Interleukin-1β has atheroprotective effects in advanced atherosclerotic lesions of mice

Abstract

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1β antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe-/- mice with advanced atherosclerosis were treated with anti-IL-1β or IgG control antibodies. Surprisingly, we found that IL-1β antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1β antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1β has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

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Fig. 1: IL-1β antibody treatment results in systemic and local downregulation of IL-1 signaling and pro-inflammatory pathways.
Fig. 2: IL-1β inhibition induces multiple detrimental changes in the pathogenesis of late-stage atherosclerotic lesions, including loss of an SMC-rich fibrous cap and inhibition of beneficial outward remodeling.
Fig. 3: A three-week course of IL-1β antibody treatment results in an increased number of macrophages within the fibrous cap.
Fig. 4: IL-1β inhibition for three-weeks results in increased proliferation of local macrophages but no change in monocyte trafficking or apoptosis.
Fig. 5: IL-1 signaling within SMCs is required for SMC investment into the lesion and the fibrous cap.
Fig. 6: IL-1β inhibition is associated with polarization of fibrous cap macrophages to an M2 phenotype.

References

  1. 1.

    Yahagi, K. et al. Pathophysiology of native coronary, vein graft, and in-stent atherosclerosis. Nat. Rev. Cardiol. 13, 79–98 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Kolodgie, F. D. et al. Pathologic assessment of the vulnerable human coronary plaque. Heart 90, 1385–1391 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Virmani, R., Kolodgie, F. D., Burke, A. P., Farb, A. & Schwartz, S. M. Lessons from sudden coronary death: A comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 20, 1262–1275 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Davies, M. J., Richardson, P. D., Woolf, N., Katz, D. R. & Mann, J. Risk of thrombosis in human atherosclerotic plaques: Role of extracellular lipid, macrophage, and smooth muscle cell content. Br. Heart J. 69, 377–381 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Gomez, D. & Owens, G. K. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc. Res. 95, 156–164 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Cherepanova, O. A. et al. Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. Nat. Med. 22, 657–665 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Shankman, L. S. et al. KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat. Med. 21, 628–637 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Feil, S. et al. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ. Res. 115, 662–667 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Sui, Y. et al. IKKbeta links vascular inflammation to obesity and atherosclerosis. J. Exp. Med. 211, 869–886 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Tabas, I. & Glass, C. K. Anti-inflammatory therapy in chronic disease: Challenges and opportunities. Science 339, 166–172 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Libby, P., Tabas, I., Fredman, G. & Fisher, E. A. Inflammation and its resolution as determinants of acute coronary syndromes. Circ. Res. 114, 1867–1879 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Hansson, G. K., Libby, P. & Tabas, I. Inflammation and plaque vulnerability. J. Intern. Med. 278, 483–493 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Kirii, H. et al. Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23, 656–660 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chi, H., Messas, E., Levine, R. A., Graves, D. T. & Amar, S. Interleukin-1 receptor signaling mediates atherosclerosis associated with bacterial exposure and/or a high-fat diet in a murine apolipoprotein E heterozygote model: Pharmacotherapeutic implications. Circulation 110, 1678–1685 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Isoda, K. et al. Lack of interleukin-1 receptor antagonist modulates plaque composition in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 24, 1068–1073 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Bhaskar, V. et al. Monoclonal antibodies targeting IL-1 beta reduce biomarkers of atherosclerosis in vitro and inhibit atherosclerotic plaque formation in Apolipoprotein E-deficient mice. Atherosclerosis 216, 313–320 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Ridker, P. M. et al. Effects of interleukin-1beta inhibition with canakinumab on hemoglobin A1c, lipids, C-reactive protein, interleukin-6, and fibrinogen: A phase IIb randomized, placebo-controlled trial. Circulation 126, 2739–2748 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ridker, P. M., Thuren, T., Zalewski, A. & Libby, P. Interleukin-1beta inhibition and the prevention of recurrent cardiovascular events: Rationale and design of the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS). Am. Heart J. 162, 597–605 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Ridker, P. M. et al. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N. Engl. J. Med. 377, 1119–1131 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Ridker, P. M. et al. Relationship of C-reactive protein reduction to cardiovascular event reduction following treatment with canakinumab: A secondary analysis from the CANTOS randomised controlled trial. Lancet 391, 319–328 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Chamberlain, J. et al. Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding. PLoS One 4, e5073 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Shemesh, S. et al. Interleukin-1 receptor type-1 in non-hematopoietic cells is the target for the pro-atherogenic effects of interleukin-1 in apoE-deficient mice. Atherosclerosis 222, 329–336 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Alexander, M. R., Murgai, M., Moehle, C. W. & Owens, G. K. Interleukin-1beta modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-kappaB-dependent mechanisms. Physiol. Genom. 44, 417–429 (2012).

    Article  CAS  Google Scholar 

  24. 24.

    Libby, P., Warner, S. J. & Friedman, G. B. Interleukin 1: A mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J. Clin. Invest. 81, 487–498 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Loppnow, H. & Libby, P. Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6. J. Clin. Invest. 85, 731–738 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Baylis, R. A., Gomez, D. & Owens, G. K. Shifting the focus of preclinical, murine atherosclerosis studies from prevention to late-stage intervention. Circ. Res. 120, 775–777 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Gomez, D., Shankman, L. S., Nguyen, A. T. & Owens, G. K. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat. Methods 10, 171–177 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Amento, E. P., Ehsani, N., Palmer, H. & Libby, P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler. Thromb. 11, 1223–1230 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Alexander, M. R. et al. Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice. J. Clin. Invest. 122, 70–79 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tacke, F. et al. Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery. J. Exp. Med. 203, 583–597 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Potteaux, S. et al. Suppressed monocyte recruitment drives macrophage removal from atherosclerotic plaques of Apoe-/- mice during disease regression. J. Clin. Invest. 121, 2025–2036 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Haka, A. S., Potteaux, S., Fraser, H., Randolph, G. J. & Maxfield, F. R. Quantitative analysis of monocyte subpopulations in murine atherosclerotic plaques by multiphoton microscopy. PLoS One 7, e44823 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Abdulaal, W. H. et al. Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system. Eur. J. Immunol. 46, 912–918 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 8, 265–277 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Vadiveloo, P. K., Stanton, H. R., Cochran, F. W. & Hamilton, J. A. Interleukin-4 inhibits human smooth muscle cell proliferation. Artery 21, 161–181 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Hawker, K. M., Johnson, P. R., Hughes, J. M. & Black, J. L. Interleukin-4 inhibits mitogen-induced proliferation of human airway smooth muscle cells in culture. Am. J. Physiol. 275, L469–477 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Jenkins, S. J. et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332, 1284–1288 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Jenkins, S. J. et al. IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. J. Exp. Med. 210, 2477–2491 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Sica, A. & Mantovani, A. Macrophage plasticity and polarization: In vivo veritas. J. Clin. Invest. 122, 787–795 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Zhao, X. N., Li, Y. N. & Wang, Y. T. Interleukin-4 regulates macrophage polarization via the MAPK signaling pathway to protect against atherosclerosis. Genet. Mol. Res. 15, gmr.15017348 (2016).

  42. 42.

    Tabas, I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat. Rev. Immunol. 10, 36–46 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Cai, B. et al. MerTK cleavage limits proresolving mediator biosynthesis and exacerbates tissue inflammation. Proc. Natl Acad. Sci. USA 113, 6526–6531 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    van Gils, J. M. et al. The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. Nat. Immunol. 13, 136–143 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Wanschel, A. et al. Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention. Arterioscler. Thromb. Vasc. Biol. 33, 886–893 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Robbins, C. S. et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat. Med. 19, 1166–1172 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Baylis, R. A., Gomez, D., Mallat, Z., Pasterkamp, G. & Owens, G. K. The CANTOS trial: One important step for clinical cardiology but a giant leap for vascular biology. Arterioscler. Thromb. Vasc. Biol. 37, e174–e177 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Harrington, R. A. Targeting inflammation in coronary artery disease. N. Engl. J. Med. 377, 1197–1198 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Ibanez, B. & Fuster, V. CANTOS: A gigantic proof-of-concept trial. Circ. Res. 121, 1320–1322 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Nov, O. et al. Interleukin-1beta regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability. PLoS One 8, e53626 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Howard, C. et al. Safety and tolerability of canakinumab, an IL-1beta inhibitor, in type 2 diabetes mellitus patients: A pooled analysis of three randomised double-blind studies. Cardiovasc. Diabetol. 13, 94 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Everett, B. M. et al. Anti-inflammatory therapy with canakinumab for the prevention and management of diabetes. J. Am. Coll. Cardiol. 71, 2402–2404 (2018).

    Article  CAS  Google Scholar 

  54. 54.

    Topol, E. J. Failing the public health—Rofecoxib, Merck, and the FDA. N. Engl. J. Med. 351, 1707–1709 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Morton, A. C. et al. The effect of interleukin-1 receptor antagonist therapy on markers of inflammation in non-ST elevation acute coronary syndromes: The MRC-ILA Heart Study. Eur. Heart J. 36, 377–384 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Interleukin 1 Genetics Consortium. Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: a Mendelian randomisation analysis. Lancet Diabetes Endocrinol. 3, 243–253 (2015).

    Article  CAS  Google Scholar 

  57. 57.

    Wirth, A. et al. G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat. Med. 14, 64–68 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  Google Scholar 

  59. 59.

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  Google Scholar 

  60. 60.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Luo, W., Friedman, M. S., Shedden, K., Hankenson, K. D. & Woolf, P. J. GAGE: Generally applicable gene set enrichment for pathway analysis. BMC Bioinforma. 10, 161 (2009).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the Owens laboratory members for their input. We thank P. Libby, M. Nahrendorf, and P. Swirski for their constructive discussion during the project completion. We thank E. Greene for her assistance in generating the SMC-specific Il1r1 knockout mice, A. Nguyen for performing retro-orbital injections, and M. McCanna for technical support. We thank the University of Pittsburgh Center for Biologic Imaging for their assistance with confocal microscopy. This work was supported by NIH grants R01 HL121008, R01 HL132904, and R01 HL136314 to G.K.O. D.G. was supported by Scientific Development Grant 15SDG25860021 from the American Heart Association. R.A.B. was supported by NIH grant F30 HL136188. B.G.D. was supported by a Predoctoral Fellowship from the American Heart Association (14PRE20380659). C.S.H. was supported by K22HL117917. The generation of the IL1R1fl/fl mice was funded by FP7/EU Project MUGEN (MUGEN LSHG-CT-2005-005203) to W.M. and by MRC research (G0801296) to E.P.

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D.G. and G.K.O. originally conceived of and designed the experiments. D.G. performed experiments, analyzed data, performed statistical analysis, and wrote the manuscript. R.A.B. designed and performed experiments, analyzed data, and contributed to manuscript writing. B.G.D. performed staining and data analysis. A.A.C.N. performed staining and data analysis of necrotic core and Ter119 data. G.F.A. analyzed the RNA-seq dataset. S.M. performed staining and analyzed data. C.S.H. performed calcification staining. A.W., W.M., S.E.F., and E.P. provided Il1r1fl/fl mice. G.J.R. helped with the monocyte trafficking assay. H.G. provided the IL-1β antibody and the IgG control and helped in experimental design. G.K.O. supervised the project. All co-authors read the manuscript.

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Correspondence to Gary K. Owens.

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H.G. is a full-time employee of Novartis Pharma AG, Basel.

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Gomez, D., Baylis, R.A., Durgin, B.G. et al. Interleukin-1β has atheroprotective effects in advanced atherosclerotic lesions of mice. Nat Med 24, 1418–1429 (2018). https://doi.org/10.1038/s41591-018-0124-5

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