Hepatitis B virus (HBV) infection remains one of the most significant global health challenges, representing a major public health issue due to its substantial disease burden (GBD 2019 Hepatitis B Collaborators, 2022). In 2016, the World Health Organization (WHO) established the goal of eliminating viral hepatitis as a public health threat by 2030 (World Health Organization, 2016). In alignment with this objective, researchers worldwide have made considerable strides in the clinical diagnosis and treatment of chronic hepatitis B (CHB). However, achieving clinical cure for hepatitis B continues to present a formidable challenge, necessitating further research to overcome existing barriers.

In this context, the serological clearance of HBV DNA has emerged as a secondary target, with evidence suggesting that this clearance can significantly reduce the incidence of liver cirrhosis and hepatocellular carcinoma, thereby improving long-term patient outcomes (Liu et al., 2014). Advances in antiviral therapies have made serological of HBV DNA clearance more attainable; nonetheless, a subset of patients still experiences poor serological response, especially those with alanine aminotransferase (ALT) levels at the lower limit of normal prior to treatment.

Recent changes in dietary habits and lifestyle have led to a shifting spectrum of liver diseases, notably an increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) (Riazi et al., 2022). In light of the lack of breakthroughs in curing chronic hepatitis B, the population of CHB patients coexisting with MASLD has been steadily rising (Mak et al., 2024). This unique overlap has garnered attention from researchers, with studies indicating that CHB patients with MASLD may have a higher likelihood of achieving HBV DNA clearance during antiviral therapy (Huang et al., 2024). Conversely, other research has suggested that MASLD may not serve as a favorable factor for HBV DNA clearance in CHB patients (Yang and Wei, 2022). Despite these divergent viewpoints, the molecular and biological mechanisms underlying these observations remain underexplored.

Single-cell RNA sequencing (scRNA-seq) has emerged as an innovative technique that can provide high-resolution insights into the specific cellular populations influencing disease pathophysiology. Through single-cell transcriptomic analysis, we may elucidate the distinct immune microenvironment in the livers of CHB patients with MASLD, potentially revealing novel therapeutic targets for curing CHB.

In this retrospective study, we analyzed 403 patients with ALT levels below the normal threshold who underwent liver biopsy at our institution and subsequently received antiviral therapy with nucleos(t)ide analogs (NAs), with at least one-year follow-up data. Among these, 177 patients were diagnosed with MASLD. We compared the rates of serological HBV DNA clearance after one year of antiviral treatment, revealing that CHB patients with MASLD exhibited a higher probability of achieving HBV DNA clearance. To further investigate the underlying mechanisms, we merged publicly available single-cell transcriptomic data from CHB patients with MASLD. This analysis revealed an increased proportion of CD8+ Teff-GZMH cells in MASLD patients, suggesting heightened T cell activity, along with enhanced interactions among CD8+ Tc-PDCD1 and CD8+ Trm-CD69 cells, particularly within the active CCL-CCR pathways. These findings may elucidate why coexisting MASLD serves as a favorable factor for HBV DNA clearance in CHB patients and could provide pivotal insights and potential targets for future clinical research aimed at curing CHB.

Therefore, we hypothesize that the co-existence of MASLD creates a unique intrahepatic immune microenvironment, characterized by the activation of tissue-resident memory CD8+ T cells (TRM) and enhanced chemokine signaling via CCL-CCR pathways, which promotes immune-mediated clearance of HBV DNA. This study integrates clinical outcomes from a cohort of CHB patients with normal ALT and MASLD with single-cell transcriptomic analysis to investigate this mechanistic hypothesis and identify potential immune drivers of viral clearance.

CHB remains a significant global public health challenge, and the WHO’s goal of eliminating viral hepatitis as a public health threat by 2030 places increasing demands on current treatment strategies (World Health Organization, 2016). Meanwhile, the rising prevalence of MASLD has resulted in a growing number of patients with concurrent CHB and MASLD (Mak et al., 2024). This co-occurrence presents a novel scenario for antiviral treatment in CHB patients, raising substantial interest in its potential impact on HBV DNA clearance. However, current research findings on this topic presents conflicting results.

In our study, we retrospectively analyzed a cohort of 403 CHB patients with ALT levels below the normal threshold who underwent liver biopsy at our institution and subsequently received antiviral therapy with NAs, with at least one-year of follow-up. Among these patients, 177 were diagnosed with MASLD. Baseline clinical characteristics revealed that CHB patients with MASLD exhibited significantly higher BMI (24.27 ± 2.64 vs. 21.21 ± 2.52), ALT (27.89 ± 7.63 vs. 23.09 ± 8.42), GGT (28.17 ± 15.19 vs. 21.15 ± 15.32), and Triglycerides (1.73 ± 0.99 vs. 1.18 ± 0.87), along with lower levels of HDL (1.16 ± 0.28 vs. 1.40 ± 0.33). Elevated BMI and triglyceride levels, combined with reduced HDL, suggest a higher likelihood of metabolic dysfunction in patients with MASLD. Additionally, higher baseline ALT and GGT levels indicate that CHB patients with MASLD may experience greater hepatic inflammation. Histological and biochemical analysis corroborated these findings, revealing that patients with both CHB and MASLD had higher rates of significant liver inflammation (63.84% vs. 51.77%, P=0.015), significant fibrosis (38.98% vs. 27.43%, P=0.014), and significant histological damage (68.36% vs. 58.85%, P=0.05). Previous studies have established that appropriate levels of intrahepatic inflammation can enhance the immune system’s capacity to clear infected cells, thereby improving the efficacy of antiviral therapies (Fanning et al., 2019). Notably, the results of one-year follow-up demonstrated that CHB patients with MASLD achieved a higher HBV DNA clearance rate (74.58% vs. 58.85%, P=0.002), even when their ALT levels were at the lower limit of normal. Logistic regression analysis further confirmed that with the presence of MASLD was an independent predictor of HBV DNA clearance at one year (OR: 1.977, P=0.00190). This finding aligns with the recent WHO guidelines for the prevention, diagnosis, care, and treatment of individuals with chronic hepatitis B infection, which recommend antiviral therapy in the presence of MASLD, regardless of ALT, HBV DNA, or APRI scores (World Health Organization, 2024). Nevertheless, the mechanisms underlying the enhanced antiviral treatment response in CHB patients with MASLD remain insufficiently understood. Some studies have suggested that MASLD may inhibit HBV DNA replication via the TLR4/MyD88/IFNβ signaling pathway in animal models of CHB (Zhang et al., 2015). Other research proposes that metabolic stress may attenuate the progression of chronic hepatitis B (Tourkochristou et al., 2022). However, these studies primarily focused on bulk tissue molecular biology. Given the complexity of the intrahepatic immune microenvironment in CHB patients with MASLD, there is a notable gap in research exploring immune characteristics at the single-cell level. Given the limited understanding of the intrahepatic immune microenvironment in CHB patients with MASLD and the unclear mechanisms underlying HBV DNA clearance in this population, we analyzed data from GSE192740 (Krenkel et al., 2020), a single-cell transcriptomic dataset of CHB patients, and GSE182159 (Zhang et al., 2023), a dataset of MASLD patients. After merging and batch effect correction, we identified a total of 116,795 cells categorized into five major cell types. Notably, the proportion of myeloid cells was higher in the MASLD samples compared to other groups. These myeloid cells, which include liver-resident Kupffer cells and monocytes recruited from the bloodstream, play a critical role in the regulation of various liver diseases (Khan et al., 2024). The increased proportion of myeloid cells in the MASLD group may reflect heightened intrahepatic inflammation relative to the CHB cohort without MASLD. T cells, as the predominant effector cells, constituted the largest cell population in our single-cell data across all groups. We identified nine subtypes of T cells, and interestingly, the MASLD group exhibited a marked increase in CD8+ Teff-GZMH cells and a decrease in CD8+ TRM-CD69 cells. Further Cellchat analysis of cell interaction differences between the MASLD and non-MASLD groups revealed that although the CD8+ TRM-CD69 cells constituted the smallest population in the MASLD group, their intercellular interactions were remarkably active, both in terms of number and strength. TRM cells are a recently characterized subset of memory T cells that reside at infection sites, maintaining a highly activated state and are implicated in the pathogenesis of various diseases, including infections, cancers, and autoimmune diseases (Christo et al., 2024). Virus-specific liver tissue-resident memory T (TRM) cells play a critical role in controlling viral replication by directly targeting infected hepatocytes and orchestrating local immune responses within the liver microenvironment. These cells contribute to the functional cure for HBV patients by maintaining a sustained antiviral state, even in the absence of continuous antiviral therapy. Liver TRM cells, characterized by their unique ability to persist in the liver parenchyma without recirculating, provide long-term viral control through their rapid reactivation upon antigen re-exposure and their capacity to secrete antiviral cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). This localized immune surveillance mechanism is essential for preventing viral rebound and achieving durable immune-mediated control of HBV infection (Dusséaux et al., 2017; Pallett et al., 2017; Lim et al., 2019).

Furthermore, our analysis indicated that the CD8+ TRM-CD69 cells in the MASLD group engaged in heightened interactions with other cell types, particularly with CD3+NKT-FCGR3A cells. Notably, we identified significant signaling changes in the interactions between CD8+ TRM-CD69 and various T cell subtypes, with the CCL3-CCR1, CCL3-CCR5, CCL4-CCR5, and CCL5-CCR3 pathways showing the most pronounced increases. The expression levels of CCL3, CCL4, and CCL5 were also significantly elevated in MASLD compared to the non-MASLD group. Chemokines are a subclass of cytokines responsible for the transport of immune cells and the development of lymphoid tissues (Chen et al., 2018). Based on their primary protein structure and the positions of the first two cysteine residues, chemokines can be categorized into four main classes: C, CC, CXC, and CX3C, each participating in a variety of immune regulatory functions (Huang et al., 2008; Fox et al., 2015). Specifically, CCL3 and CCL4, also known as MIP-1α and MIP-1β (macrophage inflammatory proteins), are critical in attracting various immune cell types, including monocytes, T cells, and natural killer (NK) cells, to sites of inflammation and injury. CCL5, known as RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted), is another multifunctional chemokine that can recruit several types of immune cells, including T cells, monocytes, eosinophils, and basophils. The heightened presence of CCL3, CCL4, and CCL5 correlates with increased immune cell recruitment and activation within the liver microenvironment, suggesting a robust inflammatory response conducive to viral elimination.

It is noteworthy that in this study, the percentage of TRM cells in the livers of patients with MASLD was lower than that in other groups. However, this lower frequency does not imply that these TRM cells are “quiescent.” On the contrary, Cellchat analysis strongly suggests that they remain highly active. This phenomenon may be attributed to the MASLD liver microenvironment—characterized by lipotoxicity, insulin resistance, endoplasmic reticulum stress, and gut dysbiosis-derived metabolites—which serves as a potent chronic low-grade inflammatory stimulus (Wang et al., 2025). Such an environment efficiently activates TRM cells and induces them to produce soluble mediators. Meanwhile, the inflammatory microenvironment in MASLD may also involve robust negative feedback mechanisms that restrain excessive TRM expansion without necessarily suppressing their potent short-term effector functions. Consequently, although their proportion is relatively low, their functional activity remains high.

In contrast, in the hepatitis B group persistent viral antigens provide a powerful signal driving the expansion and long-term maintenance of antigen-specific TRM cells, resulting in a higher TRM frequency (Liu et al., 2023). However, while HBV involves continuous high-level exposure to viral antigens (PAMPs), MASLD is driven by metabolically stressed damage-associated molecular patterns (DAMPs). The persistent high-load PAMP stimulation in HBV may lead to TRM exhaustion and functional impairment, whereas the non-specific inflammatory stimulation in MASLD can activate a broader repertoire of TRM clones, potentially endowing them with enhanced functional activity.

In our differential gene enrichment analysis of T cells, we also observed significant enrichment in pathways such as the TNF signaling pathway and the T cell receptor signaling pathway, both of which are closely linked to tissue inflammation and T cell recruitment. The active inflammation observed in MASLD tissue may be associated with an increased rate of HBV DNA clearance after one year. A sufficiently vigorous immune response could enhance virus elimination. These findings suggest that the resident CD8+ TRM-CD69 T cells in the liver tissue of patients with CHB and MASLD may activate the immune system via CCL-CCR pathways, thereby promoting immune cell recruitment and enhancing HBV clearance.

Consistent with our findings, we observed a notable presence of Monocyte-THBS1 cells within myeloid cell subpopulations in the MASLD cohort. Monocytes are a type of immune cell that can respond to invading pathogens or tissue damage in the bloodstream by migrating to the site of injury. They play a critical role in regulating the inflammatory response, presenting antigens, and interacting with other immune cells such as the CD8+ TRM-CD69 cells identified in our earlier research, thereby influencing the progression of diseases. In patients with chronic hepatitis B (CHB) complicated by MASLD, the heightened activity of CD8+ TRM-CD69 cells, coupled with an increased abundance of Monocyte-THBS1 cells, likely facilitates a synergistic effect that enhances immune responses and promotes the clearance of HBV. Furthermore, our analysis of differentially expressed genes in myeloid cells revealed significant enrichment in the T cell receptor signaling pathway. These observations lend robust support to our hypothesis that tissue-resident memory CD8+ T cells may play a crucial role in enhancing HBV DNA clearance in CHB patients with MASLD complications and normal ALT levels, potentially mediated through the CCL-CCR pathways activated by Monocyte-THBS1 cells.

However, there were several limitations to our study. First, the clinical cohort was based on a single-center sample, which may introduce bias. Additionally, the lack of single-cell transcriptomic data specifically from liver tissues of CHB patients with MASLD limits our ability to fully capture the immune characteristics of this population. Future validation through cellular, animal, and clinical studies is warranted to corroborate our findings and explore their implications, which will be a focal point in our upcoming research endeavors.