FIGURE LEGENDS
Figure 1. Tenofovir alafenamide (TAF) treatment ameliorates liver injury in the nonalcoholic steatohepatitis (NASH) mouse model.(A) Experimental schedule for the NASH mouse model using streptozotocin (STZ) injection and high-fat, high-cholesterol (HFHC) diet. C57BL/6J mice were injected with 0.2 mg STZ and fed HFHC diet or carbohydrate (CHO) diet and treated with mock or TAF. NASH resulted from the HFHC diet after STZ subcutaneous injection for 4 weeks (n = 6-10). (B) Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TRIG) levels in STZ-injected, HFHC-fed NASH experimental animals treated with mock or TAF. (C) Representative histological analyses of liver sections stained with haematoxylin and eosin, Sirius red, anti-lipopolysaccharide (LPS) and anti-toll-like receptor 4 (TLR4) expression in STZ-injected mock-treated HFHC diet-fed and STZ-injected TAF-treated HFHC diet-fed mice livers. Original magnification: 50×. (D) Comparison of the frequency of LPS- or TLR4-positive cells between STZ-injected, HFHC diet-fed NASH experimental animals treated with mock or TAF. Morphometric analysis of Sirius Red-stained liver section was performed from 5 fields of 5 liver sections per group. Comparison of NASH Activity Score (NAS) from mouse liver specimens among each treatment group. (E) Experimental schedule for the NASH mouse model using choline-deficient, L-amino acid-defined, high-fat (CDAHF) diet-fed mock or TAF. C57BL/6J mice were fed a CDAHF or CHO diet for 6 weeks (n = 7-10). (F) Serum ALT, AST, and TRIG levels in CDAHF diet-fed NASH experimental animals treated with mock or TAF. *p<0.05, **p <0.001, ***p<0.001.
Figure 2. TAF decreases the number of activated mononuclear phagocytes (MPs) in the STZ-injected, HFHC diet-fed NASH mouse liver.(A) Representative dot plot describing the proportions of recruited/resident MPs. Data are representative of five independent experiments. (B) Recruited (CD11bhighF4/80low) and resident (CD11blowF4/80high) MP cell number per g liver weight and cell percentage in the mock or TAF-treated STZ-injected, HFHC diet-fed NASH mouse model was counted using flow cytometry. (C) Mean fluorescence intensity (MFI) of programmed death ligand 1 (PD-L1), IAIE and MER proto-oncogene, and tyrosine kinase (MerTK) in CD11b+F4/80+ cells. (D) Frequency of PD-L1+/IAIE+/MerTK+intrahepatic MP cells in the STZ-injected, HFHC diet-fed NASH mouse model treated with mock or TAF. (E) Relative mRNA expression of inflammatory genes in mock or TAF-treated NASH mouse liver. *p<0.05, **p <0.001, ***p<0.001.
Figure 3. TAF decreases the number of activated MPs in the choline-deficient, L-amino acid-defined, high-fat (CDAHF) diet-fed NASH mouse liver. (A) Representative dot plot describing the proportions of recruited/resident MPs. Data are representative of five independent experiments. (B) Recruited (CD11bhighF4/80low) and resident (CD11blowF4/80high) MP cell number per g liver weight and cell percentage in the mock or TAF-treated CDAHF diet-fed NASH mouse model was counted using flow cytometry. (C) MFI of PD-L1, IAIE and MerTK in CD11b+ F4/80+ cells. (D) Frequency of PD-L1+/IAIE+/MerTK+intrahepatic MP cells in the CDAHF diet-fed NASH mouse model treated with mock or TAF. *p <0.05, **p<0.001, ***p <0.001.
Figure 4. Ex vivo effect of TAF on MPs isolated from the livers of the thioacetamide (TAA)-induced liver injury mouse model. (A) The liver injury mouse model experimental schedule using TAA injection (n = 5). Mice were intraperitoneally injected with 100 or 150 mg/kg TAA three times a week. (B) Representative histological analyses of liver sections stained with H&E, Sirius red in TAA-treated mouse liver. Original magnification: 50×. Morphometric analysis of Sirius Red-stained liver section was done from 5 fields of 5 liver sections per group. (C)Ex vivo experimental scheme of MPs isolated from the liver injury mouse model treated with mock or TAF. The mice livers were perfused, and MPs were isolated using gradient centrifugation. (D) Frequency of PD-L1+ cells among isolated CD11b+F4/80+ cells after treatment with mock or TAF. (E) MFI of PD-L1 in isolated CD11b+F4/80+ cells after mock or TAF treatment. *p <0.05, **p <0.01, ***p <0.001.