL4 (Th2 cytokine), produced by NKT cells, inhibits HBV RNA and HBsAg production (47). Activated Th17 cells primarily secrete cytokines such as IL17A, IL6, and TNF and also recruit macrophages, neutrophils, and lymphocytes to induce local inflammation (48–51). IL6 (Th17 cytokine) exerts direct antiviral effects by activating the NF-κB pathway in infected hepatocytes (52, 53). A cross-sectional study found that high levels of IL17A were associated with spontaneous HBsAg loss (48), which is consistent with our study. IL-17 could effectively inhibit HBV replication in a noncytopathic manner (54). IL4 has been confirmed to inhibit the secretion of IL6 and TNF in vivo and in vitro ( 55). In this study, IL2, IL4, IL6, and IL17A (Th17 cytokine) in the Add-on group showed the same change trend, increasing at the early stage and decreasing at the later stage, especially in patients with good response, suggesting that the dynamic balance between Th1/Th2/Th17 immunity may play a key role in functional cure under combination therapy.

Especially, the timing of CCL3 and IL1B elevation may be associated with the response of antiviral therapy. During the combination therapy, patients with an increase in IL1B and CCL3 at week 24 did not respond as well as those with an increase at week 12, but the non-responders had almost no significant increase in CCL3. IL1B is a pro-inflammatory and antiviral cytokine (52, 56), produced mainly by inflammatory macrophages in the liver (52). HBV modulates liver macrophage function to impair the production of IL1B to maintain the infection status (52). It is speculated that the late or insignificant increase in IL1B may be a manifestation of an inhibitory immune microenvironment, which is not conducive to the functional cure of CHB.

Compared with the CR and PR groups, CCL5 and CXCL10 were maintained at high levels after the early increase in the NR group during combined treatment. CXCL10 is a potent chemoattractant of activated T cells, leading to immune activation (48). Sonneveld MJ et al. observed that higher levels of CXCL10 before PEG-IFN therapy may be beneficial for obtaining HBeAg loss (57). However, Wong, G.L. et al. reported that lower levels of CXCL10 can predict HBsAg loss (58), which was consistent with our studies. In fact, 41 HBeAg-positive and 45 HBeAg-negative patients were enrolled and received 48 weeks of PEG-IFN-α plus adefovir. And S B Willemse et al. figured that higher baseline CXCL10 levels appeared to be associated with favorable responses in HBeAg-positive patients but not in HBeAg-negative patients (13). Therefore, the relationship between cytokines and the response may be influenced by HBV replication levels. However, in this study, CXCL10 did not show significant changes during NAs treatment, which may be a manifestation of the lack of an effective immune response.

We noted that the level of IFNG was higher in patients with good responses than in patients with partial responses and poor responses under combination therapy. But patients treated with NAs had higher levels of IFNG and worse responses. This phenomenon may be related to the duration of high levels of IFNG. It has been reported that the presence of long-term IFNG is associated with chronic inflammation and contributes to the occurrence of tumors (59). In the Add-on group, the IFNG level increased and then decreased. It is suggested that long-term high levels of pro-inflammatory cytokines are not conducive to the functional cure, and the dynamic balance between pro-inflammatory and anti-inflammatory cytokines plays a vital role in treating CHB.

CHB is characterized by immunosuppression, decreased anti-inflammatory cytokines expression, and increased anti-inflammatory cytokines expression. The ability of NK cells to secrete cytokines and the HBV-specific immune response was partially restored after the immune tolerance was broken (60). The PEG-IFN-α treatment restored immunity and suppressed the production of immunosuppressive cytokines (61). Li, M. H. et al. (61) enrolled patients with CHB and treated them with NAs and PEG-IFN-α, respectively. It was found that the level of IL10 in both groups increased with time, and the increase was more evident in the NAs group, which was consistent with our study. In our study, the level of IL10 increased in both the NAs and Add-on treatment, especially during NA treatment.

The different correlations of cytokines among patients with different responses to PEG-IFN-α treatment are due to different profiles of cytokine change. Ning Qin et al. found that PEG-IFN-α enhanced the inhibition of NK cells on Treg cells, and this inhibition was associated with a significant decline in HBsAg (46). Nishio, A. et al. found that patients with early NK cell activation after PEG-IFN-α treatment had greater HBsAg decline (62). Our study suggests that PEG-IFN-α treatment induces different immune microenvironment changes, which may be the mechanism of different outcomes. But the underlying mechanism needs further exploration. There was a significant inverse correlation between IL1B and CCL5 in nonresponsive patients. IL1B is a proinflammatory cytokine associated with innate immunity. CCL5 is mainly produced by CD8⁺T cells and can chemotaxis and activate T lymphocytes, dendritic cells, and natural killer cells (63). The negative correlation between the two cytokines in non-responders may indicate that the coordinated interaction between innate and specific immunity contributes to viral clearance.

In summary, our study found that the pattern of serum cytokine dynamics correlates with the response to the sequential combination therapy of PEG-IFN-α and NAs. A mild and harmonious interaction of Th1/Th2/Th17 cytokines is associated with a favorable virological response, and sustained high levels of certain inflammatory cytokines are not conducive to HBsAg clearance. There were some deficiencies in this study. First, we did not detect the variation in the number and function of cytokine-related immune cells. Second, cytokines and HBsAg in liver tissue were not detected. However, these preliminary results provide clues for further revealing the immune mechanism of antiviral therapy and developing new therapeutic targets.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study protocol was approved by the Ethics Committee of the Fifth Medical Center of Chinese PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.

JF contributed to conception and design of the study. X-YJ, XL, S-NZ, X-NZ, C-BZ participated in data collection. W-XW performed the statistical analysis. W-XW, RJ wrote the first draft of the manuscript. F-SW and JF revised the manuscript. All authors contributed to the article and approved the submitted version.