Hypermethylation of the glutathione peroxidase 4 gene promoter is associated with the occurrence of immune tolerance phase in chronic hepatitis B

Chronic HBV infection can lead to intrahepatic inflammation, and the repeated cycle of liver inflammatory injury and repair results in the development of liver fibrosis, irreversible cirrhosis, and even HCC [1]. Effective antiviral therapy has significant implications for improving patient prognosis as it can inhibit HBV replication, normalize liver enzyme levels, reduce liver inflammation, reverse fibrosis progression, and reduce the risk of liver cancer [4]. However, the current status of HBV diagnosis and treatment is not optimistic,and achieving the WHO’s goal of eliminating viral hepatitis by 2030 remains a challenging task. Currently, “functional cure” is considered the ultimate treatment endpoint for chronic HBV infection. This refers to sustained loss of HBsAg along with negative results on HBV DNA tests, sustained normal transaminase levels, and improvement in liver histological conditions after antiviral treatment [20]. Approved treatment options for CHB include nucleos(t)ide analogues and long-acting interferon. Nucleoside drugs are widely used due to their ability to inhibit reverse transcriptase activity effectively and suppress HBV replication while being well-tolerated. However, achieving clearance of HBsAg is difficult with a very low probability (only 0.5%) of attaining a functional cure [21]; most patients require long-term or lifelong medication to continuously suppress viral replication [22]. Additionally, the long-term use of nucleoside drugs carries the risk of drug resistance. Different from nucleos(t)ide analogues, long-acting interferon treatment time is fixed, usually 48 weeks, and HBsAg clearance rate after treatment can reach 3–5% [23, 24], although it improves the HBsAg clearance rate, the side effects of interferon treatment are more, so it is rarely used alone in clinical practice. In general, although the existing antiviral treatment can bring benefits to patients, overall it is still difficult to achieve HBsAg clearance, and the functional cure for HBV still does not yet meet the clinical needs [25, 26]. Based on the existing antiviral drugs, constantly searching for new therapeutic targets and developing new treatment strategies are expected to achieve the goal of eliminating HBV.

To achieve a functional cure for HBV, a combination of drugs with distinct mechanisms is imperative [27]. In terms of immunotherapy, overcoming immune tolerance following HBV infection and stimulating the body to reestablish an anti-HBV immune response present urgent scientific challenges [28]. PPR agonists have exhibited remarkable efficacy in tumor treatment, thus holding the potential for playing a pivotal role in antiviral therapy, particularly against HBV. At present, in the field of CHB treatment, pattern recognition agonists for different targets, including Toll-like receptor agonists, and RIG − 1 agonists, now has entered the stage of HBV drug clinical trials and achieved good results. Activating the body’s natural immune response is likely to achieve functional cure of HBV [27, 29]. However, it is also necessary to be alert that the innate immune response is usually not targeted, which may cause the “cytokine storm” and related adverse reactions [30]. Additionally, various STING agonists are under investigation. Existing research indicates that compared to Toll-like receptor agonists, STING agonists mainly induce immune cells to synthesize IFN-β, and produce lower levels of inflammatory cytokines such as interleukins, which suggests that STING agonists have less risk of inflammatory factor storm and better safety when inducing anti-HBV response [31]. In the mice model of chronic hepatitis with persistent infection of HBV preplasmid-induced recombinant cccDNA (rcccDNA), other researchers have discovered that activation of the STING signaling pathway can inhibit HBV replication by inhibiting ccc DNA-mediated transcription in hepatocytes and epigenetic modifications involved in HBV replication while also reducing liver tissue damage and fibrosis levels [32]. Overall, STING signaling pathways play a significant role in the chronic HBV infection process by disrupting immune tolerance.

. Cellular homeostasis can regulate the activation of the cGAS-STING signaling pathway. For example, cellular manganese (Mn²⁺) ions enhance the sensitivity of cGAS to double-stranded DNA (dsDNA), thereby promoting cGAMP production [33]. Intracellular zinc ion (Zn²⁺) promotes the phase separation of cGAS and DNA binding, which is necessary for maintaining the activation of the cGAS-STING signaling pathway [34]. Redox homeostasis is an important component of cellular homeostasis and a physiological self-correction mechanism to cope with different stress microenvironments such as viral infection and tumors. GPX4 is a kind of glutathione (GSH) and selenium-dependent glutathione peroxidase, a key regulator to the ferroptosis regulation pathway, by scavenging free radicals, participating in the hydrolysis of lipid peroxides, which protect cells from accumulation of lipid peroxides, reduce OS damage, is an antioxidant molecule [35]. Studies have shown that GPX4 is involved in a variety of biological functions, including neuronal loss, autophagy, cell repair, inflammation, ferroptosis, apoptosis and OS [36]. Indirect or direct inhibition of GPX4 leads to insufficient lipid peroxides clearance, accumulation of intracellular lipid peroxides, OS damage and ferroptosis. That is, impaired GPX4 activity is generally positively correlated with increased OS and regulatory cell death (RCD) in disease [37].

Studies have demonstrated the involvement of GPX4 in the pathogenesis and progression of various diseases, including inflammatory disorders, autoimmune conditions, neurodegenerative ailments, ischemia-reperfusion(I/R) injury, tumors, and so on. Notably, GPX4 expression in myeloid-like cells, particularly macrophages, serves to restrict CASP1-CASP11-mediated inflammasome activation and GSDMD-dependent pyroptosis. This mechanism effectively prevents cytokine storm and septic death during bacterial infections [38]. Additionally, GPX4 inhibits the inflammatory NF-κB pathway activation and the production of proinflammatory mediators (e.g., IL1B and PTGS2/COX2) across diverse experimental models, such as angiogenesis and hair follicle development [39, 40]. A mouse model with conditional knockout of GPX4 in T cells displays an accumulated membrane lipid peroxide phenotype, resulting in an intrinsic defect in protection against acute lymphocytic choriomeningitis virus and Leishmania infection [41]. GPX4 deficiency in the pancreas or small intestine can lead to pancreatitis [42] and inflammatory bowel disease [43]. Furthermore, GPX4 ablation in neutrophils exacerbates the development of autoimmune diseases, such as systemic lupus erythematosus, by inducing ferroptosis. Collectively, these conditional GPX4 depletion models suggest a broad regulatory role for GPX4 in the prevention of inflammatory and autoimmune diseases. In the nervous system, GPX4 promotes postnatal neuronal differentiation in mice [44]. Overexpression of GPX4 can prevent I/R injury in some tissues by limiting lipid peroxidation. Loss or inhibition of GPX4 expression accelerates I/R injury. For example, down-regulation of GPX4 expression by Mir135b-3p promotes I/R injury in cardiomyocytes [45]. GPX4 plays a dual role in tumorigenesis and progression. Lack of GPX4 causes cell death and tissue damage. On the one hand, chronic inflammation may occur, leading to DAMP-mediated macrophage polarization in the tumor microenvironment and promoting tumor growth. On the other hand, acute cell death caused by GPX4 depletion can eliminate tumor cells, release DAMPs, and activate antitumor immunity. Numerous studies have shown that GPX4 inhibitors enhance sensitivity to chemotherapy, radiotherapy, and immunotherapy by inducing ferroptosis [46]. For example, inhibition of GPX4 to enhance ferroptosis can increase the sensitivity of HCC to sorafenib [47], Epstein-Barr virus(EBV)-infected nasopharyngeal carcinoma to platinum [48], etc. But, because GPX4 is widely expressed in anti-tumor immune cells such as T cells, dendritic cells, and neutrophils and in immunosuppressive cells such as Tregs, non-selective targeting of GPX4 in preclinical cancer therapy may lead to immune side effects that suppress anti-tumor immunity.

Of course, GPX4 is also involved in the occurrence and development of some liver diseases. One study found that GPX4 is critical for hepatocyte survival and proper liver function [49]. Targeting GPX4 may become a new way to treat a variety of liver diseases. For instance, inhibiting GPX4 activity induces ferroptosis in HCC cells [47]; targeting GPX4 can mitigate ferroptosis to treat metabolic-associated fatty liver disease [50]. Activation of GPX4 by Maresin1 (MaR1) alleviates liver injury while reducing the expression of ROS, MDA, and inflammatory factors [51]. However, there is no relevant study on GPX4 in CHB. Considering that GPX4 is widely expressed in immune cells and is the first line of defense of the immune system against inflammation, we chose PBMCs for detection. Moreover, some related studies have identified impaired expression of the GPX4 gene in PBMCs from breast cancer patients which could serve as a biomarker indicating an increased risk for breast cancer [52], indicating that the expression of GPX4 in PBMCs is indeed related to some particular disease.

DNA methylation, a chemical modification of DNA, is a type of epigenetics. DNA methylation is the acquisition of methyl groups by DNA methyltransferases (DNMTs), which in turn affects gene expression without changing the DNA sequence. DNA methylation can inhibit gene expression, leading to lower gene expression levels. DNA methylation is often observed in chronic inflammatory diseases such as cholangitis, inflammatory bowel disease, hepatitis, and liver fibrosis. Studies have pointed out that at the same time, the abnormal expression of DNMTs associated with HBV persistent infection shows that abnormal gene promoter methylation exists in patients with CHB [53]. Studies have pointed out that OS can lead to epigenetic changes, which may be associated with the occurrence and development of CHB. Now there is no research on GPX4 promoter methylation status in PBMCs from CHB patients and its relationship with immune tolerance.

In this study, we reported that CHB patients had lower GPX4 expression and higher methylation levels in PBMCs and serum than HCs. And, among CHB patients, those in the immune tolerance phase had lower GPX4 expression levels and higher methylation levels in PBMCs or serum than those in the immune activation phase and reactivation phase. The results indicate that there is a decrease in GPX4 expression in PBMCs of CHB patients caused by hypermethylation, which is most significant in the immune tolerance phase among all clinical stages, suggesting that this phenomenon may be involved in the development of immune tolerance leading to HBV chronic infection in CHB. In addition, the patients in the immune tolerance phase and inactive low replication phase had lower STING mRNA expression levels in PBMCs and lower serum levels of IFN-β than those in the immune activation phase and reactivation phase, and there was no significant difference with HCs, which suggests that STING may be involved in mediating the immune process of PBMCs against HBV, and may be a key target for breaking immune tolerance after HBV infection. However, the corresponding antiviral response was not stimulated by the high viral load in the immune tolerance phase, and the levels of STING and IFN-β were similar to those in healthy people. It has been found that the cellular redox homeostasis maintained by GPX4 is required for STING activation. GPX4 deficiency enhances cellular lipid peroxidation, thereby specifically inhibiting the cGAS-STING pathway [16]. Therefore, we hypothesized that the lower expression levels of STING and IFN-β in the immune tolerance phase may be associated with the lower expression of GPX4. Therefore, we analyzed the correlation of GPX4 mRNA expression level and methylation level with STING mRNA expression level in PBMCs and serum IFN-β level in CHB patients. The results showed that GPX4 mRNA level was significantly positively correlated with STING mRNA expression level in PBMCs and serum IFN-β level, and GPX4 methylation level was significantly negatively correlated with STING mRNA expression level in PBMCs and serum IFN-β level. This supports our speculation. Then, we detected the OS markers in the peripheral blood serum of CHB patients and analyzed the correlation between them and GPX4 promoter methylation level. The results showed that GPX4 promoter methylation level was significantly negatively correlated with serum SOD level and significantly positively correlated with serum ROS and MDA levels, which was consistent with OS caused by GPX4 inhibition. Next, we analyzed the correlation between serum levels of inflammatory factors and GPX4 promoter methylation levels in the peripheral blood of CHB patients. The results indicated that GPX4 promoter methylation level was significantly positively correlated with serum TNF-α, IL-1β, and IL-6 levels, consistent with the role of GPX4 in inhibiting inflammation. Meanwhile, we analyzed the relationship between GPX4 promoter methylation level in PBMCs and the viral load of CHB patients and found a clear positive correlation between them. So we speculate that HBV infection may inhibit the expression of GPX4, but further studies are needed to verify this hypothesis. These results support the hypothesis that GPX4 plays an important role in the pathogenesis and immune tolerance of CHB. In conclusion, we have reasons to assume that the high methylation level of the GPX4 promoter plays an important role in the pathogenesis and immune tolerance of CHB, inhibits the antiviral effect of STING, and promotes the development of immune tolerance in CHB patients. Understanding this function of GPX4 may help DNMT inhibitors combined with various antioxidants and STING agonists to treat CHB, and improve the antiviral effect, to achieve the functional cure of CHB. However, it is noteworthy that the methylation status of the GPX4 promoter was only detected in PBMCs and not in liver tissue, mainly because of the difficulty in obtaining liver tissue specimens from CHB patients. In addition, PBMCs, including monocytes, T cells, B cells, and NK cells, are considered to be the first line of defense of the immune system against inflammation, and previous studies have reported that various diseases can affect gene expression of PBMCs through host immunity or inflammatory response.

Our study has some limitations. First, the sample size was relatively small and all patients were from a single center, which may have led to selection bias. In addition, the possibility of false positive clinicopathological features cannot be ignored. Second, we only evaluated the correlation between GPX4 and the expression levels of related cytokines, and the specific regulatory relationship between them is unknown. In conclusion, the GPX4 promoter was hypermethylated in CHB patients, especially in the immune tolerance phase. The methylation level of the GPX4 promoter is correlated with the expression levels of STING, IFN-β, and related cytokines, suggesting that GPX4 may play an important role in the pathogenesis and immune tolerance of CHB, which may provide new ideas for achieving functional cure of CHB. But the further studies are needed to clarify the exact role of GPX4 in CHB pathogenesis and its potential as a therapeutic target.