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Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis

Nature Cardiovascular Research volume 2, pages 572–586 (2023)Cite this article

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Abstract

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

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Fig. 1: Increased monocytosis in Tet2 CH mice is seen with reversal by IL-6R antibody administration.
Fig. 2: IL-6R antagonism ameliorates atherosclerosis in Tet2 CH mice.
Fig. 3: IL-6 increases CSF1R expression and apoptosis resistance in Tet2-deficient macrophages.
Fig. 4: Accelerated atherosclerosis in Tet2 CH mice is reversed by PLX3397.
Fig. 5: Human ESC-derived TET2-deficient macrophages show increased CSF1R expression and resistance to apoptosis after IL-6 treatment.

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Data availability

The data supporting the findings of this study are available within the paper and its supplementary information. Data regarding the single cell RNA sequence of CSF1R expression can be accessed at https://zenodo.org/record/7876218#.ZEvhOHbMIVA. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (nos. HL-118567 and HL-148071 to N.W.; no. HL155431 to A.R.T.) and the Leducq Foundation (no. TNE-18CVD04 to A.R.T.). P.L. receives funding support from the National Heart, Lung, and Blood Institute (nos. 1R01HL134892 and 1R01HL163099-01), the RRM Charitable Fund and the Simard Fund.

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Author notes

  1. These authors contributed equally: Wenli Liu, Nan Wang, Alan R. Tall.

Authors and Affiliations

  1. Division of Molecular Medicine, Department of Medicine, Columbia University, New York, NY, USA

    Wenli Liu, Mustafa Yalcinkaya, Ranran Wang, Tong Xiao, Ira Tabas, Nan Wang & Alan R. Tall

  2. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Inés Fernández Maestre, Pablo Sánchez Vela & Ross Levine

  3. Louis V. Gerstner Jr Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Inés Fernández Maestre

  4. Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Malgorzata Olszewska & Eirini P. Papapetrou

  5. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Malgorzata Olszewska & Eirini P. Papapetrou

  6. Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Malgorzata Olszewska & Eirini P. Papapetrou

  7. Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    Patrick B. Ampomah

  8. Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

    J. Brett Heimlich

  9. Division of Genomic Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

    Alexander G. Bick

  10. Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Peter Libby

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Contributions

W.L. designed and performed the experiments, analyzed the data and wrote the manuscript. M.Y. performed the experiments and analyzed the data. I.F.M. and P.S.V. provided the mouse bone marrow for the transplantation experiments and wrote the manuscript. M.O. and E.P.P. designed and conducted the experiments related to human ESCs and iPSC-derived macrophages. P.B.A. performed the experiments and analyzed the data. J.B.H. and A.G.B. provided the human TET2 CH RNA-seq data. R.W. and T.X. performed the experiments and analyzed the data. R.L., E.P.P. and I.T. shared the reagents, designed the experiments and provided scientific feedback about the manuscript. N.W. and A.R.T. designed the experiments and wrote the manuscript.

Corresponding authors

Correspondence to Wenli Liu, Nan Wang or Alan R. Tall.

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Competing interests

A.R.T. is a consultant for Amgen, CSL Behring, AstraZeneca and Foresite Laboratories, and is on the scientific advisory board of Staten Biotechnology, Fortico Biotech and Beren Therapeutics. P.L. is an unpaid consultant to, or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, MedImmune, Merck, Moderna, Novo Nordisk, Novartis, Pfizer and Sanofi Regeneron. P.L. is a member of the scientific advisory board for Amgen, Caristo Diagnostics, Cartesian Therapeutics, CSL Behring, DalCor Pharmaceuticals, Dewpoint Therapeutics, Elucid Bioimaging, Kancera, Kowa Pharmaceuticals, Olatec Therapeutics, MedImmune, Novartis, PlaqueTec, TenSixteen Bio, Soley Thereapeutics and XBiotech. His laboratory has received research funding in the last 2 years from Novartis, Novo Nordisk and Genentech. P.L. is on the board of directors of XBiotech. He has a financial interest in XBiotech, a company developing therapeutic human antibodies, in TenSixteen Bio, a company targeting somatic mosaicism and CHIP to discover and develop new therapeutics to treat age-related diseases, and in Soley Therapeutics, a biotechnology company that is combining artificial intelligence with molecular and cellular response detection to discover and develop new drugs, currently focusing on cancer therapeutics. His interests were reviewed and are managed by Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. The other authors declare no competing interests.

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Nature Cardiovascular Research thanks Kathryn Moore, Siddhartha Jaiswal and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1

a. Ldlr−/− male mice were transplanted with WT or Tet2KO male mice bone marrow; after 6 weeks reconstitution, WT→Ldlr−/− (n = 9) and Tet2KO→Ldlr−/− (n = 10) mice were fed a western diet for 12 weeks. Bar graph shows total lesion area in the aortic root. Two-tailed unpaired t-test. b-c. WT and Tet2KO macrophage were pretreated with or without IL-1β (100 μg/ml) blocking antibodies for 1 hr, then treated with LPS (20 ng/ml) for 4 hours followed by nigericin (5 μg/ml) for another 30 mins. Secreted IL-1β and IL-6 were determined by ELISA. n = 5 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. d. Bone marrow derived macrophages were pre-incubated with IL-6R blocking antibodies (100 μg/ml) for 1 hour and then challenged with or without IL-6 (25 ng/ml). qPCR was used to measure the mRNA level of Socs3 expression. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. e-g. Peripheral blood white blood cells counts, RBC counts and platelet counts after 12 weeks western diet. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. h-l. Body weight, plasma cholesterol and triglycerides, liver/body weight ratio, spleen/body weight ratio after 12 weeks western diet. m. Immunoblot of isolated splenic monocytes and neutrophil Caspase1, cleaved IL-1β and β-actin after 12 weeks western diet. n = 6 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test or two-tailed unpaired t-test. n. Immunofluorescence staining of macrophage (anti-Mac2, Green), TREM2 (Red) in aortic roots and quantification of TREM2+Mac2+ cells as the percentage of total Mac2+ cells per lesion area in aortic root cross-sections. Scale Bar, 50μm. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. o. Immunofluorescence staining of macrophage (anti-Mac2, Green), IL-1β expression (Red) in aortic roots and quantification of IL-1β mean fluorescence intensity per lesion area in aortic root cross-sections. Scale Bar, 50μm. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. p. Quantification of CSF1R expression in monocytes of Ctrl and Tet2+/− mice. n = 5 mice in ctrl group. n = 6 mice in Tet2 + /- group. Two-tailed unpaired t-test. All the Data are Mean ± SEM.

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Extended Data Fig. 2

a. Gating strategy for LSK cells and Hematopoietic progenitor cells (HPC, the first row), LSK (Sca1+c-Kit+) subpopulation (middle row) and HPC (Sca1−c-Kit+) subpopulation (the third row). For the LSK subpopulation, CD150+CD48−, CD150−CD48−, CD150−CD48+ LSK fractions are long term HSC (LT-HSC), short term HSC (ST-HSC) and multipotent progenitors (MPP) respectively. For the HPC subpopulation, the cells are defined as common myeloid progenitors (Lin−Sca1−c-Kit+CD34+CD16/32int; CMP), granulocyte and macrophage progenitors (Lin−Sca1−c-Kit+CD34+CD16/32hi, GMP). b. Hematopoietic stem and progenitor cells in BM. n = 12 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. c-h. CSF1R expression on stem and progenitor cells as determined by flow cytometry shown as mean fluorescence intensity and percentage of positive cells. n = 12 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. All the Data are Mean ± SEM.

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Extended Data Fig. 3

a. Immunoblot and quantification of p-STAT3/STAT3 with or without IL-6 (25 ng/ml). n = 3 independent experiments. Two-tailed unpaired t-test. b. BMDMs were pretreated with trichostatin A (TSA, 100 nM) or DMSO for 1 hour and then incubated with IL-6 (25 ng/ml) for 2 hours. The level of nuclear STAT3 acetylation was determined by western blot. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. c. BMDMs were incubated with IL-6 (25 ng/ml) for indicated time. Methylation levels of the STAT3 binding site in the Csf1r promoter region of macrophages were analyzed. n = 3 independent experiments. Two-tailed unpaired t-test. d. Immunoblot and quantification of DNMT1, DNMT3A and DNMT3B expression in BMDMs with or without IL-6 (25 ng/ml) treatment for 4 hours. n = 5 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. e. BMDMs were pretreated with 5-Azacytidine (5-aza, 1 μM) or DMSO for 1 hour and then incubated with or without IL-6 (25 ng/ml) for 8 hours. mRNA level of Csf1r was tested by qPCR. n = 4 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. f. BMDMs were pretreated with 5-aza (1 μM) or DMSO for 1 hour and then incubated with IL-6 (25 ng/ml) for 4 hours. STAT3 binds to the Csf1r promoter area was revealed by ChIP-qPCR assay. Shown is percentage of input. n = 4 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. g. BMDMs were pretreated with TSA (100 nM) or DMSO for 1 hour and then incubated with (25 ng/ml) for 4 hours. Methylation levels of the STAT3 binding site in the Csf1r promoter region of macrophages. n = 5 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. All the Data are Mean ± SEM.

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Extended Data Fig. 4

Immunoblot of CSF1R and cleaved Caspase 3 in macrophage with or without IL-6 (25 ng/ml) treatment for 48 hours. Bar graph shows the quantification of immunoblots. n = 5 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. All the Data are Mean ± SEM.

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Extended Data Fig. 5

Ctrl and Tet2CH mice were fed a western diet for 7 weeks followed by another 5 weeks with or without PLX3397 (200 mg/kg) formulated WD feeding. a. Food intake per day per mouse during 5 weeks PLX3397 WD feeding period. n = 3 independent cages per group. One-way ANOVA followed by Tukey’s multiple-comparison test. b. Plasma cholesterol levels. n = 11 mice in Ctrl+Control group, n = 12 mice in Tet2CH+Control group, n = 13 mice in Ctrl+PLX3397 group, n = 13 mice in Tet2CH + PLX3397 group. One-way ANOVA followed by Tukey’s multiple-comparison test. c-f. Body weight, liver/body weight ratio, spleen/body weight ratio and peripheral blood white blood cells counts, RBC counts and platelet counts after 12 weeks western diet. n = 11 mice in Ctrl+Control group, n = 12 mice in Tet2CH+Control group, n = 13 mice in Ctrl+PLX3397 group, n = 13 mice in Tet2CH + PLX3397 group. One-way ANOVA followed by Tukey’s multiple-comparison test. g. Control and Tet2KO BMDMs were treated with or without oxidized LDL (oxLDL, 50 μg/ml), acetylated LDL (acLDL, 50 μg/ml) or cholesterol crystal (CHO-C,100 μg/ml) overnight and then challenged with LPS (20 ng/ml) for 4 hours with fresh media. Secreted IL-6 level was quantified by ELISA. n = 4 independent experiments. Two-tailed unpaired t-test. All the Data are Mean ± SEM.

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Extended Data Fig. 6

a. Flow cytometric analysis of cell surface molecular expression on human embryonic stem cell-derived TET2 deficient macrophages and its isogenic control in day 24. n = 3 independent experiments. Two-tailed unpaired t-test. b. Strategy for gating and confirmation of induced pluripotent stem cell (iPSC) with TET2 haploinsufficiency (TET+/−) and isogenic control for the progenitors (CD34+) and lineage-committed (CD34 + CD45+) cells differentiation on Day1. c. Control and TET2+/− iPSCs were treated with or without hIL-6 (25 ng/ml) for 48 hours. CSF1R expression was analyzed by flow cytometry. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. d. Flow cytometric analysis of cell surface molecular expression on iPSC derived TET+/− and isogenic control macrophage after 24 days of differentiation. n = 3 independent experiments. Two-tailed unpaired t-test. e. Control and TET2+/− iPSC-derived macrophages were treated with or without hIL-6 (25 ng/ml) for 48 hours. CSF1R expression was quantified by flow cytometry. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. f. Immunoblot and quantification of CSF1R and cleaved Caspase 3 in control and TET2+/− iPSC-derived macrophages with or without hIL-6 (25 ng/ml) treatment for 48 hours. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. g. TET2+/− and its isogenc control iPSCs were treated with or without oxidized LDL (oxLDL, 50 μg/ml), acetylated LDL (acLDL, 50 μg/ml) or cholesterol crystal (CHO-C,100 μg/ml) overnight and then challenged with LPS (20 ng/ml) for 4 hours with fresh media. Secreted IL-6 level was quantified by ELISA. n = 4 independent experiments. Two-tailed unpaired t-test. All the Data are Mean ± SEM.

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Extended Data Fig. 7 A schematic summary of the main findings.

The schema illustrating proposed mechanisms underlying the benefit of IL-6R or CSF1R inhibition in Tet2 CH-accelerated atherosclerosis.

Supplementary information

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Source Data Figs. 2, 3 and 5 and Extended Data Figs. 1, 3, 4 and 6.

Uncropped immunoblots for Figs. 2, 3 and 5 and Extended Data Figs. 1, 3, 4 and 6.

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Liu, W., Yalcinkaya, M., Maestre, I.F. et al. Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis. Nat Cardiovasc Res 2, 572–586 (2023). https://doi.org/10.1038/s44161-023-00281-3

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