The Nr4a family regulates intrahepatic Treg proliferation and liver fibrosis in MASLD models
Daisuke Aki, … , Setsuko Mise-Omata, Akihiko Yoshimura
Daisuke Aki, … , Setsuko Mise-Omata, Akihiko Yoshimura
Published October 15, 2024
Citation Information: J Clin Invest. 2024;134(23):e175305. https://doi.org/10.1172/JCI175305.
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Research Article Immunology

The Nr4a family regulates intrahepatic Treg proliferation and liver fibrosis in MASLD models

Abstract

Metabolic dysfunction–associated steatotic hepatitis (MASH) is a chronic progressive liver disease that is highly prevalent worldwide. MASH is characterized by hepatic steatosis, inflammation, fibrosis, and liver damage, which eventually result in liver dysfunction due to cirrhosis or hepatocellular carcinoma. However, the cellular and molecular mechanisms underlying MASH progression remain largely unknown. Here, we found an increase of the Nr4a family of orphan nuclear receptor expression in intrahepatic T cells from mice with diet-induced MASH. Loss of Nr4a1 and Nr4a2 in T cell (dKO) ameliorated liver cell death and fibrosis, thereby mitigating liver dysfunction in MASH mice. dKO resulted in reduction of infiltrated macrophages and Th1/Th17 cells, whereas it led to a massive accumulation of Tregs in the liver of MASH mice. Combined single-cell RNA transcriptomic and TCR sequencing analysis revealed that intrahepatic dKO Tregs exhibited enhanced T cell immunoreceptor with Ig and ITIM domains (TIGIT) and IL-10 expression and were clonally expanded during MASH progression. Mechanistically, we found that dKO Tregs expressed high levels of basic leucine zipper ATF-like transcription factor (Batf), which promotes Treg cell proliferation and function upon TCR stimulation. Collectively, our findings not only provide an insight into the impact of intrahepatic Treg cells on MASH pathogenesis, but also suggest a therapeutic potential of targeting of the Nr4a family to treat the disease.

Authors

Daisuke Aki, Taeko Hayakawa, Tanakorn Srirat, Shigeyuki Shichino, Minako Ito, Shin-Ichiroh Saitoh, Setsuko Mise-Omata, Akihiko Yoshimura

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Figure 8

The loss of Nr4a1 and Nr4a2 in T cell promotes Treg function.

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The loss of Nr4a1 and Nr4a2 in T cell promotes Treg function. (A) Repres...
(A) Representative CTV intensity histograms normalized to mode in WT responder cells (left) and percentages of CTVlo (middle) and CTVhi (right) cells. Hepatic CD4+CD25+Tigit+ Tregs were sorted from WT and dKO mice fed CD for 8 weeks. CTV-labeled naive CD4+ T cells from the spleen of Ly5.1 mice were activated with Dynabeads Mouse T cell activator CD3/CD28 in the presence of sorted Tregs at a ratio of 5:1 (responder cells:Tregs). The cells were gated on CD45.1+CD4+ cells (n = 6 per group) (B) Schematic representation of the experimental procedure of MASH induction mouse model with Treg-specific inducible deletion of Nr4a1 and Nr4a2. (C) Representative Sirius red staining of liver sections from female mice. Original magnification, ×10. Scale bars: 100 μm. Quantification of Sirius red staining area (%) per field (right) in MASH-induced WT and iFoxp3dKO mice (n = 4 female per group). (D–F) Representative flow cytometry plots (left) and percentages (right) of macrophages (CD45+CD11bhiF4/80int) (D), Th17 cells (CD45+CD4+IL17a+) (E), and Tregs (CD45+CD4+Foxp3+) (F) in the liver of MASH-induced WT and iFoxp3dKO mice (n = 7 per group). (G) Representative flow cytometry plots (left) and percentages (right) of IL-10+ in dKO splenic Tregs transduced with a shRNA targeting Luciferase or Batf. After resting culture, the cells were stimulated with PMA and ionomycin for 5 hours in the presence of Golgi plug before staining IL-10. The cells were gated on GFP+CD4+ cells (n = 5 per group). P values were calculated using unpaired 2-tailed Student’s t test or Mann-Whitney U test (A and C–G). *P < 0.05; **P < 0.01.