Abstract

Background: Nonalcoholic steatohepatitis (NASH) is a progressive condition characterized by excessive inflammation, which may lead to fibrosis. To date, the contribution of T cells to NASH remains largely unexplored. Our unpublished data suggest that CD4 T cells with pathogenic Th1 phenotype accumulate in murine NASH livers. Therefore, we investigated the role of CD4 T lymphocytes in NASH development.

Methods: Whole-body CD4 knockout (CD4^-/-) and wild-type (WT) mice were fed chow or NASH-inducing diet high in fat, fructose, and cholesterol (FFC) for 24 weeks. Insulin resistance and metabolic phenotype were evaluated at weeks 16 and 18, respectively. Liver injury, inflammation, and fibrosis, along with gut microbiome were evaluated after 24 weeks of feeding.

Results: CD4 deletion in FFC-fed mice did not affect food intake, weight gain, lean and fat mass, insulin resistance, and metabolic profile (e.g., metabolic rate, energy expenditure) as assessed by CLAMS (Comprehensive Lab Animal Monitoring System). However, liver weight and liver injury (assessed by plasma alanine aminotransferase) were significantly reduced in FFC-fed CD4^-/- mice compared to WT, despite no changes in liver steatosis (measured by triglyceride levels). Mouse intrahepatic leukocytes were isolated and subjected to immunoprofiling by mass cytometry (CyTOF) and flow sorting of monocyte-derived macrophages (MoMF). Transcriptomic analysis in isolated MoMF using NanoString technology revealed that scar-associated macrophage markers (Trem2 and CD9) were less abundant in FFC-fed CD4^-/- mice. Concordantly, liver hydroxyproline assay and Sirius red staining demonstrated a striking reduction in hepatic collagen deposition in FFC-fed CD4^-/- mice, suggesting that CD4 T cells are required for liver fibrogenesis in NASH. The decrease in inflammatory and fibrogenic response was further validated by IHC for galectin-3 (macrophage marker) and αSMA (a marker for activated hepatic stellate cells). Finally, FFC-associated microbiota alterations, such as increases in bacterial genes linked with glycolysis, glucogenesis, and lysine biosynthesis, were not observed in FFC-fed CD4^-/- mice. Also, the gut microbiota alpha diversity in FFC-fed CD4^-/- mice resembled that of chow-fed controls, exhibiting similar Firmicutes/Bacteroidetes ratios.

Conclusion: Genetic loss of CD4 T cells in a murine NASH model results in a reduction in liver injury, inflammation, and fibrosis, independent of the metabolic syndrome. Together, we allude that CD4 T cells contribute to NASH pathogenesis.