Fig. 1: Elevated TIM3 in macrophages is positively associated with NASH fibrosis development in mice.

A Representative HE- and Sirius red-stained sections from liver tissue of mice fed a negative control diet (NCD) and methionine and choline-deficient (MCD) diet for 5 weeks, and NCD and high-fat diet (HFD) for 26 weeks (n = 6/group). Quantification of Sirius red-positive areas from 6 regions. B qPCR was used to measure the mRNA expression levels of the immune checkpoint molecules PD-1, PD-L1, Havcr2, LAG3, CTLA4, and TIGIT from the liver tissue of mice in A. C, D Representative immunofluorescence staining sections from liver tissue of mice in A. Quantification of TIM3-positive F4/80-positive cells. E Isolated hepatic F4/80^highCD11b^low Kupffer cells (KCs) and F4/80^lowCD11b^high monocyte-derived macrophages (MDMs), from mice treated with NCD and MCD for 5 weeks were analyzed for TIM3 expression by flow cytometry. Quantification of the percentage of TIM3-positive cells (n = 6/group). F TIM3 protein expression levels in primary KCs isolated from NCD-fed WT mice and RAW264.7 cells treated with 0.5 mM palmitic acid (PA) for 24 h. The relative protein expression was normalized to the level of β-actin. G–L Myeloid Havcr2 knockdown (Havcr2^fl/+; Lyz2-cre) mice and Lyz2-cre mice were fed an MCD diet for 5 weeks (n = 7/group). IHC staining and Sirius red staining showed fibrosis in WT and Havcr2 knockdown mice (H). The mRNA expression levels of α-SMA, Col1a1, Col1a2, and Timp-1 in liver tissues of mice were determined by qPCR (I). The NAFLD activity score (NAS) and fibrosis score of liver tissues were quantified (J, K). The serum ALT and AST levels are shown in L. Data are presented as the mean ± SEM; significance determined by Student’s unpaired t-test (A, C, D, F, H, J–L) and two-way ANOVA with Bonferroni’s multiple comparisons test (B, E, I). *p < 0.05, **p < 0.01, ***p < 0.001.