Discussion
In this study, we confirmed that TAF alleviated NASH in two different NASH mouse models by decreasing the activity of recruited and intrahepatic MPs by blocking the AKT pathway. Our results demonstrated that liver damage and IAIE+/PD-L1+/MerTK+intrahepatic MP number were lower in TAF-treated NASH mice. Moreover, LPS-stimulated TAF-treated CD14+ human monocytes expressed lower PD-L1+HLA-DR+ levels than LPS-stimulated mock-treated ones. These results suggest that TAF decreased the numbers of recruited and intrahepatic MPs in the NASH liver. Moreover, pAKT levels were decreased in TAF-treated NASH mice livers and isolated peripheral human CD14+ monocytes, suggesting that TAF blocks AKT phosphorylation in MPs.
During NASH, recruited and intrahepatic MPs differentiate into pro-inflammatory MPs (Sung, 2021). The single-cell transcriptome analysis of NASH mouse livers showed that genes most specific to MPs were primarily NASH-induced genes (Xiong et al., 2019). Another recent single cell analysis also demonstrated that the recruited and intrahepatic MPs exhibit distinct inflammatory phenotypes during NAFLD progression (Krenkel et al., 2020). Other studies also demonstrated that in NASH/ALD livers, CD68+ MP numbers increased with the progression of fibrosis (Pil Soo Sung et al., 2022), and Trem2+CD9+ MPs derived from the recruited MPs display a profibrogenic phenotype (Ramachandran et al., 2019). Therefore, it is critical to investigate the mechanisms that target the activated MPs to treat NASH.
The NASH mouse models show pathological features similar to that of human NASH (Saito et al., 2015). Therefore, we established a NASH mouse model using a combination of chemical (STZ) and dietary interventions with HFHC diets. A recent study on human NAFLD livers suggested that gut-derived LPS induces liver damage by activating MPs via the TLR-4 pathway (Carpino et al., 2020). Our IHC results showed that LPS and TLR-4 expression was significantly reduced in the TAF-treated HFHC diet-fed mice compared to the mock-treated ones. A previous study revealed that TAF mitigates ALT levels in chronic hepatitis B virus infection maybe due to metabolic factors associated with NAFLD (Sripongpun et al., 2022). The NASH mouse model showed elevated serum ALT, AST, and TRIG levels, indicating severe liver damage. After TAF administration in the NASH mouse model, serum ALT, AST, and TRIG levels were remarkably restored to normal ranges. CD11bhighF4/80low +, used as a recruited MP marker, exhibited a higher percentage in the mock-treated HFHC diet group and was markedly decreased in the TAF-treated group. Previous studies have revealed that TDF, an antiviral agent used to treat chronic hepatitis B, has additional immune-modulatory effects (S. W. Lee et al., 2021; Zhao et al., 2020). To investigate anti-inflammatory effects of TAF on inflamed intrahepatic MPs, MPs were isolated from a TAA-induced liver injury mouse model and treated with TAF ex vivo . The activation status of the isolated MPs was significantly reduced after TAF treatment, suggesting that TAF has anti-inflammatory effects.
Akt is known as a key player in signal transduction and regulation of cellular processes. The PI3K/AKT pathway regulates MP survival, migration, proliferation, and inflammatory signals and promotes the development of inflammation (Linton, Moslehi, & Babaev, 2019). The PI3K/Akt pathway is activated by TLR4 or other cytokines, chemokines, and Fc receptors. TLR activation ultimately affects the activation state of macrophages (Lopez-Pelaez, Soria-Castro, Bosca, Fernandez, & Alemany, 2011). Moreover, LPS increases the phosphorylation of Akt (S. J. Lee, Seo, & Kim, 2015). Our data demonstrated that AKT phosphorylation was inhibited by TAF administration in a NASH mouse model. Moreover, pAKT levels were decreased in TAF-treated activated peripheral human CD14+ monocytes, suggesting that TAF blocks AKT phosphorylation in MPs. Further human studies are necessary to discover the safe and effective anti-inflammatory dose of TAF in patients with NASH.