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Oct 12, 2023

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Nature Aging (2023)Cite this

Nature Aging (2023)Cite this article

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Regulatory T (Treg) cells modulate several aging-related liver diseases. However, the molecular mechanisms regulating Treg function in this context are unknown. Here we identified a long noncoding RNA, Altre (aging liver Treg-expressed non-protein-coding RNA), which was specifically expressed in the nucleus of Treg cells and increased with aging. Treg-specific deletion of Altre did not affect Treg homeostasis and function in young mice but caused Treg metabolic dysfunction, inflammatory liver microenvironment, liver fibrosis and liver cancer in aged mice. Depletion of Altre reduced Treg mitochondrial integrity and respiratory capacity, and induced reactive oxygen species accumulation, thus increasing intrahepatic Treg apoptosis in aged mice. Moreover, lipidomic analysis identified a specific lipid species driving Treg aging and apoptosis in the aging liver microenvironment. Mechanistically, Altre interacts with Yin Yang 1 to orchestrate its occupation on chromatin, thereby regulating the expression of a group of mitochondrial genes, and maintaining optimal mitochondrial function and Treg fitness in the liver of aged mice. In conclusion, the Treg-specific nuclear long noncoding RNA Altre maintains the immune-metabolic homeostasis of the aged liver through Yin Yang 1-regulated optimal mitochondrial function and the Treg-sustained liver immune microenvironment. Thus, Altre is a potential therapeutic target for the treatment of liver diseases affecting older adults.

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All data needed to evaluate the conclusions in this article are present in the paper or the supplementary materials (or both). The bulk RNA-seq data generated in this study are available at the Gene Expression Omnibus (accession nos. GSE227799, GSE227837, GSE229374 and GSE227838). The Nanopore long-read sequencing data are available from the Genome Sequence Archive (https://bigd.big.ac.cn/gsa/browse/) under accession no. CRA010415. The MS proteomics data are available via the PRIDE database (http://www.proteomexchange.org) under accession no. PXD041040. The loxP Altre mouse strain can be provided by H.-B.L. pending scientific review and a completed materials transfer agreement. Requests for those mouse lines should be submitted to H.-B.L.

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We thank W. Li for helping with the mass spectrometry analysis; J. Zhao and J. Hu for their help with genotyping and sample collection; P. Ranney and C. Hughes for their initial help and suggestions in the generation of the Altre loxP mice; X. Wang and H. Li for their contribution to the key discussion; all members of the Li laboratories for discussions and suggestions; and the sequencing and flow cytometry core facilities at Shanghai Institute of Immunology for their support. This work was supported by the National Natural Science Foundation of China (nos. 82341017, 82030042, 32070917 and 82111540277 to H.-B.L.; no. 82202017 to C.D.; no. 82271756 to J.Z.), the Chongqing International Institute for Immunology (no. 2021YJC01 to H.-B.L.), the Ministry of Science and Technology of China (no. 2021YFA1100800 to H.-B.L.), the Shanghai Science and Technology Commission (nos. 20JC1417400, 201409005500 and 20JC1410100 to H.-B.L.; no. 20ZR1472900 to Y.Y.; no. 22QA1408100 to J.Z.), the Shanghai Municipal Health Commission (nos. 2022XD047 and 2022JC001 to H.-B.L.), the Innovative Research Team of High-Level Local Universities in Shanghai (no. SHSMU-ZDCX20212501 to H.-B.L.; no. SHSMU-ZDCX20212500 to J.Z.), and the China Postdoctoral Science Foundation (no. 2021M702160 to C.D.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

These authors contributed equally: Chenbo Ding, Zhibin Yu, Esen Sefik, Jing Zhou.

Medical Center on Aging, Center for Immune-Related Diseases at Shanghai Institute of Immunology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Chenbo Ding, Zhibin Yu, Jing Zhou, Gaoyang Wang, Bin Li, Youqiong Ye & Hua-Bing Li

Shanghai Jiao Tong University School of Medicine-Yale Institute for Immune Metabolism, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Chenbo Ding, Zhibin Yu, Jing Zhou & Hua-Bing Li

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

Esen Sefik, Eleanna Kaffe & Richard A. Flavell

Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA

Esen Sefik & Richard A. Flavell

Department of Geriatrics, Medical Center on Aging of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Weiguo Hu & Hua-Bing Li

Chongqing International Institute for Immunology, Chongqing, China

Hua-Bing Li

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H.-B.L. conceived, supervised and directed the study. C.D. and Z.Y. performed all the main experiments with help from E.S. and J.Z. J.Z. and E.K. provided expert advice on liver biology. E.S. and Y.Y. provided help with the RNA-seq. Y.Y. performed the bulk RNA-seq and MS analyses. G.W. performed the Nanopore long-read sequencing analysis. C.D., Z.Y. and Y.Y. analyzed the data. B.L., W.H., R.A.F. and Y.Y. contributed to key discussions. C.D. drafted and revised the paper. H.-B.L. wrote and revised the paper. All authors discussed the results and commented on the paper.

Correspondence to Hua-Bing Li.

R.A.F. is a consultant for GSK and Zai Lab; H.-B.L. is a consultant for CARsgen Therapeutics. The other authors declare no competing interests.

Nature Aging thanks Nikolai Timchenko, Ye Zheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

a, Foxp3 and Gm16157 mRNAs level in conventional T (Tconv) and Treg cells isolated from FOXP3-IRES-mRFP reporter mice (n = 4). Two-tailed t-test. b, Relative level of Gm16157 in different T cell subsets based on our previous CRISPR screen data (GSE153862). c, Percent input of Altre in cytoplasm nuclearplasm and chromatin, Hprt is protein-coding gene as cytoplasma positive control, Neat1 and Malat1 are long non-coding RNAs as nuclearplasm and chromatin positive controls (n = 3 biologically independent cells). Data are mean ± s.d.

Source data

Nanopore long-read sequencing analysis of the lncRNA Altre.

a, The two lox sites were inserted into the third and fourth exons of the Altre locus by embryonic stem cell recombination technology. b, Representative flow cytometry gating strategy for thymocyte. c, Representative flow cytometry gating strategy for CD4+ T cell and CD8+ T cell in spleen, lymph nodes, liver, VAT (visceral adipose tissue), and colon. d, Representative flow cytometry plots of CD4+ and CD8+ subsets in the thymus (up panel) and spleen (down panel) from Altref/f and Altref/f Cd4Cre mice (2 months old; n = 3). e, Statistical analysis of the percentage of different T cell subsets in the thymus and spleen from young Altref/f and Altref/f Cd4Cre mice as in d. Two-tailed t-test. f, The relative level of Altre and its neighboring genes Igf1r, Pgpep1l, and Fam169b were analyzed by qRT-PCR in Treg cells from Altref/f and Altref/f Cd4Cre mice (n = 4). Two-tailed t-test. g, Statistical analysis of the percentage of CD45+CD4+CD25+Foxp3+ Treg cells in the thymus, spleen, liver, and VAT from Altref/f and Altref/f Cd4Cre mice (2 months old; Altref/f, n = 5; Altref/f Cd4Cre, n = 6). Two-tailed t-test. h, The relative level of Il10, Pdcd1, Ctla4, and Il2ra were analyzed by qRT-PCR in Treg cells from young Altref/f and Altref/f Cd4Cre mice (2 months old; n = 4). Two-tailed t-test. Data are mean ± s.d. NS, non-significant.

Source data

a, Representative flow cytometry plots of naïve T cells differentiation into Treg (n = 3 biologically independent cells) and Th17 (n = 4 biologically independent cells) effector subsets. b, Statistical analysis of frequency for the gated population as in a. Two-tailed t-test. Data are mean ± s.d. c, Representative flow cytometry (left panel) and statistical analysis (right panel) of suppression of naïve T cells proliferation mediated by Treg cells, as determined by flow cytometry using CellTrace Violet dilution, for peripheral (pool of splenic and lymph node) Treg cells isolated from Altref/f and Altref/f Cd4Cre mice (2 months old; n = 3) and co-cultured with WT naïve T cells at the indicated ratios, two-way ANOVA. Data are mean ± s.d. d, EAE scoring of Altref/f and Altref/f Cd4Cre mice (2 months old; Altref/f, n = 9; Altref/f Cd4Cre, n = 10), two-way ANOVA. Data are mean ± s.e.m. e, Tumor size analysis of Altref/f and Altref/f Cd4Cre mice (2 months old; n = 7), two-tailed Mann–Whitney U test. Data are mean ± s.e.m. f, Body weight changes after CD45RBhi naïve T cells and Treg cells adoptive transfer into Rag2−/− host mice (n = 5), two-way ANOVA. Treg cells isolated from Altref/f and Altref/f Cd4Cre young mice (2 months old). Data are mean ± s.d. NS, non-significant.

Source data

a, Relative level of Altre in Tregs isolated from several tissues (spleen, liver, colon, and VAT) in 8-week-old mice (n = 3). b, Relative level of Altre in Tregs isolated from several tissues (spleen, liver, colon, and VAT) in 6-month-old mice (n = 3). c, Relative level of Altre in Tregs isolated from several tissues (spleen, liver, colon, and VAT) in 14-month-old mice (n = 3). Data are mean ± s.d.

Source data

a, Body weight changes after CD45RBhi naïve T cells and Tregs adoptive transfer into Rag2−/− host mice (n = 6), two-way ANOVA. Tregs were isolated from Altre-WT and Altre-KO young mice (2 months old). b, The relative level of Altre neighbored genes Igf1r, Pgpep1l, and Fam169b was analyzed by qRT-PCR in Tregs from young Altre-WT and Altre-KO mice (2 months old; n = 3). Two-tailed t-test. c, The relative level of Altre neighbored genes Igf1r, Pgpep1l, and Fam169b was analyzed by qRT-PCR in Tregs from aged Altre-WT and Altre-KO mice (14 months old; n = 4). Two-tailed t-test. Data are mean ± s.d. NS, non-significant.

Source data

a, Representative flow cytometry plots of CD44 and CD62L staining in the liver CD4+ T cells isolated from Altre-WT and Altre-KO mice (14 months old). b, Statistical analysis of the percentage of CD62L+CD44−, CD62L+CD44+, and CD62L−CD44+ subsets in CD4+ T cell in the liver from Altre-WT and Altre-KO aged mice (n = 6) as in a. Two-tailed t-test. c, Representative flow cytometry plots of CD44 and CD62L staining in the liver CD8+ T cells isolated from Altre-WT and Altre-KO mice (14 months old). d, Statistical analysis of the percentage of CD62L+CD44−, CD62L+CD44+, and CD62L−CD44+ subsets in CD8+ T cell in the liver from Altre-WT and Altre-KO aged mice (n = 6) as in c. Two-tailed t-test. e, Statistical analysis of the percentage of CD45+CD4+ T cells in the spleen, liver, and VAT from Altre-WT and Altre-KO mice (14 months old; n = 6). Two-tailed t-test. f, Statistical analysis of the percentage of CD45+CD8+ T cells in the spleen (n = 6), liver (n = 6), and VAT (n = 5) from Altre-WT and Altre-KO aged mice (14 months old). Two-tailed t-test. g, Statistical analysis of the percentage of CD45+ γδ T cells in the spleen (Altre-WT, n = 6; Altre-KO, n = 6), liver (Altre-WT, n = 5; Altre-KO, n = 6), and VAT (Altre-WT, n = 4; Altre-KO, n = 4) from aged mice (14 months old). Two-tailed t-test. Data are mean ± s.d. NS, non-significant.

Source data

a, Statistical analysis of the percentage of IFN-γ+ in CD45+CD4+ T cells in the spleen (Altre-WT, n = 6; Altre-KO, n = 5) and liver (Altre-WT, n = 6; Altre-KO, n = 6) from aged mice (14 months old). Two-tailed t-test. b, Statistical analysis of the percentage of IFN-γ+ in CD45+CD8+ T cells in the spleen (Altre-WT, n = 6; Altre-KO, n = 5) and liver (Altre-WT, n = 6; Altre-KO, n = 6) from aged mice (14 months old). Two-tailed t-test. c, Statistical analysis of the percentage of IL-17A+ in CD45+CD4+ T cells in the spleen (n = 6) and liver (n = 5) from Altre-WT and Altre-KO mice (14 months old). Two-tailed t-test. d, Statistical analysis of the percentage of IL-17A+ in CD45+CD8+ T cells in the spleens (Altre-WT, n = 4; Altre-KO, n = 6) and livers (Altre-WT, n = 4; Altre-KO, n = 4) from aged mice (14 months old). Two-tailed t-test. Data are mean ± s.d. NS, non-significant.

Source data

a, Representative flow cytometry plots of aged Altre-KO Tregs (n = 6), aged Altre-KO Tregs infected with NC (n = 3) and mouse Altre-overexpression lentivirus vectors (n = 3), and then performed mitochondrial membrane potential (MMP) analysis by using the cationic fluorescent dye JC-10. Treg cells were isolated from Altre-KO mice (14 months old). b, Statistical analysis of mean fluorescence intensity (MFI) of JC-10 in aged Altre-KO Tregs, aged Altre-KO Tregs infected with negative control (NC) and Altre-overexpression lentivirus vectors as in a. Two-tailed t-test. Data are mean ± s.d.

Source data

a, Representative hematoxylin and eosin (H&E) and oil-red stained livers from Altre-WT and Altre-KO mice with the high fat diet for 8 months. Circline indicates a typic infiltrating area. b, Quantification of the corresponding histology scores based on oil-red staining (n = 13). Scale bar, 25 μm. Two-tailed t-test. c, Statistical analysis of frequency for cell early apoptosis in young Tregs co-cultured with phosphatidylethanolamine (PE 18:0/20:4) (n = 4 biologically independent cells). Two-tailed t-test. d, Statistical analysis of frequency for cell early apoptosis in young Tregs co-cultured with polycarbonate (PC 16:0/20:4) (n = 4 biologically independent cells). Two-tailed t-test. e, Statistical analysis of frequency for cell early apoptosis in young Tregs co-cultured with polycarbonate (PC 18:0/20:4) (n = 4 biologically independent cells). Two-tailed t-test. f, The relative level of YY1 was analyzed by qRT-PCR in Tregs infected with negative control (NC), YY1-sgRNA1, YY1-sgRNA2, and YY1-sgRNA3 lentivirus vectors. Tregs were isolated from WT mice (2 months old; n = 3). Two-tailed t-test. Data are mean ± s.d. NS, non-significant.

Source data

Altre isoform sequences from Nanopore sequencing.

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Ding, C., Yu, Z., Sefik, E. et al. A Treg-specific long noncoding RNA maintains immune-metabolic homeostasis in aging liver. Nat Aging (2023). https://doi.org/10.1038/s43587-023-00428-8

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Received: 27 July 2022

Accepted: 28 April 2023

Published: 05 June 2023

DOI: https://doi.org/10.1038/s43587-023-00428-8

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