Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

Nature Nanotechnology volume 10, pages 361–369 (2015)Cite this article

Subjects

This article has been updated

Abstract

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1’, whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Characterization of endogenous mineral in the intestinal lumen.
Figure 2: Phenotypic and nanomineral characterization of SED cells in murine and human Peyer's patches.
Figure 3: Electron microscopy characterization of murine endogenous nanomineral and three-dimensional nanotomography.
Figure 4: AMCP nanomineral uptake from the gut lumen into Peyer's patches is substantially impeded in the absence of M cells in the follicle-associated epithelium.
Figure 5: Endogenous nanomineral is co-localized with luminal peptidoglycan and dietary antigen in Peyer's patch APC.
Figure 6: Peptidoglycan signalling is required for PD-L1 expression on nanomineral-positive APCs of the Peyer's patch SED and mesenteric lymph nodes.

Similar content being viewed by others

Change history

  • 17 March 2015

    In the version of this Article originally published online, in the Methods, in the first sentence of the section 'Nuclear microscopy', ref. 7 should have been ref. 2, and in the Reference list ref. 6 was cited out of order; it should have been ref. 31. These errors have now been corrected in all versions of the Article and the references have been re-numbered.

References

  1. Powell, J. J., Thoree, V. & Pele, L. C. Dietary microparticles and their impact on tolerance and immune responsiveness of the gastrointestinal tract. Br. J. Nutr. 98(Suppl 1), S59–S63 (2007).

    Article  CAS  Google Scholar 

  2. Gomez-Morilla, I., Thoree, V., Powell, J. J., Kirkby, K. J. & Grime, G. W. Identification and quantitative analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy. Nucl. Instrum. Methods Phys. Res. B 249, 665–669 (2006).

    Article  CAS  Google Scholar 

  3. Powell, J. J., Faria, N., Thomas-McKay, E. & Pele, L. C. Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract. J. Autoimmun. 34, J226–J233 (2010).

    Article  CAS  Google Scholar 

  4. Lentner, C. (ed.) Geigy Scientific Tables 8th edn, Vol. 1 (CIBA-GEIGY Ltd, 1981).

    Google Scholar 

  5. Schedl, H. P., Osbaldiston, G. W. & Mills, I. H. Absorption, secretion, and precipitation of calcium in the small intestine of the dog. Am J Physiol 214, 814–819 (1968).

    Article  CAS  Google Scholar 

  6. Jung, C., Hugot, J. P. & Barreau, F. Peyer's patches: the immune sensors of the intestine. Int. J. Inflam. 2010, 823710 (2010).

    Article  Google Scholar 

  7. Mowat, A. M. & Bain, C. C. Mucosal macrophages in intestinal homeostasis and inflammation. J. Innate Immun. 3, 550–564 (2011).

    Article  Google Scholar 

  8. Pele, L. C. et al. Low dietary calcium levels modulate mucosal caspase expression and increase disease activity in mice with dextran sulfate sodium induced colitis. J. Nutr. 137, 2475–2480 (2007).

    Article  CAS  Google Scholar 

  9. Civitelli, R. & Ziambaras, K. Calcium and phosphate homeostasis: concerted interplay of new regulators. J. Endocrinol. Invest. 34, 3–7 (2011).

    Article  CAS  Google Scholar 

  10. Neutra, M. R. & Kraehenbuhl, J. P. Transepithelial transport and mucosal defence I: the role of M cells. Trends Cell Biol. 2, 134–138 (1992).

    Article  CAS  Google Scholar 

  11. Knoop, K. A. et al. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J. Immunol. 183, 5738–5747 (2009).

    Article  CAS  Google Scholar 

  12. Kimura, S. et al. Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches. Mucosal Immunol. http://www.nature.com/mi/journal/vaop/ncurrent/full/mi201499a.html (2014).

  13. Masahata, K. et al. Generation of colonic IgA-secreting cells in the caecal patch. Nature Commun. 5, 3704 (2014).

    Article  Google Scholar 

  14. Szentkuti, L. Light microscopical observations on luminally administered dyes, dextrans, nanospheres and microspheres in the pre-epithelial mucus gel layer of the rat distal colon. J. Control. Rel. 46, 233–242 (1997).

    Article  CAS  Google Scholar 

  15. Uskokovic, V. & Uskokovic, D. P. Nanosized hydroxyapatite and other calcium phosphates: chemistry of formation and application as drug and gene delivery agents. J. Biomed. Mater. Res B 96, 152–191 (2011).

    Article  Google Scholar 

  16. Dorozhkin, S. V. Nanosized and nanocrystalline calcium orthophosphates. Acta Biomater. 6, 715–734 (2010).

    Article  CAS  Google Scholar 

  17. Blander, J. M. & Medzhitov, R. Regulation of phagosome maturation by signals from toll-like receptors. Science 304, 1014–1018 (2004).

    Article  CAS  Google Scholar 

  18. Blander, J. M. & Medzhitov, R. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440, 808–812 (2006).

    Article  CAS  Google Scholar 

  19. Chong, C. S. et al. Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery. J. Control. Rel. 102, 85–99 (2005).

    Article  CAS  Google Scholar 

  20. Heit, A., Schmitz, F., Haas, T., Busch, D. H. & Wagner, H. Antigen co-encapsulated with adjuvants efficiently drive protective T cell immunity. Eur. J. Immunol. 37, 2063–2074 (2007).

    Article  CAS  Google Scholar 

  21. Schlosser, E. et al. TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses. Vaccine 26, 1626–1637 (2008).

    Article  CAS  Google Scholar 

  22. Klasen, I. S. et al. The presence of peptidoglycan–polysaccharide complexes in the bowel wall and the cellular responses to these complexes in Crohn's disease. Clin. Immunol. Immunopathol. 71, 303–308 (1994).

    Article  CAS  Google Scholar 

  23. Schrijver, I. A. et al. Bacterial peptidoglycan and immune reactivity in the central nervous system in multiple sclerosis. Brain 124, 1544–1554 (2001).

    Article  CAS  Google Scholar 

  24. Boskey, A. L. & Posner, A. S. Magnesium stabilization of amorphous calcium phosphate: a kinetic study. Mater. Res. Bull. 9, 907–916 (1974).

    Article  CAS  Google Scholar 

  25. Termine, J. D., Peckauskas, R. A. & Posner, A. S. Calcium phosphate formation in vitro. II. Effects of environment on amorphous-crystalline transformation. Arch. Biochem. Biophys. 140, 318–325 (1970).

    Article  CAS  Google Scholar 

  26. Hewitt, R. E. et al. Immuno-inhibitory PD-L1 can be induced by a peptidoglycan/NOD2 mediated pathway in primary monocytic cells and is deficient in Crohn's patients with homozygous NOD2 mutations. Clin. Immunol. 143, 162–169 (2012).

    Article  CAS  Google Scholar 

  27. Davies, J. M., MacSharry, J. & Shanahan, F. Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells. Immunology 131, 438–448 (2010).

    Article  CAS  Google Scholar 

  28. Stroo, I. et al. Phenotyping of Nod1/2 double deficient mice and characterization of Nod1/2 in systemic inflammation and associated renal disease. Biol. Open. 1, 1239–1247 (2012).

    Article  CAS  Google Scholar 

  29. Francisco, L. M., Sage, P. T. & Sharpe, A. H. The PD-1 pathway in tolerance and autoimmunity. Immunol. Rev. 236, 219–242 (2010).

    Article  CAS  Google Scholar 

  30. Van Heel, D. A. et al. Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn's disease. Lancet 365, 1794–1796 (2005).

    Article  CAS  Google Scholar 

  31. Gullberg, E. & Soderholm, J. D. Peyer's patches and M cells as potential sites of the inflammatory onset in Crohn's disease. Ann. NY Acad. Sci. 1072, 218–232 (2006).

    Article  CAS  Google Scholar 

  32. Fukaya, T. et al. Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph node dendritic cells in the generation of antigen-specific CD4+Foxp3+ regulatory T cells in the establishment of oral tolerance. Blood 116, 2266–2276 (2010).

    Article  CAS  Google Scholar 

  33. Scandiuzzi, L. et al. Tissue-expressed B7-H1 critically controls intestinal inflammation. Cell. Rep. 6, 625–632 (2014).

    Article  CAS  Google Scholar 

  34. Reynoso, E. D. et al. Intestinal tolerance is converted to autoimmune enteritis upon PD-1 ligand blockade. J. Immunol. 182, 2102–2112 (2009).

    Article  CAS  Google Scholar 

  35. Awaad, A., Nakamura, M. & Ishimura, K. Imaging of size-dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles. Nanomedicine 8, 627–636 (2012).

    Article  CAS  Google Scholar 

  36. Sass, W., Dreyer, H. P. & Seifert, J. Rapid insorption of small particles in the gut. Am. J. Gastroenterol. 85, 255–260 (1990).

    CAS  Google Scholar 

  37. Davies, K. M., Rafferty, K. & Heaney, R. P. Determinants of endogenous calcium entry into the gut. Am. J. Clin. Nutr. 80, 919–923 (2004).

    Article  CAS  Google Scholar 

  38. Cross, K. J., Huq, N. L., Palamara, J. E., Perich, J. W. & Reynolds, E. C. Physicochemical characterization of casein phosphopeptide–amorphous calcium phosphate nanocomplexes. J. Biol. Chem. 280, 15362–15369 (2005).

    Article  CAS  Google Scholar 

  39. De Kruif, C. G., Huppertz, T., Urban, V. S. & Petukhov, A. V. Casein micelles and their internal structure. Adv. Colloid Interface Sci. 171–172, 36–52 (2012).

    Article  Google Scholar 

  40. McGann, T. C. et al. Amorphous calcium phosphate in casein micelles of bovine milk. Calcif. Tissue Int. 35, 821–823 (1983).

    Article  CAS  Google Scholar 

  41. Minniti, F. et al. Breast-milk characteristics protecting against allergy. Endocr. Metab. Immune Disord. Drug Targets 14, 9–15 (2014).

    Article  CAS  Google Scholar 

  42. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  43. Bilton, M., Brown, A. P. & Milne, S. J. Investigating the optimum conditions for the formation of calcium oxide, used for CO2 sequestration, by thermal decomposition of calcium acetate. Electron Microscopy and Analysis Group Conference 2011 (Emag 2011) 371 ( 2012).

  44. Bres, E. F., Hutchison, J. L., Senger, B., Voegel, J. C. & Frank, R. M. HREM study of irradiation damage in human dental enamel crystals. Ultramicroscopy 35, 305–322 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the UK Medical Research Council (grant no. U105960399) for their continued support and the UK Engineering and Physical Sciences Research Council for support of the Ion Beam Centre as a UK National Facility (GR/R50097). The authors acknowledge the Dairy Council and the Sir Halley Stewart Trust for support. Work in the laboratory of J.D.L is supported by the Dutch MS Research Foundation. The authors thank M-J. Melief for help in modifying in situ techniques for the detection of peptidoglycan and I. Stroo for providing snap-frozen NOD1/2−/− samples from mice originally from S.E.G.'s laboratory. The authors thank J. Kaufman, A. Wyllie and P. Mastroeni (all University of Cambridge) for brainstorming and advice. J.C.H-G. and P.A.M. acknowledge financial support from the European Union's Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative (reference no. 312483-ESTEEM2). P.A.M. also acknowledges financial support from the European Research Council (reference 291,522 3DIMAGE). N.A.M. and D.S.D. were supported by projects BB/J014762/1 and BB/K021257/1 and institute strategic programme grant funding from the Biological and Biotechnological Research Council. I.R.W. and D.R. were supported by grants from the National Institutes of Health (DK064730 and AI111388).

Author information

Authors and Affiliations

  1. Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge, CB1 9NL, UK

    Jonathan J. Powell, Emma Thomas-McKay, Vinay Thoree, Jack Robertson, Rachel E. Hewitt, Carolin T. Haas, Sylvaine F. A. Bruggraber, Richard P. H. Thompson & Laetitia C. Pele

  2. Department of Physiology, Cambridge Advanced Imaging Centre, Development and Neuroscience, University of Cambridge, Cambridge, CB2 3DY, UK

    Jeremy N. Skepper

  3. Institute for Materials Research, SPEME, University of Leeds, Leeds, LS2 9JT, UK

    Andy Brown

  4. Departamento de Ciencia de los Materiales e Ingenieria Metalúrgica y Química Inorganica, Facultad de Ciencias, Universidad de Cádiz, Campus Universitario Rio San Pedro, Puerto Real (Cádiz), 11.510, Spain

    Juan Carlos Hernandez-Garrido

  5. Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK

    Paul A. Midgley

  6. Technische Universitaet Dresden, Fakultaet Maschinenwesen, Institut fuer Stroemungsmechanik, Dresden, 01062, Germany

    Inmaculada Gomez-Morilla

  7. Ion Beam Centre, Advanced Technology Institute, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK

    Geoffrey W. Grime & Karen J. Kirkby

  8. Institute of Cancer Sciences, The University of Manchester, 27 Palatine Road, Withington, M20 3LJ, Manchester, UK

    Karen J. Kirkby

  9. The Roslin Institute and Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK

    Neil A. Mabbott & David S. Donaldson

  10. Department of Pathology, Emory University School of Medicine, 615 Michael Street, Atlanta, 30322, Georgia, USA

    Ifor R. Williams & Daniel Rios

  11. Department of Laboratory Medicine and Pathobiology, 1 King's College Circle, Toronto, M5S 1A8, Canada

    Stephen E. Girardin

  12. Department of Immunology, Erasmus MC, University Medical Centre and MS Centre ErasMS, PO Box 2040, Rotterdam, 3000 CA, The Netherlands

    Jon D. Laman

  13. Department of Neuroscience, Section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

    Jon D. Laman

  14. Department of Internal Medicine IV and Institute of Medical Microbiology and Hygiene, University Medical Center, Freiburg, 79106, Germany

    Yakup Tanriver

  15. Immunoregulation Laboratory, MRC Centre for Transplantation, King's College London, Guys' Hospital, London, SE1 9RT, UK

    Giovanna Lombardi & Robert Lechler

  16. Royal College of Physicians, 11 St Andrews Place, Regent's Park, London, NW1 4LE, UK

    Richard P. H. Thompson

Authors
  1. Jonathan J. Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emma Thomas-McKay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vinay Thoree
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jack Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rachel E. Hewitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeremy N. Skepper
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andy Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Juan Carlos Hernandez-Garrido
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Paul A. Midgley
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Inmaculada Gomez-Morilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Geoffrey W. Grime
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Karen J. Kirkby
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Neil A. Mabbott
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. David S. Donaldson
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Ifor R. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Daniel Rios
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Stephen E. Girardin
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Carolin T. Haas
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Sylvaine F. A. Bruggraber
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Jon D. Laman
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Yakup Tanriver
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Giovanna Lombardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Robert Lechler
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Richard P. H. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Laetitia C. Pele
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.J.P. developed the overall hypothesis and led the work and, with L.C.P., was involved in specific study design, data interpretation and writing of the paper. I.G-M., G.W.G. and K.J.K. developed the necessary techniques for, and undertook with V.T. and J.J.P., PIXE/nuclear microscopy studies. J.R. and V.T. developed and optimised immunostaining and carried out cell phenotyping for the confocal studies, which were carried out with J.N.S., who also prepared the samples for HAADF STEM imaging. E.T-M., J.C.H-G. and P.A.M. were responsible for the HAADF STEM tomography. E.T-M. assisted A.B. in undertaking TEM/STEM analyses, while V.T. and E.T-M. worked with J.N.S. to carry out SEM analyses. R.L. and R.P.H.T. provided mentorship in the early development of the work/hypothesis. G.L. and Y.T. undertook the initial feeding study with labelled OVA. S.E.G. developed and characterised NOD1/2−/− mice and supported sample analysis of their Peyer's patches. N.A.M., D.S.D., I.R.W. and D.R. designed and undertook the studies to determine the impact of M-cell deficiency on AMCP uptake. J.D.L. provided the antibody and expertise for 2E9 staining and interpretation and, with A.B., S.F.A.B., R.E.H. and C.T.H., provided rigorous data review and discussion with J.J.P. and L.C.P. All authors contributed to data interpretation and to the writing and critical review of the manuscript.

Corresponding author

Correspondence to Jonathan J. Powell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 431 kb)

Supplementary information

Supplementary Movie 1 (AVI 48685 kb)

Supplementary information

Supplementary Movie 2 (MPG 7407 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Powell, J., Thomas-McKay, E., Thoree, V. et al. An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells. Nature Nanotech 10, 361–369 (2015). https://doi.org/10.1038/nnano.2015.19

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2015.19

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing