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.

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

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 options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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

Authors

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

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

Further reading

Search

Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research