Targeted delivery of antigen to intestinal dendritic cells induces oral tolerance and prevents autoimmune diabetes in NOD mice
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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.
KeywordsAutoimmune diabetes Dendritic cells Nanoparticles NOD mice Oral tolerance Oral vaccination
Carboxyfluorescein succinimidyl ester
Confocal laser scanning microscopy
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
RGD- and mannose-modified chitosan
Simulated gastric fluid
Simulated intestinal fluid
Regulatory T cell
The exact cause of type 1 diabetes remains elusive. Usually, it is considered to be a chronic autoimmune disease in which pancreatic beta cells are destroyed or damaged by the body’s own immune system, causing insulin deficiency . In individuals with type 1 diabetes, glycaemic control is achieved by daily injections or continuous s.c. infusion of insulin. However, there is currently no cure for the condition. Attempts to prevent or cure type 1 diabetes using immunotherapy that are being investigated in clinical trials include non-antigen-specific therapy (e.g. T/B cell depleting strategies, anti-inflammatory strategies or cell therapy strategies) and antigen-specific therapy (e.g. oral/nasal insulin, alum-formulated GAD65, proinsulin peptides or DiaPep277) [2, 3].
Antigen-specific therapy has been considered the ‘Holy Grail’ of immunotherapy for type 1 diabetes because it targets only beta cell reactive T cells without impairing beneficial immune responses. Peptide P277 is a 24 amino acid fragment of heat shock protein (HSP)60/65 that has been found to be an autoantigen for diabetogenic T cell clones in NOD mice and individuals with type 1 diabetes [4, 5]. In previous work, we successfully constructed a heat shock protein 65-6×P277 (H6P) vaccine, based on autoantigen P277, against autoimmune diabetes in NOD mice . For vaccination, autoantigens can be delivered via different routes, including intraperitoneal, intravenous, oral and intranasal administration . The administration route of a vaccine plays an important role in inducing an antigen-specific immune response and influences clinical efficacy . It is commonly considered that oral administration of antigens is more efficient for tolerance induction, because the intestinal immune system has a predisposition to induce tolerance  and its immunosuppressive environment can affect the lymphocytes [10, 11, 12]. Dendritic cells (DCs) are professional antigen-presenting cells with the capacity to instigate either inflammatory or anti-inflammatory adaptive immunity. The direction of the response is influenced by DC phenotype: either an activated phenotype or, conversely, a tolerogenic phenotype . It has been reported that DCs from gut Peyer’s patches tend to induce differentiation of T helper (Th)2 cells and secretion of anti-inflammatory cytokines [14, 15, 16] and are prone to inducing regulatory T cells (Tregs), which migrate and suppress damaging immune responses and secrete antigen non-specific cytokines such as TGF-β, contributing to bystander suppression .
In this study, we attempted to induce immune tolerance in NOD mice through the oral delivery of H6P to the DCs in gut Peyer’s patches. However, it is difficult for oral antigens to reach gut DCs because of the poor stability of antigens in the gastrointestinal tract and the absorption barriers presented by the intestinal mucus layer and the epithelial cell layer , which leads to low treatment efficiency. M cells are specialised epithelial cells that are mainly located in the follicle-associated epithelium of Peyer’s patches  and have high transcytotic capabilities to transport a broad range of materials from the intestinal lumen to the DCs in Peyer’s patches . The specific location and function of M cells make them an attractive target for oral vaccine delivery to DCs. Thus, we developed a delivery system based on the M cell targeting peptide arginylglycylaspartic acid (RGD)  and DC-targeting ligand mannose -modified chitosan (CS) nanoparticles (NPs), to protect and facilitate H6P antigen delivery to gut DCs, hoping to improve treatment efficiency. In this study, we aimed to investigate whether oral H6P-loaded targeting NPs could deliver H6P to DCs in gut Peyer’s patches and prevent autoimmune diabetes through the induction of T cell tolerance.
Four-week-old female NOD/LtJ mice were purchased from Beijing HFK Bioscience (Beijing, China). Six- to 8-week-old female BALB/c mice were obtained from Qinglongshan Laboratory Animal Center (Nanjing, China). Mice were housed in a pathogen-free facility with temperature (23 ± 1°C) and light (12 h light/dark cycle) control, and had free access to water and standard mouse chow. Animal care and all experiments in this study were conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and approved by the China Pharmaceutical University Institutional Animal Care and Use Committee. The investigators were not blinded to the experimental groups, unless otherwise noted.
Full details of the reagents used in this study are provided in the electronic supplementary material (ESM) Methods.
Synthesis of RGD- and mannose-modified chitosan
Mannose-modified chitosan (MCS) was synthesised as previously described . RGD-modified chitosan (RCS) or RGD- and mannose-modified chitosan (RMCS) were synthesised by using a crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate, to conjugate RGD to the amino groups of CS or MCS . Details are provided in the ESM Methods.
Preparation and characterisation of NPs
NPs were prepared by electrostatic self-assembly of oppositely charged polysaccharides at room temperature. Details are provided in the ESM Methods. The particle size, zeta potential value, association efficiency and drug loading of NPs were characterised, and details are provided in the ESM Methods.
In vitro release of H6P from H6P-loaded RMCS NPs
The H6P release profile of H6P-loaded RMCS (H6P/RMCS) NPs was separately evaluated in 0.1 mol/l HCl (pH 1.0) and PBS (pH 6.8). Details are provided in the ESM Methods.
Stability of H6P/RMCS NPs
The stability of H6P/RMCS NPs in simulated gastric fluid (SGF; containing pepsin, pH 1.2, USP 38) and simulated intestinal fluid (SIF; containing pancreatin, pH 6.8, USP 38) was tested by measuring changes in particle size and fluorescence resonance energy transfer (FRET) efficiency after a 6 h incubation. Further details are provided in the ESM Methods.
Structural integrity of the H6P antigen
The structural integrity of H6P after encapsulation in RMCS NPs was assessed by SDS-PAGE as previously described , with some modifications. The protective effects of RMCS NPs on H6P against degradation in SGF (containing pepsin, pH 1.2, USP 38) and SIF (containing pancreatin, pH 6.8, USP 38) were also assessed in vitro using SDS-PAGE. Details are provided in the ESM Methods.
Evaluation of FITC-labelled H6P delivery efficiency to Peyer’s patches
The delivery efficiencies of FITC-labelled H6P (FITC-H6P) loaded in CS NPs or RCS NPs were evaluated using confocal laser scanning microscopy (CLSM) as previously described , with some modifications. Details are provided in the ESM Methods.
Evaluation of FITC-H6P uptake by DCs in Peyer’s patches
The uptake of FITC-H6P loaded in CS NPs, MCS NPs or RMCS NPs by DCs in Peyer’s patches was evaluated using flow cytometry. FITC-H6P solution was used as a control. Details are provided in the ESM Methods.
Four-week-old female NOD mice were randomly divided into four groups (ten mice per group). Mice in three groups were given an oral gavage of H6P solution (H6P intra-gastric [i.g].), blank RMCS NPs (RMCS NPs i.g.) or H6P/RMCS NPs (H6P/RMCS NPs i.g.), respectively. Mice in the fourth group were given an s.c. injection of H6P solution (H6P s.c.). Each mouse was immunised with a dose of 100 μg H6P at 4, 5, 6, 7, 8, 9 and 10 weeks of age.
Analytical measurements and histological analysis
The blood glucose levels of NOD mice were monitored from 7 to 23 weeks of age. Mice were diagnosed as having diabetes once the blood glucose level was higher than 11 mmol/l for two consecutive days. Serum insulin levels were determined using a commercial ELISA kit. Insulitis and insulin immunohistochemistry were analysed as previously reported [27, 28]. Further details are provided in the ESM Methods.
Determination of H6P-specific antibodies
The presence of H6P-specific antibodies was determined using ELISA. Details are provided in the ESM Methods.
Four-week-old female NOD mice were randomly divided into three groups (four mice per group) and immunised with RMCS NPs i.g., H6P/RMCS NPs i.g. or H6P s.c. The vaccination process was as described in the ‘Vaccination’ section above. Four weeks after the last administration, T cell proliferation responses to H6P were assessed using a carboxyfluorescein succinimidyl ester (CFSE)-based method; cytokine secretion after H6P stimulation was determined using commercial ELISA kits; and the presence of Tregs and Th1 cells in pancreatic draining lymph nodes was analysed using flow cytometry. Details are provided in the ESM Methods.
Data were evaluated using SPSS Statistics version 17 (SPSS, Chicago, IL, USA). Data are expressed as means ± SD. Statistical significance was determined using the independent-samples t test. The log rank test was used to compare diabetes incidence curves. p < 0.05 indicated statistical significance. No inclusion or exclusion criteria were applied.
Synthesis and characterisation of RMCS
Preparation and characterisation of H6P/RMCS NPs
Characteristics of RMCS NPs and H6P/RMCS NPs
Zeta potential (mV)
Association efficiency (%)
Drug loading (%)
312.1 ± 5.6
33.5 ± 0.4
322.5 ± 6.1
34.9 ± 0.5
91.5 ± 2.7
11.2 ± 2.6
The in vitro release profiles of H6P from H6P/RMCS NPs at pH 1.0 and pH 6.8 are shown in Fig. 2b. The results indicated that less than 4.1% H6P was released from H6P/RMCS NPs at pH 1.0 (simulating the pH of the stomach). At pH 6.8, however, a burst release of H6P from H6P/RMCS NPs was observed within 0.5 h, followed by sustained release.
The stability of H6P/RMCS NPs in SGF or SIF was evaluated by particle size and FRET analysis, respectively. The particle size of H6P/RMCS NPs remained almost unchanged in SGF, but increased to 524.8 ± 10.9 nm within 0.5 h in SIF (Fig. 2c). Although the swelling or aggregation of NPs can be monitored by changes in particle size, any leakage of the encapsulated H6P may not be precisely reflected. FRET analysis can cope with this issue without any pretreatment. Since a FRET signal occurs only when the two probes are in close proximity (<10 nm), any leakage of FITC-H6P reduces FRET efficiency . The fluorescence spectrum of FRET RMCS NPs is shown in Fig. 2d. The emission intensity of FITC decreased at 518 nm and the emission intensity of rhodamine B isothiocyanate increased at 580 nm, implying energy transfer from the donor to the acceptor. As shown in Fig. 2e, the FRET efficiency of NPs increased in SGF, suggesting the formation of more tightly packed NPs that protected the loaded H6P from degradation. In SIF, the FRET efficiency of NPs decreased obviously within 0.5 h, and then decreased gradually. The results suggest that FITC-H6P started to be released from NPs in SIF.
Structural integrity of H6P
The first prerequisite for effective oral protein delivery is to protect the protein from the harsh conditions in the gastrointestinal environment. As shown in Fig. 3, native H6P was degraded in SGF (Fig. 3b, lane 3) or SIF (Fig. 3c, lane 3). However, H6P released from H6P/RMCS NPs after pretreatment with SGF (Fig. 3b, lane 4) or SIF (Fig. 3c, lane 4) showed a target H6P band and no degradation bands. The results suggest that H6P/RMCS NPs effectively protect H6P from degradation in SGF or SIF.
RCS NPs increased FITC-H6P delivery efficiency to Peyer’s patches
Evaluation of FITC-H6P uptake by DCs in Peyer’s patches
Oral vaccination with H6P/RMCS NPs prevented autoimmune diabetes in NOD mice
Oral vaccination with H6P/RMCS NPs activated an H6P-specific Th2-type humoral immune response
To identify the type of immune response, the serum isotype levels of IgG1 and IgG2a were determined. The IgG2a isotype is associated with a Th1-type response, while the IgG1 isotype is associated with a Th2-type response . As shown in Fig. 7b, the anti-H6P antibodies in the H6P/RMCS NPs i.g. group and the H6P s.c. group were almost exclusively of the IgG1 subclass. The results suggest that vaccination with H6P activated an H6P-specific Th2-type response.
Spontaneous T cell proliferation in response to H6P was downregulated and the secretion of Th1-type cytokines was inhibited after H6P/RMCS NPs vaccination
Splenocytes isolated from NOD mice were stimulated in vitro with H6P and the secreted cytokines were measured. As shown in Fig. 8c–f, splenocytes from mice treated with H6P/RMCS NPs i.g. produced higher levels of Th2-type cytokines (IL-4, IL-10) and lower levels of Th1-type cytokines (IFN-γ, IL-2) than those from mice treated with blank RMCS NPs i.g. (all p < 0.01). TGF-β plays a pivotal role in the induction of Tregs from naive T cells to treat autoimmune diseases [32, 33]. As shown in Fig. 8g, splenocytes from mice treated with H6P/RMCS NPs i.g. mice produced higher levels of TGF-β than those from mice treated with blank RMCS NPs i.g. (p < 0.01). These results suggest that the prevention of autoimmune diabetes is associated with the inhibition of Th1-type cytokine secretion and upregulation of Th2-type cytokines.
Oral vaccination with H6P/RMCS NPs increased the frequency of Tregs and decreased the frequency of Th1 cells
For type 1 diabetes immunotherapy, oral immunisation is an attractive route of vaccination. A number of preclinical studies have successfully prevented or treated autoimmune diabetes through the oral delivery of autoantigen [35, 36, 37]. However, the experimental animals in these studies needed to take a large dose of antigen for a long time and the therapeutic effect was limited. In NOD mice, oral administration of 1 mg insulin twice a week for 5 weeks and then weekly until 1 year of age has been reported to reduce the incidence of diabetes, but could not completely prevent the onset of diabetes ; in children genetically susceptible to type 1 diabetes, only a high oral dose of antigen (67.5 mg of insulin daily for 3–12 months) induced an immune response . Therefore, we believe that low delivery efficiency of the antigen may be the main reason for the low efficiency of oral antigen therapy. In a previous study, genetically modified Lactococcus lactis that expressed H6P was used as a vector for intestinal delivery of H6P, but the recombinant L. lactis was administered for 36 weeks and 16.7–25% of NOD mice still developed diabetes . In the current study, we observed that the onset of diabetes in NOD mice was 100% prevented through the oral administration of H6P/RMCS NPs once a week for 7 weeks. This ideal outcome may be attributed to the high antigen delivery efficiency of targeting H6P/RMCS NPs. The vaccination dose of H6P was chosen based on our previous study . We chose to immunise the NOD mice from 4 to 10 weeks of age (i.e. once weekly for 7 weeks), before the onset of diabetes, because this was a prevention study and we thought that that multiple drug deliveries would be associated with a better outcome.
β1 integrins are overexpressed at the apical surface of human and mouse M cells. In addition, it has been demonstrated that grafting RGD peptide that targets β1 integrins onto NPs significantly increases their transport by M cells . Mannose receptor is a 175 kDa transmembrane protein that is highly expressed on antigen-presenting cells such as DCs. It mediates the endocytosis, processing and presentation of antigens that expose mannose residues. Mannose-modified NPs have been widely used for DC-targeted delivery and show enhanced efficiency over other systems . In this study, RGD and mannose were used to modify CS, and we observed that the in vivo uptake of H6P by DCs in Peyer’s patches was enhanced significantly after its encapsulation in RMCS NPs. The characteristics of the DCs that take up the antigen (e.g. whether they are immature, activated or tolerogenic DCs) remain to be analysed in future studies. A further area of future study is identifying the distribution of the remaining antigen, whether in gut-associated lymphoid tissue or within the circulation.
Mechanisms of antigen-induced tolerance include deletion and/or anergy of effector T cells, immune deviation (Th1 to Th2) and induction of Tregs . How these mechanisms are interrelated is not completely understood. Tregs and immune deviation could be outcomes of anergy; alternatively, Tregs could induce anergy in effector T cells. In this study, oral H6P/RMCS NPs increased the secretion of Th2-type cytokines and attenuated the secretion of Th1-type cytokines. These results are in line with those of a previous report, in which vaccination of NOD mice with H6P against autoimmune diabetes was associated with a Th1 to Th2 cytokine shift . In addition, in the current study, oral H6P/RMCS NPs were associated with high levels of anti-H6P IgG1 antibody. This could be induced by HSP65 in the H6P antigen, which has about 50% homology with the human homologue HSP60 . HSP60 can induce the proliferation of B cells . The activated B cells have been reported to not only produce antibody, but also to inhibit spontaneous Th1 autoimmunity . Whether the H6P/RMCS NPs vaccination promotes the generation of H6P-specific IgA in the gut lumen was not analysed in the current study. Because the H6P antigen is incorporated into NPs, we think that anti-H6P IgA is unlikely to interfere with the uptake of NPs upon repeated administration. In recent years, the central role of CD4+CD25+FOXP3+ Tregs in maintaining peripheral immune tolerance has been confirmed . Of clinical importance is the ability of Tregs to exert antigen-non-specific ‘bystander’ suppression. In addition, bystander suppression does not depend on the ‘tolerising’ autoantigen necessarily being the primary driver of pathology. Several studies have successfully prevented or treated diabetes in animal models by inducing antigen-specific Tregs [36, 45]. In this study, we observed that oral H6P/RMCS NPs increased the frequency of CD4+CD25+FOXP3+ Tregs in pancreatic draining lymph nodes at 4 weeks after immunisation. However, whether these Tregs endure for a long time remains to be further studied. In addition, the characteristics of these Tregs (e.g. whether they are de novo induced or an expansion of pre-existing Tregs) is unclear. Of note, s.c. injection of H6P did not increase the frequency of Tregs. These results are in line with the theory that either systemic or mucosal administration of antigen can induce tolerance, but that the mucosal route appears more likely to induce Treg responses . Overall, the mechanisms of action of oral H6P/RMCS NPs against autoimmune diabetes in NOD mice may involve immune deviation (Th1 to Th2) and induction of Tregs. More detailed studies need to be performed to further elucidate the mechanism of action of H6P/RMCS NPs, including T cell responses in the gut-associated lymphoid tissues, pancreatic draining lymph nodes and pancreas at different time points.
In this study, we demonstrated that H6P/RMCS NPs efficiently deliver H6P to DCs in Peyer’s patches, induce antigen-specific T cell tolerance and prevent the onset of autoimmune diabetes in NOD mice. The results suggest that RMCS NPs are suitable for encapsulating biological macromolecules and may serve as a promising platform for oral delivery of autoantigens to induce tolerance.
We thank Y. Xing (manager of flow cytometry at the China Pharmaceutical University) for her scientific advice and technical assistance with flow cytometry.
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.
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