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 6

Loss of Nr4a1 and Nr4a2 in T cells promotes Treg proliferation.

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Loss of Nr4a1 and Nr4a2 in T cells promotes Treg proliferation. (A) Perc...
(A) Percentages of Foxp3-expressing cells in sorted splenic CD4+CD25+ Tregs (day 0) and cultured cells for 5 days (day 5) (n = 5 per group). (B) UMAP presentation showing Nr4a3 expression within Treg_Foxp3 cluster in WT and dKO hepatic CD4+ T cells from mice fed CD for 8 weeks. (C) Percentages of Annexin V+ WT and dKO splenic Tregs cultured after 3 days (n = 3 per group). (D) Representative CTV intensity histograms (left) and percentages of CTVlo (middle) and CTVhi (right) cells in WT and dKO splenic Tregs. Sorted Tregs were labeled and cultured for 3 days (n = 6 per group). (E) Representative flow cytometry analysis of cell cycle distribution (left) and quantification (right) in cultured WT and dKO splenic Tregs. After 3 days, cultured cells were labeled with Vybrant DyeCycle violet (n = 4 per group) and analyzed. (F) Representative CTV intensity histograms (left) and percentages of CTVlo (middle) and CTVhi (right) cells in splenic Tregs transduced with an empty vector or a vector encoding Nr4a2. Sorted Tregs were retrovirally transduced, rested, labeled with CTV, and then restimulated with α-CD3 and α-CD28 antibodies for 3 days. The cells were gated on GFP+CD4+ cells (n = 6 per group). (G) Schematic representation of adaptive transfer of WT and dKO splenic CD4+ T cells into MASH-induced Rag2–/– mice. (H) The ratio of hepatic CD4+Foxp3+ cell frequencies (post) from recipient mice fed CD to splenic CD4+Foxp3+ cell frequencies (input) from WT and dKO mice (n = 5 per group). P values were calculated using unpaired 2-tailed Student’s t test or Mann-Whitney U test (A, C–E, and H) or paired 2-tailed Student’s t test (F). *P < 0.05; **P < 0.01; ***P < 0.001.