Abstract
Certain somatic mutations provide a fitness advantage to hematopoietic stem cells and lead to clonal expansion of mutant blood cells, known as clonal hematopoiesis (CH). Among the most common CH mutations, ASXL1 mutations pose the highest risk for cardiovascular diseases (CVDs), yet the mechanisms by which they contribute to CVDs are unclear. Here we show that hematopoietic cells harboring C-terminally truncated ASXL1 mutant (ASXL1-MT) accelerate the development of atherosclerosis in Ldlr–/– mice. Transcriptome analyses of plaque cells showed that monocytes and macrophages expressing ASXL1-MT exhibit inflammatory signatures. Mechanistically, we demonstrate that wild-type ASXL1 has an unexpected non-epigenetic role by suppressing innate immune signaling through the inhibition of IRAK1–TAK1 interaction in the cytoplasm. This regulatory function is lost in ASXL1-MT, resulting in NF-κB activation. Inhibition of IRAK1/4 alleviated atherosclerosis driven by ASXL1-MT and decreased inflammatory monocytes. The present work provides a mechanistic and cellular explanation linking ASXL1 mutations, CH and CVDs.
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Data availability
RNA-seq data are available in the Gene Expression Omnibus (GEO) under accession number GSE279450. scRNA-seq data have been deposited in the GEO under accession number GSE279601. The mass spectrometry data have been deposited in the jPOST44 repository under accession number JPST003417. Source data are provided with this paper.
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Acknowledgements
We thank S. Shikata (Graduate School of Frontier Sciences, University of Tokyo) for her expert technical assistance. We thank the IMSUT FACS Core for assisting with flow cytometry analyses, the IMSUT Laboratory Animal Research Centre for housing mice and the IMSUT Pathology Core Laboratories for assisting with pathological analyses of murine organs. We also thank D. Oikawa (Osaka Metropolitan University), F. Tokunaga (Osaka Metropolitan University) and M. Yamamoto (Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo) for giving the plasmids. We are also immensely grateful to Y. Wang (University of Tsukuba) for supporting the analysis of the atherosclerotic lesions. This work was supported by Grant-in-Aid for Scientific Research (A) (no. 20H00537) (to T.K.), Grant-in-Aid for Scientific Research on Innovative Areas (no. 19H04756) (to T.K.), the Tokyo Biochemical Research Foundation (to T.K.), Grant-in-Aid for Scientific Research (B) (no. 19H03685) (to S.G.), the Japan Foundation for Aging and Health (to S.G.) and the Koyanagi Foundation (to S.G.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Authors and Affiliations
Contributions
N.S. designed and performed most of the experiments, analyzed and interpreted the data and wrote the paper. S.G. conceived the project and interpreted the data. Y.-H.C., M.M., T.F., X.L., K.Y., S.A., R.T., T.Y. and Y.T. assisted with the experiments. T.S. and K.M. provided the BaκB cells and advised on the data interpretation. M.S. and H.S. provided the Ldlr−/− mice and advised on the analysis of the atherosclerotic lesions. T.D. and Y.O. assisted in the analysis and staining of the atherosclerotic lesions. S.K. and A.I. performed the scRNA-seq and bulk RNA-seq experiments. K.U. performed the LC–MS analyses. C.L. and E.M. provided the IRAK1/4 inhibitors R191 and R221. T.I. assisted with the statistical analysis of scRNA-seq. J.-i.I. provided plasmids and advised on the analysis of the innate immune pathway. H.H. generated the ASXL1-MT KI mice. S.G. and T.K. secured the research budget, guided the research and wrote the paper, with N.S.
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E.M. and C.L. own stock in and are employees of Rigel Pharmaceuticals, Inc. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Mutant ASXL1 induces expansion of inflammatory monocytes in response to a high-fat diet.
a: The experimental design for competitive transplantation assays with high-fat diet (HFD) feeding. Briefly, bone marrow cells (BMCs) from Vav-cre ASXL1-MT KI mice and littermate control mice were mixed at a 1:1 ratio, followed by transplantation into lethally irradiated Ly5.1 recipient mice. Four weeks later to allow for hematopoietic reconstitution, mice were separated into a HFD-fed group and control diet-fed group. PBCs, peripheral blood cells. b: The levels of the chimerism of GFP positive ASXL1-MT cells in peripheral blood were analysed at day 0 and 4 weeks after the transplantation (n = 18). c: Fold change of the chimerism of GFP + ASXL1-MT cells in peripheral blood compared with the chimerism at the start of HFD or control diet feeding (n = 9 (5 female and 4 male) per each group, respectively). Data are the mean ± s.e.m. Data were analysed by mixed-effects analysis (REML) and Šídák’s multiple comparisons test. Row (month) Factor x Column (Diets) Factor; P = 0.0110. Šídák’s multiple comparisons test; *†P = 0.0300, **P = 0.0074, *‡P = 0.0317, *§P = 0.0404, *||P = 0.0247. d: The frequencies of neutrophils, inflammatory monocytes, and patrolling monocytes in PBCs of GFP-control or GFP + ASXL1-MT cells in mice fed a control diet or HFD at 6 months after transplantation (n = 9 for each group). The percentage of neutrophils, inflammatory monocytes, and patrolling monocytes derived from ASXL1-MT KI bone marrow (MT) or littermate-control bone marrow (WT) in control diet-fed mice or HFD-fed mice in the competitive transplantation assay after 5 months of feeding (n = 7 mice per group). See also Extended_data_Fig. S1. Data are the mean ± s.d. Paired two-tailed t-tests (c: comparison between MT and WT) and unpaired two-tailed t-tests (b and c: comparison between HFD-fed mice and control diet-fed mice) were used for the statistical analysis. Neutrophils: P = 0.0357 (control diet-fed MT vs HFD-fed MT), P = 0.0002 (HFD-fed WT vs HFD-fed MT); Inflammatory Monocytes: P = 0.0002 (control diet-fed WT vs control diet-fed MT), P < 0.0001 (HFD-fed WT vs HFD-fed MT), P = 0.0047 (control diet-fed MT vs HFD-fed MT); B cells: P = 0.0136 (control diet-fed WT vs control diet-fed MT), P < 0.0001 (HFD-fed WT vs HFD-fed MT), respectively. e: The experimental design of HFD- or control diet-feeding with ASXL1-MT KI mice or littermate control wild-type mice. f: The body weight of control or Vav-cre ASXL1-MT KI mice fed a control diet or HFD at the indicated days after transplantation (left: n = 4 male, right: n = 5 female). Data are the mean ± s.e.m. g: The absolute number of white blood cells (WBC), haemoglobin (Hb), platelets, neutrophils, inflammatory monocytes, and patrolling monocytes in the peripheral blood of control or Vav-cre ASXL1-MT KI mice after 5 months of feeding on a control diet or HFD. n = 5 (2 female, 3 male), 5 (3 female, 2 male), 4 (2 female, 3 male), 6 (3 female, 3 male) for WT mice fed the control diet, Vav-cre ASXL1-MT KI mice fed the control diet, WT mice fed HFD, and Vav-cre ASXL1-MT KI mice fed HFD, respectively. Data are the mean ± s.d. Data were assessed by one-way ANOVA with Tukey–Kramer’s post-hoc test.
Extended Data Fig. 2 HFD accelerates the expansion of ASXL1-MT cells in the myeloid population.
a: The levels of the chimerism of CD45.2+ donor cells in peripheral blood 4-week after the transplantation (n = 8, respectively). Unpaired t-tests were used for the statistical analysis. b, c: Levels of donor chimerism of CD45.2+ cells in B cells (c) and T cells (d) at the indicated weeks after the start of HFHCD feeding following the competitive transplantation (n = 8, 8, 7 Mx1-cre−/− control, n = 8, 8, 8 Mx1-cre+/− ASXL1-MT KI for weeks 0, 6, and 12, respectively). Data are the mean ± s.e.m.; two-way ANOVA with Šídák’s multiple comparison test. d–h: The counts of white blood cells (WBC) (d), haemoglobin (e), red blood cells (RBC) (f), platelets (g), and BMCs (h) in Ldlr−/− mice after 12 weeks of feeding on HFHCD. Unpaired two-tailed t-tests. i–l: The weights of body (i), spleen (j), liver (k) and kidneys (l) in Ldlr−/− mice after 12 weeks of feeding on HFHCD. e-m: Data are presented as the mean ± s.d. (n = 7 Mx1-cre−/− control, n = 8 Mx1-cre+/− ASXL1-MT KI, respectively). Unpaired two-tailed t-tests.
Extended Data Fig. 3 Mutant ASXL1-derived haematopoiesis promotes atherosclerosis in Ldlr−/− mice.
a: Representative images of the whole aorta stained with Sudan IV from female Ldlr−/− mice with Vav-cre ASXL1-MT KI or control bone marrow after 12 weeks of HFHCD feeding. b: Quantification of the lesion area in the whole aorta (n = 5 female mice per group). c, d: Quantification of the lesion area in the aortic root (A) and whole aorta (B) in male Ldlr−/− mice with WT or Vav-cre ASXL1-MT KI bone marrow after 14 weeks of HFHCD feeding. (n = 5 WT, 6 Vav-cre ASXL1-MT.) e, f: Body weight and major organ weight of female (e) and male (f) Ldlr−/− mice transplanted with WT or Vav-cre ASXL1-MT KI bone marrow at the time of the atherosclerotic lesion analysis. (n = 5 WT female, 6 ASXL1-MT female, 5 WT male, 6 ASXL1-MT male.) g: The frequencies of neutrophils, total monocytes, inflammatory monocytes, and patrolling monocytes in peripheral blood cells in Ldlr−/− female mice fed the HFHCD for 12 weeks (n = 9 for each group). Data are shown as the mean ± s.d.; Unpaired two-tailed t-tests with Welch’s correction (b–g).
Extended Data Fig. 4 Effects of mutant ASXL1 expression on myeloid cells in the atherosclerotic condition.
a, b: Representative FACS histograms of GFP expression in peritoneal macrophages (a) and peripheral blood myeloid cells (b) from LysM-cre ASXL1-MT KI mice. Blue shows the expression of CD11bneg lymphocytes as a negative control. c: Quantification of the lesion area in the aortic root of Ldlr−/− mice with WT or LysM-cre +/– ASXL1-MT KI bone marrow after 14 weeks of HFHCD feeding (n = 12 mice per group). d–k: The counts of white blood cells (WBC) (d), red blood cells (RBC) (e), platelets (f), neutrophils (g), inflammatory monocytes (h), patrolling monocytes (i), B cells (j) and T cells (k) in Ldlr−/− mice with LysM-cre+/– ASXL1-MT KI or control bone marrow after 14 weeks of HFHCD feeding (n = 6 mice per group). Data are shown as the mean ± s.d.; Unpaired two-tailed t-tests (c–k).
Extended Data Fig. 5 scRNA-seq clustering of cells dissociated from aorta of Ldlr−/− mice.
a: Heat map of the top 10 differentially expressed genes enriched in each cluster. b: Table showing the top ten differentially expressed genes in each cluster with cell annotations. c: Feature plot of the representative marker gene in each cluster to determine cell annotations.
Extended Data Fig. 6 Mutant ASXL1-derived monocytes/macrophages show inflammatory-biased features.
a: Bar graph of the cell counts of each cluster in scRNA-seq analysis of plaque cells. UMAP plot of CD45+ cells from aortas of Ldlr−/− mice with 100% bone marrow of the indicated genotypes (1st experiment: control 1 mouse, ASXL1-MT 1 mouse; 2nd experiment: control 2 mice pooled; ASXL1-MT 2 mice pooled). Data are the mean ± s.d.; Data were analysed by Two-way ANOVA and Šídák’s multiple comparisons test. Column (mutation) Factor; P = 0.0094. Šídák’s multiple comparisons test; P = 0.0341 (Res-like macrophages), P = 0.0415 (Granulocytes-1). b: Violin plots of Cxcl12 in Cluster 2 Res-like macrophages. c: Feature plots of the 1st experimental pair merged with Cxcr4 (red) and Cxcl12 (green). d: Dot plots of the gene set enrichment analysis (GSEA) of inflammatory macrophages (Cluster 0 and 4) and of monocytes (Cluster 10) from aorta of Ldlr–/– mice. The significance test is single tail test on the appropriate (positive/negative) side of the null distribution. e, f: Bulk RNA-seq data of plaque CD11b+ F4/80+ macrophages from mice transplanted with ASXL1-MT BMCs or mice transplanted with control BMCs (n = 3 per group). Relative mRNA expression (FPKM) of regulatory genes for Toll-like receptor (TLR)/IL-1 receptor signalling (e) and of inflammasome/Map3k genes (f) in aortic macrophages derived from ASXL1-MT-KI bone marrow versus control. Data are shown as the mean ± s.d.; Unpaired t-tests (e, f).
Extended Data Fig. 7 Mutant ASXL1 confers macrophage hypersensitivity towards inflammation.
a: Quantitative RT-PCR analysis of the proinflammatory cytokine genes Tnf, Il-6, and Il-1b in WT and ASXL1-MT KI BMDMs treated with lipopolysaccharide (LPS) (1 μg/mL) for the indicated lengths of time. Values and error bars represent the mean ± s.d. of triplicates and are representative of at least two independent experiments. Unpaired two-tailed t-tests were used for the statistical analyses. Il6: P = 0.0004 (2 h), P < 0.0001 (4 h); Il1b: P = 0.0148 (4 h), respectively. b: Quantitative RT-PCR analysis of the proinflammatory cytokine genes Tnf, Il-6, Il-1b, and Nlrp3 in WT and ASXL1-MT KI BMDMs treated with IL-β (10 ng/mL), IL-18 (100 μg/mL) or oxidized low-density lipoprotein (oxLDL) (25 μg/mL) for 24 h. Values and error bars represent the mean ± s.d. of triplicates. Unpaired two-tailed t-tests were used for the statistical analyses. c–f: BMDMs derived from an ASXL1-MT KI mouse or control mouse were plated in triplicates and treated with 100 ng/mL LPS combined for 4 h combined with a final 30 min incubation with 5 mM ATP (pH 7.0). Western blot analysis (c) and quantification of pro-IL-1β, pro-caspase-1, cleaved caspase-1, and α-tubulin in WCL, and of cleaved IL-1β and cleaved caspase-1 in supernatant were assessed by ImageJ (d, f). Levels of Il-1β in culture supernatants of BMDMs derived from an ASXL1-MT KI mouse or control mouse were assessed by ELISA (e). Values and error bars represent the mean ± s.d. of triplicates. Unpaired two-tailed t-tests were used for the statistical analyses. The samples were derived from the same experiment and blots were processed in parallel (c). g: Quantitative RT-PCR analysis of Asxl1 gene and the proinflammatory cytokine genes Tnf, Il-6, and Il-1b in lentivirus-mediated Asxl1 KO BMDMs derived from Rosa26-LSL-Cas9-KI mice. The lentivirus with non-targeting single guide RNA (sgNT) was used as the control. BMDMs were treated with 100 ng/mL LPS for 2 h. Values and error bars represent the mean ± s.d. of triplicates. Unpaired two-tailed t-tests were used for the statistical analyses. h: BaκB cells expressing Cas9 and TLR5 were treated with lentivirus-mediated Asxl1 KO, followed by stimulation with flagellin (5 ng/mL) for 18 h. GFP induction was assessed by flow cytometry as a reporter of NF-κB. The results are represented by MFI of more than 1 × 104 cells which were cultured and treated in different three wells. Data are the mean of MFI ± s.d. and were assessed by one-way ANOVA with Tukey–Kramer’s post-hoc test. P < 0.0001 (unstimulated); P < 0.0001 (stimulated), respectively.
Extended Data Fig. 8 ASXL1 interacts with inflammatory factors including IRAK1-TAK1 complex.
a: Scheme of liquid chromatography-mass spectrometry (LC-MS) with immunoprecipitated-ASXL1 in 293 T cells. 293 T cells were transfected with N-terminus 3x FLAG-tagged ASXL1-WT or N-terminus 3x FLAG-tagged ASXL1-MT. Binding proteins were analysed by LC-MS. b, c: Pathway enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) 2021 Human data sets with IP-MS using ASXL1-WT-specific binding proteins in 293 T cells. The top 10 enriched pathways (b) and top 20 genes included in each enriched pathway (c) are shown. For the pathway analysis, proteins that bind to ASXL1-WT 10-fold more than ASXL1-MT were used. Bars represent –log10 (p-value) of interacting proteins that appear in the designated pathway. The terms of the infectious disease gene sets including TLR signalling are coloured red. d: Scheme of endogenous-ASXL1 tagging with 3x-FLAG-P2A-Blasticidin S deaminase (BSD) in THP-1 cells using CRISPR-Cas9. THP-1 cells expressing Cas9 were simultaneously transfected with the appropriate amount of donor plasmid and gRNA plasmid using Lonza 4D-Nucleofector. e: A western blot of ASXL1 with immunoprecipitated samples for the RIME assay. f: The top 20 genes included in top 10 enriched pathway by Enrichr-based KEGG 2021 Human pathway enrichment analysis using 3xFLAG-tagged endogenous ASXL-specific binding proteins in THP-1 cells. The terms of the infectious disease gene sets including TLR signalling are coloured red. The pathway enrichment analyses were performed using Enrichr (https://maayanlab.cloud/Enrichr/)46,47,48.
Extended Data Fig. 9 ASXL1 interacts with major components of the innate immune pathway in the cytoplasm.
a, b: 293 T cells were transfected with a 3xFLAG-ASXL1-WT, 3xFLAG-ASXL1-MT, or 3xFLAG-ASXL1-CT expression plasmid together with a plasmid encoding HA-TAB2 (a) or HA-TAB3 (b). Cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with anti-HA antibody. WCL was also prepared as input and analysed by immunoblotting with anti-HA antibody and anti-FLAG antibody. Representative blots from two replicates are shown. c: 293 T cells were transfected with a 3xFLAG-ASXL1-WT or 3xFLAG-ASXL1-MT expression plasmid together with a plasmid encoding HA-Tak1. Cell lysates were immunoprecipitated with anti-HA antibody, and Tak1-bound ASXL1 were detected by immunoblotting with anti-FLAG antibody. d: 293 T cells transfected with a 3xFLAG-ASXL1-WT or 3xFLAG-ASXL1-MT expression plasmid together with a plasmid encoding HA-tagged TAK1 or HA-tagged catalytically inactive TAK1-K63W were immunoblotted for HA (TAK1 or TAK1-K63W) on immunoprecipitated FLAG-ASXL1. WCL was prepared and analysed by immunoblotting with anti-HA, anti-FLAG, and anti-phosphorylated TAK1 (phospho-TAK1) antibodies. A representative blot from two replicates is shown. e: Immunoblots for ASXL1 in the nuclear (Nucleus) and cytoplasmic (Cytoplasm) fractions isolated from primary BMDMs from a ASXL1-MT KI mouse and cells from a littermate control mouse. f: 293 T cells were transfected with 3xFLAG-ASXL1. The cells were fixed and subjected to immunofluorescence staining. ASXL1 (red) is superimposed over nuclei stained with DAPI (blue). Representative images for vector control (top), ASXL1-WT (middle), and ASXL1-MT (bottom) are shown. Scale bars: 75 μm. Images were captured by EVOS. g: 293 T cells were transfected with a 3xFLAG-ASXL1-WT or 3xFLAG-ASXL1-MT expression plasmid together with a plasmid encoding HA-IRAK1. Nuclear (right) and cytoplasmic (left) protein fractions were separated using hypotonic buffer and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with anti-HA antibody. h: Immunoprecipitation of FLAG-ASXL1 followed by immunoblotting of IRAK1 and FLAG from THP1 cells that are stably expressing ASXL1-WT (WT), ASXL1-MT (MT), ASXL1-CT (CT), or empty vector (Mock). i: Immunoblots for p65 NF-κB in the nuclear (Nucleus) and cytoplasmic (Cytoplasm) fractions isolated from primary BMDMs from a ASXL1-MT KI mouse and cells from a littermate control mouse. BMDMs were treated with 100 ng/mL LPS or PBS for 30 minutes before lysis. Quantification of the ratio of p65 NF-κB to LaminB1 were assessed by ImageJ.
Extended Data Fig. 10 ASXL1 interacts with IRAK1 at C-terminal lesions and regulates TAK1-mediated NF-κB signalling.
a: 293 T cells were transfected with FLAG-TAK1 and HA-K63Ub and pre-treated with IRAK1/4 inhibitor R191 (100 nM) for 1 h. The K63-linked polyubiquitination of TAK1 was assessed by immunoblotting for HA (K63Ub) on immunoprecipitated FLAG-TAK1. b, c: THP-1 cells (b) and RAW264.7 cells (c) were pre-treated with IRAK1/4 inhibitor R191 (100 nM) for 30 min and stimulated with 1 μg/mL LPS for 30 min to evaluate the phosphorylation of IκB, JNK, and p38 MAPK. d: BMDMs derived from ASXL1-MT KI mice and control mice were pre-treated with IRAK1/4 inhibitor R191 (100 nM) for 1 h and stimulated with 1 μg/mL LPS for 30 minutes to evaluate the phosphorylation of TAK1(T187), IκB, JNK, and p38 MAPK. Quantification of the relative levels obtained by densitometry analysis of the images of pTAK1(T187) compared to total TAK1 are shown under pTAK1. All samples were derived from the same experiment and blots were processed in parallel (b–d). e: BMDMs derived from ASXL1-MT KI mice and control mice were pre-treated with IRAK1/4 inhibitor R191 (100 nM) for 1 h and stimulated with 100 ng/mL LPS for 5 minutes or 30 minutes to evaluate the phosphorylation of TAK1(S412), TAK1 (T187), and IκB. The relative levels of pTAK1(S412) to total TAK1 and of pTAK1(T187) to total TAK1 were assessed by ImageJ. f: Outline of the transplantations of WT or Vav-cre ASXL1-MT KI bone marrow into Ldlr−/− recipient mice followed by IRAK1/4 inhibitor (R221) treatment. One month post-transplantation, the recipient mice were separated into the high fat/high cholesterol diet (HFHCD) group and R221-containing HFHCD group. Peripheral blood cells (PBCs) were analysed after 6 weeks of feeding. Atherosclerotic lesions were analysed after 12 weeks of feeding.
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Unprocessed western blots.
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Sato, N., Goyama, S., Chang, YH. et al. Clonal hematopoiesis-related mutant ASXL1 promotes atherosclerosis in mice via dysregulated innate immunity. Nat Cardiovasc Res 3, 1568–1583 (2024). https://doi.org/10.1038/s44161-024-00579-w
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DOI: https://doi.org/10.1038/s44161-024-00579-w
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