Abstract

Background: Nonalcoholic steatohepatitis (NASH) is a progressive disease characterized by liver inflammation. Emerging evidence implicates T cells in the disease pathogenesis. Our unpublished data show that CD4 T cells contribute to NASH development; however, hepatic CD4 T cell phenotypes and functions in NASH have not yet been systematically examined.

Methods: C57BL/6J mice were fed with a chow or NASH-inducing diet high in fat, fructose, and cholesterol (FFC) for 24 weeks. Purified intrahepatic CD4 T cells were characterized by the cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq, which combines scRNA-seq with analysis of cell surface proteins), immunophenotyping by mass cytometry (CyTOF), NanoString targeted mRNA analysis (nCounter), and analyses of effector cytokines using flow cytometry following ex vivo stimulation (anti-CD3/CD28 with or without PMA/Ionomycin).

Results: To better understand the role of CD4 T cell subsets in murine NASH, we leveraged two complementary approaches: an unbiased approach by CITE-seq and CyTOF with 44 well-established T cell markers. These two single-cell analyses revealed that hepatic CD4 T cells from chow and FFC diet-fed mice are highly heterogeneous as they separated into 15 and 18 unique clusters by CITE-seq and CyTOF, respectively. Livers from chow-fed mice had a distinct population of naïve T cells, while NASH livers were significantly enriched in T-bet⁺ cells in various stages of activation, suggesting enrichment of Th1 cells. Interestingly, NASH livers were also enriched in Foxp3⁺ cells as well as regulatory T cells (defined as Foxp3⁺ CD25⁺). To compensate for the depth of sequencing in CITE-seq, we profiled mRNA expression of select 750 immune-related genes in hepatic CD4 T using NanoString technology. A total of 207 genes were differentially expressed between chow liver and NASH-derived CD4 T cells. Top upregulated genes in NASH CD4 T cells included Ifng, Havcr2 (Tim3), and Pard3, confirming the enrichment of Th1 cells in NASH livers. To further validate the Th1 effector phenotype in NASH, hepatic CD4 T cells from chow and FFC-fed mice were re-stimulated ex vivo. Upon stimulation, NASH-derived CD4 T showed markedly increased expression of IFNγ, with minimal changes in IL17a, compared to chow liver CD4 T cells. Also, frequencies of TNFα⁺ IFNγ⁺ cells were significantly increased in NASH-derived CD4 T cells.

Conclusion: These results indicate that IFNγ- and TNFα-producing Th1 cells are enriched in diet-induced murine NASH, likely promoting NASH pathogenesis.