Mitochondria, an organelle ubiquitously present in most cells, comprises a bilayer membrane structure and matrix component. Mitochondria plays a pivotal role in cellular energy production, metabolism, and signal transmission (1). Any alteration in its structural and functional integrity may lead to mitochondrial dysfunction, characterized by adenosine triphosphate (ATP) depletion, overproduction of reactive oxygen species (ROS), reduction in mitochondrial membrane potential (MMP), and damage to mitochondrial deoxyribonucleic acid (mtDNA). These changes can further perpetuate cell apoptosis and inflammatory response (2). Recent findings suggest that mitochondrial dysfunction arising from mitochondrial injury can trigger a cascade of cell injuries, including ATP depletion, ROS upsurge, inflammatory response, and apoptosis, and is instrumental in the onset and progression of various hepatic diseases such as hepatitis C (3), chronic alcoholic liver disease (4), non-alcoholic fatty liver disease (NAFLD) (5) and drug-induced liver injury (DILI) (6).

Studies have indicated that hepatitis B virus (HBV) infection may result in elevated levels of calcium ions, overproduction of ROS, and reduced ATP synthesis within the mitochondria. Additionally, HBV infection has been linked to the overactivation of the mitochondrial permeability transition pore (MPTP), leading to mitochondrial swelling and cell death (7). Yoo et al. (8) observed a correlation between high expression of MARCH5E3, a mitochondrial ubiquitin ligase, and increased survival rates among patients with hepatocellular carcinoma (HCC). This association is attributed to MARCH5E3’s interaction with the hepatitis B viral x (HBx) protein, which accumulates in the mitochondria and triggers its degradation, thereby inhibiting HBx-induced ROS overproduction, mitochondrial autophagy, and cyclooxygenase-2 expression (8, 9). The above studies confirmed that mitochondria played an important role in HBV infection. Other studies also found circulating lymphocyte activation was associated with HBV infection. These suggested the lymphocyte mitochondria may be abnormal during HBV infection.

Hepatitis B virus (HBV) does not directly inflict damage upon liver cells but does so through the modulation of the immune system (10). T cell activation, proliferation, and differentiation are fundamentally driven by shifting in cellular metabolism, with mitochondria playing a central role (11). Mitochondrial dysfunction, as seen in COX-deficient T cells, can severely impair these metabolic pathways, affect T cell responses, and lead to immunodeficiency due to compromised metabolic functionality (12). Li L et al. (13) found downregulation of the JAK1-STAT3 pathway and depolarization of mitochondria emerged as crucial factors contributing to T cell anergy. Research has demonstrated that the hepatitis B virus can integrate its genetic material into mitochondrial DNA (mtDNA), thereby impacting mitochondrial function (14). Zhou et al. identified mitochondrial dysfunction in the immune cells of individuals with chronic HBV infection, establishing a notable association between mitochondrial function in immune cells and HBV viral load, HBeAg, and HBsAg (15). Presently, limited research exists regarding the connection between mitochondria and immune cells in the context of HBV infection. The impact of HBV infection on T cell mitochondrial function, and T cell exhaustion and differentiation require further investigation. In this study, two novel mitochondrial energy metabolism indices, MM (16) and MMP^low17 were employed to comprehensively analyze the changes in mitochondrial metabolism of immune cells at various stages of HBV infection. The goal was to explore the correlation between these changes and hepatic inflammation and their potential clinical diagnostic significance. The findings from this research were anticipated to shed light on the immunological mechanisms underlying liver injury resulting from HBV infection, elucidate the variations in immune cell mitochondrial energy metabolism across different stages of HBV infection, introduce novel diagnostic markers for early liver inflammation, and identify new therapeutic targets for HBV-induced liver inflammation.

The advancements in research on the mechanism of mitochondrial injury in liver diseases have led to a shift toward intervention strategies targeting mitochondria as a new approach to treating various liver conditions (21). Key determinants of cell death include mitochondrial membrane potential and calcium homeostasis (22). The accumulation of calcium ions not only diminishes ATP production but also induces the excessive opening of the mitochondrial permeability transition pore (MPTP), leading to the collapse of mitochondrial membrane potential (23). Reduced mitochondrial membrane potential significantly contributes to mitochondrial damage, subsequently triggering intracellular ROS generation, signaling apoptosis, and releasing inflammatory factors and mtDNA, culminating in localized liver tissue inflammation (14). This process plays a pivotal pathological role in hepatocyte apoptosis and liver inflammation.

The role of immune-mediated injury in liver damage among patients with HBV infection is significant. Currently, there is limited research on the correlation between mitochondrial damage in immune cells and liver injury in these patients. Studies have revealed that the presence of HBV in immune cells can cause a reduction in the mitochondrial membrane potential of these cells, and hepatitis B antigen has been associated with membrane potential impairment (15). However, the relationship between lymphocyte mitochondrial membrane potential and liver inflammation has not been reported, and there is a lack of research where mitochondrial membrane potential serves as a clinical detection parameter for HBV infection.

In our research, it was observed that the MMP^low percentage of CD8⁺T cells exhibited consistent variations with the progression of the disease. The MMP^low percentage of CD8⁺T cells was significantly lower in the LC compared to the HC and CHB groups, and the MMP^low percentage of CD56⁺NK cells in the LC group was also lower than that in the healthy control group. Our analysis revealed that the mitochondrial mass of CD4⁺CD8⁺T cells in the CHB group was lower than that in the normal healthy control group. These findings indicate that as the disease advances, the membrane potentials of CD8⁺T cells and NK cells that dominate viral immunity are elevated, suggesting an activated state of these cells. Changes in mitochondrial mass and membrane potential are significant contributors to increased reactive oxygen species (ROS) production. Fisicaro et al. (24) identified ROS as a key marker of HBV infection inflammation, increased production of ROS by dysfunctional mitochondria can lead to DNA damage, and superoxide production (MitoSOX) can increase the function of T cells. We also analyzed the relationship between lymphocyte membrane potential and ROS. We found the ROS expression of CD45⁺ MMP^low cells was lower than MMP^high cells, and the expression of ROS in CD4⁺T cells, CD8⁺T cells and CD56⁺NK cells in patients in the inflammatory group was higher than that in the non-inflammatory group, but there were no statistical difference ( Supplementary Figure 7 ). The specimens of Supplementary Figure 7 were newly collected. Its sample size was smaller than that used in other figures. The relationship between MMP and ROS in CHB needs to be further studied and verified by larger samples. Therefore, further exploring the relationship between membrane potential and ROS or ATP, analyzing the function of MitoSOX and DNA damage in regulating T cells during HBV infection will provide a deeper understanding of the biological significance of mitochondrial function in immune cells and offer additional biomarkers for the clinical diagnosis of CHB and LC.

The relationship between immune cell membrane potential and early liver inflammation was further analyzed in light of the understanding that abnormal immune cell function is a key factor in liver injury. It was observed that changes in immune cell membrane potential may serve as a more sensitive and accurate indicator of liver inflammation than conventional markers such as ALT and AST, which are released after liver injury. The study revealed a linear negative correlation between the MMP^low ratio of CD4⁺T and CD8⁺T cells in the CHB group and AST levels. Furthermore, a positive correlation was identified between the MM of CD4⁺T cells and CD8⁺T cells and ALT levels. The MMP^low percentage of CD8^-CD4^-T cells was found to be decreased in CHB patients with liver inflammation detected via ultrasonography but normal ALT levels.

In the study, it was also observed that CD8⁺T cells, CD56⁺ NK cells, and CD4^-CD8^- T cells in the non-inflammatory group exhibited a higher proportion of low mitochondrial membrane potential. This finding may be attributed to the need for increased mitochondrial production and energy availability during lymphocyte activation. Elevated membrane potential enhances activity within the mitochondrial respiratory electron transport chain, resulting in activated lymphocytes displaying greater mitochondrial membrane potential enrichment. This enrichment supports lymphocyte activation and contributes to a more robust inflammatory response.

This result suggests that the participation of mitochondria may also be required for the polarization of T cells, Similar to the study by Xiao et al. in tumors (23). T cell mitochondrial function may be involved in immune damage by influencing T cell function (17). Different T cell subsets are involved in different stages of liver inflammation, and it is particularly noteworthy that mitochondrial dysfunction of CD3⁺CD4^-CD8^- T cells can occur in early liver inflammation. Activated CD3⁺CD4^-CD8^-T cells were divided into cytotoxic and proinflammatory subgroups (25, 26), this study further found that the MMP^low percentage of CD3⁺CD4^-CD8^-T cells can reflect early liver inflammation. The above results may be related to the involvement of CD3⁺CD4^-CD8^-T cells in innate immunity and pro-inflammatory, but there were different subsets of CD3⁺CD4^-CD8^-T (25), so further research on the changes of mitochondrial function in different subsets of CD3⁺CD4^-CD8^-T cells will further clarify the mechanism of T cells in HBV infection.

The existing studies on mitochondrial dysfunction in HBV found HBV-specific CD8⁺T cells had extensive mitochondrial alterations and increased production of ROS by dysfunctional mitochondria can lead to DNA damage (24, 27), and further studies found replenishment of NAD could restore HBV-specific CD8⁺T cell functions (28). The above and our studies all suggest the HBV infection can cause mitochondrial dysfunction, but our study focuses on factors that cause mitochondrial function changes such as potential and mass, while other studies explore the products of mitochondrial function changes such as ROS and ATP, and we also found NK and CD3⁺CD4^-CD8^-T cells had mitochondrial dysfunction which was not explored in other studies.

T cell exhaustion and NK cell function and differentiation are associated with HBV infection. Yan Jia et al. found HBV DNA polymerase upregulated the transcription of PD-L1 and suppressed T cell activity (29). HBV infection mice were used to show that metabolic abnormalities in dysfunctional CD8⁺T cells were the manifestation of prolonged antigenic stimulation (30). In the NK cell, the study also found cell phenotypic alteration and dysfunctional state post hepatitis B subviral particle stimulation in CHB patients. Mitochondria are the main organelles that control cell metabolism, and this study found that the mitochondrial potential and quality of circulating lymphocytes in CHB patients were altered. This study suggests that mitochondrial energy may be related to T cell exhaustion and NK cell function changes, which provides a direction for further revealing the immune tolerance mechanism caused by HBV infection.

The study has several limitations. Firstly, the results require validation with a larger sample size. Secondly, data from HCC patients are absent. Additionally, the relationship between mitochondrial damage and antigen-specific T-cell function during HBV infection needs further investigation, and the study of the relationship between potential and ROS and ATP were necessary to further clarify the role of lymphocyte mitochondria in HBV infection. Consequently, future research should focus on conducting multicenter studies throughout the entire HBV infection cycle, specifically examining mitochondrial aspects of immune cells. Further exploration of mitochondrial mechanisms in HBV infection using animal and cell models is warranted.

Lymphocyte-induced immune damage is an important mechanism for the occurrence of HBV-infected liver inflammation. The mitochondrial mechanism of lymphocyte activation has been discovered and has been implicated in the development of tumor immunity and autoimmune diseases. Studies have found that HBV infection can cause abnormal mitochondrial metabolism in liver cells, but there is a lack of research on lymphocyte mitochondria and HBV infection, this study conducted a systematic analysis of the percentage of lymphocyte MMP^low and the MM in different stages of HBV infection to investigate their correlation with early liver inflammation. In this study, we found that the MMP^low percentage of CD8⁺T cells and NK cells changed regularly at different stages of HBV infection. The MMP^low and MM of T cells were correlated with liver inflammatory markers such as ALT and AST. This study also aimed to establish the clinical diagnostic value of MMP^low and MM in HBV infection and found the MMP^low and MM of CD8⁺T cells could be used as a potential indicator to distinguish LC from CHB. The MMP^low of CD3⁺CD4^-CD8⁺T cells had clinical value in reflecting early liver inflammation. This study contributes to a deeper understanding of the role of T cells in liver inflammation, provides clinical data for further research on the mechanism of T cell activation and provides a potential diagnostic indicator for clinical cirrhosis and early liver injury.