HBV can form stable cccDNA in the hepatocyte nucleus after entering liver cells through receptors, which is the fundamental reason for its persistence. Presently, the existing therapeutic drugs are mainly divided into nucleoside analogs and interferon- α. Neither of the two types of drugs can effectively clear the cccDNA. Research has shown that HBV clearance is related to the host immune response. Killer cells such as NK, NKT, and CD8 + T cells release INF- γ and Granzyme - B to clear infected hepatocytes, and helper T cells secrete IL-21 and IFN- γ, promoting the killing of cells and assisting in the generation of antibodies, jointly helping the clearance of HBV.
In the study of HBV related infections, iNKT cells serve as earliest cells to secrete IFN- γ. When the host lacks iNKT cells, the recruitment and function of CD4 + and CD8 + T cells in the liver is reduced, ultimately leading to persistent HBV infection [11]. Here we observed that the percentage of iNKT cells gradually decreased as the disease progressed, specifically CD4-NKT cells that decreased gradually, and CD4 + iNKT cells that increased gradually. Thus suggesting that in the process of chronic HBV infection, HBV not only promotes virus escape by reducing the percentage of iNKT cells, but also induces a CD4+/CD4- subgroup shift in iNKT cells, reducing the function of iNKT cells and mediating host immune dysfunction. The absence of iNKT cells diminishes specific immune responses against HBV[15]. However, the mechanisms underlying the decrease in the number of iNKT cells and the phenotypic deviation involved in disease progression remain unclear. However, this can be attributed to the inhibition of iNKT cell by cellular lipid changes or the recruitment of iNKT cells to the liver [7, 16, 17]. The fact that iNKT cells are non-antigen-presenting cells they cannot directly interact with viruses, their functional activation is regulated by APC. Studies have shown that the activation of iNKT cells is influenced by NLRP3-IL-1 in liver [18]. Thus, this indirect regulation elucidates the important role of APC in regulating iNKT cell activation and participation in liver disease progression.
A recent study showed that iNKT cells can die from pyroptosis and liver inflammation [19]. The decrease in iNKT cells due to pyroptosis promotes the progression of liver inflammation, which is consistent with the phenomenon we observed in HBV-infected patients. Pyroptosis is a newly discovered type of cell death induced by the activation of caspases and mediated by the gasdermin (GSDMs) family. Inflammasomes promote the activation of caspase-1/4/5/11 and cleave GSDMs into two parts: GSDMS-NT and GSDMS-CT[20]. The N-terminal domain can punch holes in the cell membrane and enhances the capability of processing IL-1β or IL-18[21]. More and more studies have shown that in addition to TLR activation, the release of TNF-α can also promote the occurrence of pyroptosis[22, 23]. The CCR2 + monocytes in the peripheral blood are chemotactic to the liver through CCL2, and mainly secrete TNF-α in the liver [24]. In this study both CCR2 + TREM-1 + and CX3CR1 + TREM-1 + monocytes gradually increased with disease progression, TREM-1 activation in THP-1 cells reduced the number of iNKT cells. and, caspase-1 and GSDMD-NT levels in iNKT cells were increased in TREM-1 overexpressing group. Thus, suggesting that the activation of TREM-1 in monocytes can indirectly promote pyroptosis of iNKT cells which is similar to the findings from our previous study where we showed that HBV can upregulate the expression of TREM-1 and promote the release of various inflammatory factors, including TNF-α [13]. Therefore, the decrease in the number of iNKT cells induced by pyroptosis not only reduces the ability of the host to clear the virus but also promotes the progression of liver inflammation through the release of cell contents.
Previous studies have shown that iNKT cells can be activated by two methods. One is direct activation, mediated by TCR signals, and second is indirect activation, mediated by external cytokines. Directly activated iNKT cells typically secrete IFN- γ without CD4 expression, while the indirectly activated iNKT exhibits diversity. Additionally, our study observed that TREM-1 promotes cytokine secretion, induces iNKT cell dysfunction, and promotes IL-4 and IL-17 secretion in iNKT cells. Compared with CD4-iNKT, IL-4 and IL-17 secretion levels from CD4 + iNKT cells are significantly higher[25, 26]. Our research suggests that the activation of the TREM-1 signaling pathway in monocytes promotes the dysfunction of iNKT cells through the regulation of various cytokines and may participate in the process of CD4-/CD4 + iNKT imbalance.
However, this study had some limitations. Firstly, due to time limitation an animal model was not established, thus further studies need to be carried out to validate the results in vivo. Second, since this was a retrospective study there was a certain selection bias. Third, because iNKT cell count was so small, it was impossible to verify whether iNKT cells in clinical patients underwent pyroptosis through western blotting. Moreover, at the same time, in China, patients with HBV have undergone an expanded treatment program; therefore, it increasingly became difficult to find patients with liver biopsy, in order to detect the correlation between monocytes and iNKT cells in the liver, and verify the manner in which iNKT cells die in the liver. Thus, we would carry studies in animal model to verify our results.
In conclusion, in this study we observed that the dynamic changes in iNKT cell count and phenotype were closely related to HBV infection, where HBV induced the expression of TREM-1 in monocyte and secretion of TNF- α and other inflammatory cytokines to promote the pyroptosis and functional changes in iNKT cell. Consequently, disrupting the host's antiviral ability, promoting the persistence virus infection leading to chronic liver inflammation. Therefore, findings from the present study have helped us in identifying new drug targets for HBV infection treatment. However, future studies need to be carried out to further strengthen our findings that would pave way for the development of new treatment strategies for patients with HBV infections.