, Volume 61, Issue 6, pp 1384–1396 | Cite as

Targeted delivery of antigen to intestinal dendritic cells induces oral tolerance and prevents autoimmune diabetes in NOD mice

  • Yulin Chen
  • Jie Wu
  • Jiajia Wang
  • Wenjing Zhang
  • Bohui Xu
  • Xiaojun XuEmail author
  • Li ZongEmail author



The intestinal immune system is an ideal target to induce immune tolerance physiologically. However, the efficiency of oral protein antigen delivery is limited by degradation of the antigen in the gastrointestinal tract and poor uptake by antigen-presenting cells. Gut dendritic cells (DCs) are professional antigen-presenting cells that are prone to inducing antigen-specific immune tolerance. In this study, we delivered the antigen heat shock protein 65-6×P277 (H6P) directly to the gut DCs of NOD mice through oral vaccination with H6P-loaded targeting nanoparticles (NPs), and investigated the ability of this antigen to induce immune tolerance to prevent autoimmune diabetes in NOD mice.


A targeting NP delivery system was developed to encapsulate H6P, and the ability of this system to protect and facilitate H6P delivery to gut DCs was assessed. NOD mice were immunised with H6P-loaded targeting NPs orally once a week for 7 weeks and the onset of diabetes was assessed by monitoring blood glucose levels.


H6P-loaded targeting NPs protected the encapsulated H6P from degradation in the gastrointestinal tract environment and significantly increased the uptake of H6P by DCs in the gut Peyer’s patches (4.1 times higher uptake compared with the control H6P solution group). Oral vaccination with H6P-loaded targeting NPs induced antigen-specific T cell tolerance and prevented diabetes in 100% of NOD mice. Immune deviation (T helper [Th]1 to Th2) and CD4+CD25+FOXP3+ regulatory T cells were found to participate in the induction of immune tolerance.


In this study, we successfully induced antigen-specific T cell tolerance and prevented the onset of diabetes in NOD mice. To our knowledge, this is the first attempt at delivering antigen to gut DCs using targeting NPs to induce T cell tolerance.


Autoimmune diabetes Dendritic cells Nanoparticles NOD mice Oral tolerance Oral vaccination 



Carboxyfluorescein succinimidyl ester


Confocal laser scanning microscopy


Concanavalin A




Dendritic cell


FITC-labelled heat shock protein 65-6×P277


Fluorescence resonance energy transfer


Heat shock protein 65-6×P277


Heat shock protein 65-6×P277-loaded RGD- and mannose-modified chitosan


Heat shock protein




Mannose-modified chitosan




RGD-modified chitosan


Arginylglycylaspartic acid


RGD- and mannose-modified chitosan


Simulated gastric fluid


Simulated intestinal fluid


T helper


Regulatory T cell



We thank Y. Xing (manager of flow cytometry at the China Pharmaceutical University) for her scientific advice and technical assistance with flow cytometry.

Contribution statement

YLC, JW, XJX and LZ contributed to the conception and design of the study. YLC, JJW, WJZ and BHX performed the experiments and analysed the results. YLC, JW, XJX and LZ drafted the manuscript. All authors revised the manuscript critically and gave final approval of the submitted version. LZ is the guarantor of the work.


This study was supported by National Natural Science Foundation of China (No. 30973650/H3008) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_0676).

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2018_4593_MOESM1_ESM.pdf (268 kb)
ESM (PDF 267 kb)


  1. 1.
    van Belle TL, Coppieters KT, von Herrath MG (2011) Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev 91:79–118CrossRefPubMedGoogle Scholar
  2. 2.
    Lernmark A, Larsson HE (2013) Immune therapy in type 1 diabetes mellitus. Nat Rev Endocrinol 9:92–103CrossRefPubMedGoogle Scholar
  3. 3.
    Frumento D, Nasr MB, Essawy BE, D’Addio F, Zuccotti GV, Fiorina P (2017) Immunotherapy for type 1 diabetes. J Endocrinol Investig 40:803–814CrossRefGoogle Scholar
  4. 4.
    Elias D, Reshef T, Birk OS, van der Zee R, Walker MD, Cohen IR (1991) Vaccination against autoimmune mouse diabetes with a T cell epitope of the human 65-kDa heat shock protein. Proc Natl Acad Sci U S A 88:3088–3091CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Abulafia-Lapid R, Elias D, Raz I, Keren-Zur Y, Atlan H, Cohen IR (1999) T cell proliferative responses of type 1 diabetes patients and healthy individuals to human hsp60 and its peptides. J Autoimmun 12:121–129CrossRefPubMedGoogle Scholar
  6. 6.
    Jin L, Zhu A, Wang Y et al (2008) A Th1-recognized peptide P277, when tandemly repeated, enhances a Th2 immune response toward effective vaccines against autoimmune diabetes in nonobese diabetic mice. J Immunol 180:58–63CrossRefPubMedGoogle Scholar
  7. 7.
    Xu D, Prasad S, Miller SD (2013) Inducing immune tolerance: a focus on type 1 diabetes mellitus. Diabetes Manag 3:415–426CrossRefGoogle Scholar
  8. 8.
    Li AF, Escher A (2003) Intradermal or oral delivery of GAD-encoding genetic vaccines suppresses type 1 diabetes. DNA Cell Biol 22:227–232CrossRefPubMedGoogle Scholar
  9. 9.
    Mowat AM (2003) Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3:331–341CrossRefPubMedGoogle Scholar
  10. 10.
    Matzinger P, Kamala T (2011) Tissue-based class control: the other side of tolerance. Nat Rev Immunol 11:221–230CrossRefPubMedGoogle Scholar
  11. 11.
    Rimoldi M, Chieppa M, Salucci V et al (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat Immunol 6:507–514CrossRefPubMedGoogle Scholar
  12. 12.
    Mucida D, Park Y, Kim G et al (2007) Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 317:256–260CrossRefPubMedGoogle Scholar
  13. 13.
    Banchereau J, Briere F, Caux C et al (1999) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811CrossRefGoogle Scholar
  14. 14.
    Everson MP, Lemak DG, McDuffie DS, Koopman WJ, McGhee JR, Beagley KW (1998) Dendritic cells from Peyer’s patch and spleen induce different T helper cell responses. J Interf Cytokine Res 18:103–115CrossRefGoogle Scholar
  15. 15.
    Hashiguchi M, Hachimura S, Ametani A et al (2011) Naïve CD4+ T cells of Peyer’s patches produce more IL-6 than those of spleen in response to antigenic stimulation. Immunol Lett 141:109–115CrossRefPubMedGoogle Scholar
  16. 16.
    Iwasaki A, Kelsall BL (1999) Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J Exp Med 190:229–239CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Peron JP, de Oliveira AP, Rizzo LV (2009) It takes guts for tolerance: the phenomenon of oral tolerance and the regulation of autoimmune response. Autoimmun Rev 9:1–4CrossRefPubMedGoogle Scholar
  18. 18.
    Wang X, Sherman A, Liao G et al (2013) Mechanism of oral tolerance induction to therapeutic proteins. Adv Drug Deliv Rev 65:759–773CrossRefPubMedGoogle Scholar
  19. 19.
    Kraehenbuhl JP, Neutra MR (2000) Epithelial M cells: differentiation and function. Annu Rev Cell Dev Biol 16:301–332CrossRefPubMedGoogle Scholar
  20. 20.
    Davitt CJ, Lavelle EC (2015) Delivery strategies to enhance oral vaccination against enteric infections. Adv Drug Deliv Rev 91:52–69CrossRefPubMedGoogle Scholar
  21. 21.
    Garinot M, Fiévez V, Pourcelle V et al (2007) PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release 120:195–204CrossRefPubMedGoogle Scholar
  22. 22.
    Raviv L, Jaron-Mendelson M, David A (2015) Mannosylated polyion complexes for in vivo gene delivery into CD11c+ dendritic cells. Mol Pharm 12:453–462CrossRefPubMedGoogle Scholar
  23. 23.
    Yao W, Jiao Y, Luo J, Du M, Zong L (2012) Practical synthesis and characterization of mannose-modified chitosan. Int J Biol Macromol 50:821–825CrossRefPubMedGoogle Scholar
  24. 24.
    Wang C, Chen B, Zou M, Cheng G (2014) Cyclic RGD-modified chitosan/graphene oxide polymers for drug delivery and cellular imaging. Colloids Surf B Biointerfaces 122:332–340CrossRefPubMedGoogle Scholar
  25. 25.
    Biswas S, Chattopadhyay M, Sen KK, Saha MK (2015) Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice. Carbohydr Polym 121:403–410CrossRefPubMedGoogle Scholar
  26. 26.
    Primard C, Rochereau N, Luciani E et al (2010) Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells. Biomaterials 31:6060–6068CrossRefPubMedGoogle Scholar
  27. 27.
    Mariño E, Richards JL, McLeod KH et al (2017) Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes. Nat Immunol 18:552–562CrossRefPubMedGoogle Scholar
  28. 28.
    Qi F, Wu J, Yang T, Ma G, Su Z (2014) Mechanistic studies for monodisperse exenatide-loaded PLGA microspheres prepared by different methods based on SPG membrane emulsification. Acta Biomater 10:4247–4256CrossRefPubMedGoogle Scholar
  29. 29.
    Lai J, Shah BP, Garfunkel E, Lee KB (2013) Versatile fluorescence resonance energy transfer-based mesoporous silica nanoparticles for real-time monitoring of drug release. ACS Nano 7:2741–2750CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Jiang PL, Lin HJ, Wang HW et al (2015) Galactosylated liposome as a dendritic cell-targeted mucosal vaccine for inducing protective anti-tumor immunity. Acta Biomater 11:356–367CrossRefPubMedGoogle Scholar
  31. 31.
    Maassen CB, Boersma WJ, Van Holten-Neelen C, Claassen E, Laman JD (2003) Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens: implications for vaccine development. Vaccine 21:2751–2757CrossRefPubMedGoogle Scholar
  32. 32.
    Bilate AM, Lafaille JJ (2012) Induced CD4+Foxp3+ regulatory T cells in immune tolerance. Annu Rev Immunol 30:733–758CrossRefPubMedGoogle Scholar
  33. 33.
    Fei L, Wang L, Jin XM, Yan CH, Shan J, Shen XM (2009) The immunologic effect of TGF-beta1 chitosan nanoparticle plasmids on ovalbumin-induced allergic BALB/c mice. Immunobiology 214:87–99CrossRefGoogle Scholar
  34. 34.
    Sakaguchi S (2000) Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101:455–458CrossRefPubMedGoogle Scholar
  35. 35.
    Zhang ZJ, Davidson L, Eisenbarth G, Weiner HL (1991) Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. J Endocrinol Investig 88:10252–10256Google Scholar
  36. 36.
    Bergerot I, Arreaza GA, Cameron MJ et al (1999) Insulin B-chain reactive CD4+ regulatory T cells induced by oral insulin treatment protect from type 1 diabetes by blocking the cytokine secretion and pancreatic infiltration of diabetogenic effector T cells. Diabetes 48:1720–1729CrossRefPubMedGoogle Scholar
  37. 37.
    Takiishi T, Korf H, Van Belle TL et al (2012) Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice. J Clin Invest 122:1717–1725CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bonifacio E, Ziegler AG, Klingensmith G et al (2015) Effects of high-dose oral insulin on immune responses in children at high risk for type 1 diabetes: the Pre-POINT randomized clinical trial. JAMA 313:1541–1549CrossRefPubMedGoogle Scholar
  39. 39.
    Ma Y, Liu J, Hou J et al (2014) Oral administration of recombinant Lactococcus lactis expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice. PLoS One 9:e105701CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Harrison LC, Hafler DA (2000) Antigen-specific therapy for autoimmune disease. Curr Opin Immunol 12:704–711CrossRefPubMedGoogle Scholar
  41. 41.
    Jindal S, Dudani AK, Singh B, Harley CB, Gupta RS (1989) Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. Mol Cell Biol 9:2279–2283CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Cohensfady M, Nussbaum G, Pevsnerfischer M et al (2005) Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway. J Immunol 175:3594–3602CrossRefGoogle Scholar
  43. 43.
    Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL (2001) Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol 167:1081–1089CrossRefPubMedGoogle Scholar
  44. 44.
    Wing K, Sakaguchi S (2010) Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol 11:7–13CrossRefPubMedGoogle Scholar
  45. 45.
    Kasagi S, Zhang P, Che L et al (2014) In vivo-generated antigen-specific regulatory T cells treat autoimmunity without compromising antibacterial immune response. Sci Transl Med 6:241–278CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of PharmaceuticsChina Pharmaceutical UniversityNanjingPeople’s Republic of China
  2. 2.Minigene Pharmacy LaboratoryChina Pharmaceutical UniversityNanjingPeople’s Republic of China
  3. 3.State Key Laboratory of Natural MedicinesChina Pharmaceutical UniversityNanjingPeople’s Republic of China

Personalised recommendations