Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter

Journal name:
Nature Medicine
Volume:
21,
Pages:
820–826
Year published:
DOI:
doi:10.1038/nm.3890
Received
Accepted
Published online

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1, 2. Interruption of IL18 action reduces atherosclerosis in mice3, 4. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

At a glance

Figures

  1. Identification of alternative IL18-binding proteins.
    Figure 1: Identification of alternative IL18-binding proteins.

    (a) Aortic root lesion intima, Mac-3+ macrophage, CD4+ T cell and α-actin–positive SMC areas in Apoe−/− (n = 10) and Apoe−/−Il18r−/− (n = 7) mice. Data are mean ± s.e.m. (b) FACS of endothelial cells isolated from Apoe−/− or Apoe−/−Il18r−/− mice after binding with biotin-IL18 with and without an excess of unlabeled IL18 (50 or 100 ng/ml), followed by incubation with phycoerythrin (PE)-streptavidin. (c) p-Tyr detected by immunoblotting in endothelial cells isolated from Apoe−/− or Apoe−/−Il18r−/− mice treated with or without IL18 or heat-inactivated IL18 for 30 min. β-actin was used as a loading control. (d) Identification of IL18-binding proteins in endothelial cells from Apoe−/−Il18r−/− mice. SDS-PAGE silver staining was used to detect IL18-bound proteins in three sequential eluates from a column containing anti-IL18 antibody-bound protein A–agarose beads that had been loaded with cell lysates from endothelial cells treated with or without IL18.

  2. NCC expression and characterization.
    Figure 2: NCC expression and characterization.

    (a) Immunostaining of NCC in normal human (bar, 1,000 μm) and mouse aortas (bar, 50 μm). (b) Immunostaining of NCC, IL18r and CD68+ macrophages in human atherosclerotic lesions. Negative controls were rabbit and mouse (not shown) IgG. Bars, 500 μm. (c) Immunostaining of NCC in lesions from Apoe−/− mice. Macrophage, SMC and endothelial areas are boxed and shown at higher magnification. Negative controls were lesions from Apoe−/−Ncc−/− mice. Bars, 200 μm; inset bars, 50 μm. (d) Immunofluorescence staining of NCC (red) and IL18r (green) in macrophages, SMCs and endothelial cells (ECs) in atherosclerotic lesions from Apoe−/− mice. Autofluorescence refers to fluorescence without primary antibody. Bars, 50 μm. (e) RT-PCR results and immunostaining to detect NCC in peritoneal macrophages (bars, 50 μm) and splenic T cells (bars, 20 μm) isolated from Apoe−/− mice, treated with or without IL18. Macrophages from Apoe−/−Ncc−/− mice were used as a negative control for immunostaining. (f) Immunoblots to detect NCC in cytokine-treated endothelial cells and SMCs from WT mice. Doublet bands corresponding to NCC are indicated. Lysate from NCC-transfected COS-7 cells was used as a positive control. (g) IL18r immunoprecipitation followed by NCC immunoblotting to detect NCC-IL18r complexes in macrophages from Apoe−/− mice treated with or without IL18. Macrophages from Apoe−/−Ncc−/− mice were used as a negative control. (h) Left, FACS to detect binding of biotin-IL18 or heated biotin-IL18 to NCC- or vector-transfected COS-7 cells. Middle, Scatchard plot analysis of FITC-IL18 binding affinity to NCC-transfected COS-7 cells. Right, immunoblot detection of p-Tyr in NCC- or vector-transfected COS-7 cells treated as indicated. β-actin was used as a loading control. Data in e are mean ± s.e.m. from 36 independent experiments; the data in h are from 3–6 independent experiments. *P = 0.003.

  3. IL18r and NCC function in atherosclerosis.
    Figure 3: IL18r and NCC function in atherosclerosis.

    (af) Aortic root lesion intima area (a); thoracic-abdominal aorta oil red O staining (representative images are shown to the right) (b); aortic root lesion Mac-3+ macrophage content, CD4+ T cell numbers, and MHC class II–positive area (c); lesion SMC content (d); aortic root oil red O–positive area (e); and plasma IFN-γ, IL6 and IL18 levels (f) in Apoe−/− (n = 10), Apoe−/− Il18r−/− (n = 7), Apoe−/−Ncc−/− (n = 10) and Apoe−/−Ncc−/− Il18r−/− (n = 10) mice and in Apoe−/−Ncc−/−Il18r−/− (n = 10) mice receiving bone marrow from Apoe−/− (n = 11), Apoe−/−Il18r−/− (n = 10) or Apoe−/−Ncc−/−Il18r−/− (n = 10) mice, after mice consumed a Western diet for 12 weeks. Data are mean ± s.e.m. Bars, 1 cm. (g) IFN-γ and IL6 levels, determined by ELISA, in the culture medium of CD4+ T cells isolated from the indicated groups of mice after stimulation without (control) or with IL18 or IL18 plus IL12. Data in g are mean ± s.e.m. from three independent experiments.

  4. NCC mediates IL18 signaling and downstream cytokine and chemokine production in macrophages, COS-7 cells and FlpIn-293 cells.
    Figure 4: NCC mediates IL18 signaling and downstream cytokine and chemokine production in macrophages, COS-7 cells and FlpIn-293 cells.

    (a) Scatchard plot analysis of FITC-IL18 binding affinity to macrophages isolated from Apoe−/−Il18r−/− mice. (b) Immunoblot to detect p-ERK1/2 and p-p38 in macrophages treated with or without IL18 from the indicated groups of mice. (c) IFN-γ and MCP-1 levels, determined by ELISA, in the culture medium of macrophages treated with or without IL18, isolated from the indicated groups of mice. (d) Left, IL6 levels, determined by ELISA, in the culture medium of macrophages treated with or without IL18 and hydrochlorothiazide, isolated from the indicated groups of mice. Right, immunoblot detection of p-ERK1/2 in macrophages treated as indicated, isolated from Apoe−/− and Apoe−/−Il18r−/−. (e) Immunoblot to detect p-SPAK in macrophages treated with or without IL18 for the indicated periods of time, isolated from the indicated groups of mice. (f) Immunoblots to detect p-STAT3, p-p38 and p38 in vector- or NCC-transfected COS-7 cells that were treated with IL18 or IL18 plus IL12 for the indicated periods of time. (g,h) Immunoblots to detect p-ERK1/2 in COS-7 cells transfected with vector (pcDNA3.1 in g and pCI-neo in h) or the indicated NCC cDNA constructs and stimulated with or without IL18 for 15 min. (i) Immunoblots to detect p-NCC in whole cell lysates or membrane fractions from vector- or NCC-transfected COS-7 cells, stimulated with or without IL18. (j) Left, IL6 levels, determined by ELISA, in the culture medium of vector- or NCC-transfected COS-7 cells after stimulation without (control) or with IL18 or IL18 plus IL12. Middle, immunoblots to detect total NCC and p-NCC in FlpIn-293 cells without (Control) or with induction with tetracycline; induced cells were untreated or treated with IL18 or heat-inactivated IL18. Right, quantification of band densities in induced cells. Data in a are from 3–6 experiments; data in c, d and j are mean ± s.e.m. from 3–6 independent experiments. In immunoblots, total ERK1/2 (b) or β-actin (d,e,gj) were used as loading controls.

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Author information

  1. These authors contributed equally to this study.

    • Jing Wang &
    • Chongxiu Sun

Affiliations

  1. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Jing Wang,
    • Chongxiu Sun,
    • Norbert Gerdes,
    • Conglin Liu,
    • Mengyang Liao,
    • Jian Liu,
    • Michael A Shi,
    • Aina He,
    • Yi Zhou,
    • Galina K Sukhova,
    • Huimei Chen,
    • Jie Zhang,
    • Xiang Cheng,
    • Qiang Ke,
    • Peter Libby &
    • Guo-Ping Shi
  2. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Department of Pathophysiology, Peking Union Medical College, Tsinghua University, Beijing, China.

    • Jing Wang
  3. Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, China.

    • Chongxiu Sun
  4. Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians University Munich, Munich, Germany.

    • Norbert Gerdes
  5. Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

    • Conglin Liu
  6. Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.

    • Mengyang Liao &
    • Xiang Cheng
  7. Departments of Cardiology and Geriatrics, Graduate School of Medicine, Nagoya University, Nagoya, Japan.

    • Xian Wu Cheng,
    • Masafumi Kuzuya &
    • Toyoaki Murohara
  8. Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.

    • Mengmeng Jiang &
    • Gary E Shull
  9. Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, Oregon, USA.

    • Shaunessy Rogers,
    • Chao-Ling Yang &
    • David H Ellison
  10. Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.

    • Sabina Jelen &
    • René Bindels
  11. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Petr Jarolim

Contributions

J.W. and C.S. performed most of the experiments. N.G. completed the original IL18 and IL18r mutant mouse analysis. C.L., M.L., M.A.S., A.H., Y.Z., H.C., J.Z., X.C. and Q.K. performed RT-PCR, lesion analysis, cell culture and plasma ELISA. J.L. helped with the NCC cDNA cloning. G.K.S. performed immunostaining. X.W.C., M.K., T.M. and P.L. helped with experimental design, writing and data interpretation. M.J. and G.E.S. provided the NCC mutant mice. S.R., C.-L.Y. and D.H.E. provided the NCC monoclonal antibody and performed the 293 cell experiments. S.J. and R.B. made the human NCC mutant constructs. P.J. measured plasma Mg and K. G.-P.S. designed and performed the experiments and wrote the manuscript.

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The authors declare no competing financial interests.

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