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Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates

An Author Correction to this article was published on 26 July 2018

This article has been updated

Abstract

Macrophage accumulation in atherosclerosis is directly linked to the destabilization and rupture of plaque, causing acute atherothrombotic events. Circulating monocytes enter the plaque and differentiate into macrophages, where they are activated by CD4+ T lymphocytes through CD40–CD40 ligand signalling. Here, we report the development and multiparametric evaluation of a nanoimmunotherapy that moderates CD40–CD40 ligand signalling in monocytes and macrophages by blocking the interaction between CD40 and tumour necrosis factor receptor-associated factor 6 (TRAF6). We evaluated the biodistribution characteristics of the nanoimmunotherapy in apolipoprotein E-deficient (Apoe–/–) mice and in non-human primates by in vivo positron-emission tomography imaging. In Apoe–/– mice, a 1-week nanoimmunotherapy treatment regimen achieved significant anti-inflammatory effects, which was due to the impaired migration capacity of monocytes, as established by a transcriptome analysis. The rapid reduction of plaque inflammation by the TRAF6-targeted nanoimmunotherapy and its favourable toxicity profiles in both mice and non-human primates highlights the translational potential of this strategy for the treatment of atherosclerosis.

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Fig. 1: TRAF6i–HDL biodistribution and uptake.
Fig. 2: TRAF6i–HDL biodistribution in non-human primates.
Fig. 3: TRAF6i–HDL therapy decreases plaque macrophage content as assessed by histology.
Fig. 4: TRAF6i–HDL decreases plaque inflammation due to impaired Ly6Chi monocyte recruitment.
Fig. 5: TRAF6i–HDL shows effects on monocyte migration, among other affected processes.
Fig. 6: TRAF6i–HDL therapy does not elicit adverse immune or toxic effects in mice and non-human primates.

Change history

  • 26 July 2018

    In the version of this Article originally published, the surname of the author Edward A. Fisher was spelt incorrectly as ‘Fischer’. This has now been corrected.

References

  1. Swirski, F. K. & Nahrendorf, M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science 339, 161–166 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Schönbeck, U. & Libby, P. CD40 signaling and plaque instability. Circ. Res. 89, 1092–1103 (2001).

    Article  PubMed  Google Scholar 

  3. Lutgens, E. et al. Requirement for CD154 in the progression of atherosclerosis. Nat. Med. 5, 1313–1316 (1999).

    Article  PubMed  CAS  Google Scholar 

  4. Mach, F., Schönbeck, U., Sukhova, G. K., Atkinson, E. & Libby, P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 394, 200–203 (1998).

    Article  PubMed  CAS  Google Scholar 

  5. Schönbeck, U., Sukhova, G. K., Shimizu, K., Mach, F. & Libby, P. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc. Natl Acad. Sci. USA 97, 7458–7463 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Lutgens, E. et al. Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype. Proc. Natl Acad. Sci. USA 97, 7464–7469 (2000).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Lutgens, E. et al. Deficient CD40–TRAF6 signaling in leukocyte prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile. J. Exp. Med. 207, 391–404 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Zarzycka, B. et al. Discovery of small molecule CD40–TRAF6 inhibitors. J. Chem. Inf. Model. 55, 294–307 (2015).

    Article  PubMed  CAS  Google Scholar 

  9. Chatzigeorgiou, A. et al. Blocking CD40–TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance. Proc. Natl Acad. Sci. USA 111, 2686–2691 (2014).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Swirski, F. K. et al. Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata. J. Clin. Invest. 117, 195–205 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Swirski, F. K. et al. Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc. Natl Acad. Sci. USA 103, 10340–10345 (2006).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Kiener, P. A. et al. Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes. J. Immunol. 155, 4917–4925 (1995).

    PubMed  CAS  Google Scholar 

  14. Iloki Assanga, S. B. et al. Cell growth curves for different cell lines and their relationship with biological activities. Int. J. Biotechnol. Mol. Biol. Res. 4, 60–70 (2013).

    Article  CAS  Google Scholar 

  15. Imhof, B. A. & Aurrand-Lions, M. Adhesion mechanisms regulating the migration of monocytes. Nat. Rev. Immunol. 4, 432–444 (2004).

    Article  PubMed  CAS  Google Scholar 

  16. Wenger, G. D. & O’Dorisio, M. S. Induction of cAMP-dependent protein kinase I during human monocyte differentiation. J. Immunol. 134, 1836–1843 (1985).

    PubMed  CAS  Google Scholar 

  17. Chinetti-Gbaguidi, G., Colin, S. & Staels, B. Macrophage subsets in atherosclerosis. Nat. Rev. Cardiol. 12, 10–17 (2015).

    Article  PubMed  CAS  Google Scholar 

  18. Afford, S. C. et al. CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface Fas ligand expression and amplifies Fas-mediated hepatocyte death during allograft rejection. J. Exp. Med. 189, 441–446 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Bhogal, R. H. et al. Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation. PLoS ONE 7, e30867 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Tang, Y. et al. Up-regulation of the expression of costimulatory molecule CD40 in hepatocytes by hepatitis B virus X antigen. Biochem. Biophys. Res. Commun. 384, 12–17 (2009).

    Article  PubMed  CAS  Google Scholar 

  21. Kawai, T., Andrews, D., Colvin, R. B., Sachs, D. H. & Cosimi, A. B. Thromboembolic complications after treatment with monoclonal anti-body against CD40 ligand. Nat. Med. 6, 114 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. André, P. et al. CD40L stabilizes arterial thrombi by a β3 integrin-dependent mechanism. Nat. Med. 8, 247–252 (2002).

    Article  PubMed  Google Scholar 

  23. Ahonen, C. et al. The CD40–TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells. Nat. Immunol. 3, 451–456 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Duivenvoorden, R. et al. A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation. Nat. Commun. 5, 3065 (2014).

    Article  PubMed  CAS  Google Scholar 

  25. Shah, P. K. et al. Effects of recombinant apolipoprotein A-IMilano on aortic atherosclerosis in apolipoprotein E-deficient mice. Circulation 97, 780–785 (1998).

    Article  PubMed  CAS  Google Scholar 

  26. Moore, K. J., Sheedy, F. J. & Fisher, E. A. Macrophages in atherosclerosis: a dynamic balance. Nat. Rev. Immunol. 13, 709–721 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. 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  PubMed  PubMed Central  CAS  Google Scholar 

  28. Dutta, P. et al. Myocardial infarction accelerates atherosclerosis. Nature 487, 325–329 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Nissen, S. E. et al. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA 290, 2292–2300 (2003).

    Article  PubMed  CAS  Google Scholar 

  30. Tardif, J. C. et al. Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 297, 1675–1682 (2007).

    Article  PubMed  Google Scholar 

  31. Lerch, P. G. et al. Production and characterization of a reconstituted high density lipoprotein for therapeutic applications. Vox Sang. 71, 155–164 (1996).

    Article  PubMed  CAS  Google Scholar 

  32. Nykiforuk, C. L. et al. Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol. J. 9, 250–263 (2011).

    Article  PubMed  CAS  Google Scholar 

  33. Sanchez-Gaytan, B. L. et al. HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages. Bioconjug. Chem. 26, 443–451 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Degoma, E. M. & Rader, D. J. Novel HDL-directed pharmacotherapeutic strategies. Nat. Rev. Cardiol. 8, 266–277 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Tardif, J. C. et al. Effect of rHDL on Atherosclerosis-Safety and Efficacy (ERASE) Investigators. Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 297, 1675–1682 (2007).

    Article  PubMed  Google Scholar 

  36. Tardif, J. C. et al. Effects of the high-density lipoprotein mimetic agent CER-001 on coronary atherosclerosis in patients with acute coronary syndromes: a randomized trial. Eur. Heart J. 35, 3277–3286 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Kim, Y. et al. Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics. ACS Nano. 7, 9975–9983 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Pérez-Medina, C. et al. In vivo PET imaging of HDL in multiple atherosclerosis models. JACC Cardiovasc. Imaging 9, 950–961 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Ridker, P. M. et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N. Engl. J. Med. 359, 2195–2207 (2008).

    Article  PubMed  CAS  Google Scholar 

  40. Everett, B. M. et al. Rationale and design of the Cardiovascular Inflammation Reduction Trial: a test of the inflammatory hypothesis of atherothrombosis. Am. Heart J. 166, 199–207 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  42. Stone, G. W. et al. A prospective natural-history study of coronary atherosclerosis. N. Engl. J. Med. 364, 226–235 (2011).

    Article  PubMed  CAS  Google Scholar 

  43. Jonas, A. Reconstitution of high-density lipoproteins. Methods Enzymol. 128, 553–582 (1986).

    Article  PubMed  CAS  Google Scholar 

  44. Pérez-Medina, C. et al. PET imaging of tumor-associated macrophages with 89Zr-labeled high-density lipoprotein nanoparticles. J. Nucl. Med. 56, 1272–1277 (2015).

    Article  PubMed  Google Scholar 

  45. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Anders, S., Pyl, P. T. & Huber, W. HTSeq — a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

    Article  PubMed  CAS  Google Scholar 

  47. Mudge, J. M. & Harrow, J. Creating reference gene annotation for the mouse C57BL6/J genome assembly. Mamm. Genome 26, 366–378 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Ritchie, M. E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Wang, J. et al. GO-function: deriving biologically relevant functions from statistically significant functions. Brief. Bioinform. 13, 216–227 (2012).

    Article  PubMed  Google Scholar 

  50. Kanehisa, M., Goto, S., Sato, Y., Furumichi, M. & Tanabe, M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 40, D109–D114 (2012).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the Icahn School of Medicine and the following Mount Sinai’s core facilities: flow cytometry core, quantitative PCR core and TMII’s preclinical imaging core. This study was funded by National Institutes of Health grants R01 HL118440, R01 HL125703 and P01 HL131478 (all to W.J.M.M.), R01 EB009638 (to Z.A.F.) and R01 HL144072 (to W.J.M.M. and Z.A.F.), as well as by NWO grants ZonMW Veni 016156059 (to R.D.) and ZonMW Vidi 91713324 (to W.J.M.M.), and by the European Research Council (ERC Con to E.L.) and by the DFG (SFB 1123-A5 to E.L.).

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Authors and Affiliations

Authors

Contributions

R.D. and W.J.M.M. designed the study. R.D., M.L., T.B., M.M.T.v.L., M.L.S., J.T., T.T.P.S., J.K., E.S.G.S., J.O., E.A.F., R.E.T., N.K., P.R., A.K., F.K.S., M.N., Z.A.F., E.L. and W.J.M.M. designed, performed and oversaw the in vivo and ex vivo experiments. F.F., B.L.S.-G. and M.L. developed and produced TRAF6i–HDL. Flow cytometry, histology and immunostaining, laser capture microdissection, and blood chemistry experiments were performed and analysed by R.D., M.L., M.M.T.v.L. and J.M. FMT/CT was performed and analysed by R.D., Y.Y., G.W. and M.N. The RNA sequencing was performed and analysed by X.Z., B.Z., R.D. and M.L. Monocyte migration assays were performed by J.K. PET/CT and pharmacokinetic studies in mice were performed by C.P.-M., J.T. and T.R. 89Zr-PET/MRI in non-human primates was performed and analysed by T.B., M.L.S., C.P.-M. and C.C. The manuscript was written by R.D., M.L. and W.J.M.M. All authors contributed to the writing of the manuscript and approved the final draft. R.D., Z.A.F. and W.J.M.M. provided funding.

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Correspondence to Willem J. M. Mulder or Raphaël Duivenvoorden.

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Supplementary figures, tables and video captions.

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Supplementary Video 1

Three-dimensional MRI of a non-human primate.

Supplementary Video 2

Three-dimensional PET distribution of 89Zr-labelled TRAF6i–HDL at 60 minutes in a non-human primate.

Supplementary Video 3

In vitro transendothelial migration of monocytes pre-treated with TRAF6i–HD.

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Lameijer, M., Binderup, T., van Leent, M.M.T. et al. Efficacy and safety assessment of a TRAF6-targeted nanoimmunotherapy in atherosclerotic mice and non-human primates. Nat Biomed Eng 2, 279–292 (2018). https://doi.org/10.1038/s41551-018-0221-2

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