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Oral or Nasal Antigen Induces Regulatory T Cells That Suppress Arthritis and Proliferation of Arthritogenic T Cells in Joint Draining Lymph Nodes

Femke Broere^1,*, Lotte Wieten^*, Elles I. Klein Koerkamp^*, Joel A. G. van Roon, Teun Guichelaar^*, Floris P. J. G. Lafeber and Willem van Eden^*

^* Division of Immunology, Institute of Infectious Diseases and Immunology, University of Utrecht, Utrecht; and Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The propagation of mucosal tolerance as a therapeutic approach in autoimmune diseases remains a difficult goal to achieve, and therefore further mechanistic studies are necessary to develop potential clinical protocols to induce mucosal regulatory T cells (Tr cells). In this study we addressed whether oral or nasal proteoglycan induced functional Tr cells in the cartilage proteoglycan-induced chronic arthritis model. Both nasal and oral application of human proteoglycan before induction of disease suppressed arthritis severity and incidence. Tolerized mice showed enhanced numbers of IL-10 producing CD4⁺ cells in the paw-draining lymph nodes. Furthermore, CD4⁺ spleen cells displayed enhanced expression of molecules associated with Tr cells, such as IL-10, Foxp3, and TGF-β. Transfer of CD4⁺ spleen cells from mucosally tolerized donors into proteoglycan-immunized mice abolished arthritis and reduced humoral responses, indicative of Tr cells with the capacity to inhibit already induced immune responses. Tr cells were activated upon transfer, because enhanced proliferation was observed in the joint draining lymph nodes compared with activated T cells from nontolerized donors. Upon cotransfer with naive proteoglycan-specific T cells, mucosally induced Tr cells inhibited proliferation of these arthritogenic T cells in vivo. Herein we show that both oral and nasal Ag application induced Tr cells, which had a direct inhibitory effect on already established pathogenic B and T cell responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mucosal tolerance is important to prevent inadequate and pathogenic reactivity to normally innocuous Ags that enter the body via mucosal surfaces. In animal models of inflammatory diseases, both oral and nasal Ag application has been shown to protect against the induction of disease via the induction of regulatory T cells (Tr cells)² (1, 2). Mucosal tolerance has been proposed as a means to induce Ag-specific protection in inflammatory diseases, ranging from autoimmune disease to transplant rejection and allergy (3, 4). However, successful translation of prevention of disease in animal models to therapeutic application in human disease proved to be difficult (5, 6, 7, 8, 9, 10, 11). The disappointing outcome of clinical trials can be explained by several facts, such as: the trials were performed in end-stages of disease; the patient populations studied were inherently diverse; and there was a lack of pretreatment screening to determine whether the Ag was immunologically relevant in patients to be treated.

Despite these difficulties, it is unequivocal that oral tolerance can be achieved in humans (12, 13, 14, 15, 16, 17). In some cases, Ags fed suppressed in vitro T cell recall responses but failed to suppress Ag-specific IgG, IgM, and secretory IgA responses, suggesting a differential effect on T and B cell responses (12). Intriguingly, this seems not be the case in IgE-mediated allergy, as it was shown that desensitization by oral immunotherapy was highly successful (18).

Previously, it was demonstrated that relevant epitopes of the cartilage proteoglycan (PG) were recognized by T cells in the context of human class II MHC in HLA-DR4- and HLA-DQ8-transgenic (Tg) mice (19). Moreover, several studies in rheumatoid arthritis (RA) patients showed T and B cell responses against the human PG, indicating that PG may be a potential autoantigen in RA (20, 21, 22). Collectively, these studies suggest that PG may be a target of disease-associated T cell responses in patients with RA (23, 24, 25, 26) (S. E. Berlo, H. de Jong, W. van Eden, and B. J. Prakken, unpublished results). Difficulties with translating animal models into clinical trials clearly show the need to further unravel the complex mechanisms of mucosal tolerance, and to explore their application in experimental autoimmune disease models. Because orally and nasally induced tolerance might induce different Tr cells (27) we wanted to explore both forms of tolerance induction in the PG-induced arthritis (PGIA) model. PGIA is a chronic relapsing model for RA, and disease is based on a combined T and B cell response directed against joint cartilage PG (28, 29, 30).

In this model we studied the potential regulatory role of mucosally induced Tr cells and whether these cells could alter the in vivo response of potentially arthritogenic T cells.

Oral and nasal application of PG was found to suppress the induction of disease in a comparable fashion. The reduction of arthritis severity correlated with enhanced numbers of IL-10-producing Ag-specific CD4⁺ T cells in the local draining lymph nodes. Additionally, enhanced mRNA levels for IL-10 and Foxp3 in CD4⁺ splenocytes indicated the presence of increased numbers of functional Tr cells within this T cell pool, as also shown by reduced arthritis severity in acceptor mice upon CD4⁺ spleen cell transfer. Furthermore, transfer of mucosally induced Tr cells led to IL-10 production in the joint draining lymphoid tissues and reduced influx of CD4⁺ cells and neutrophils into the joints. In the same experiments, transferred (CFSE-labeled) mucosally induced CD4⁺ Tr cells exhibited enhanced proliferation in the joint draining lymph nodes and inhibited proliferation of naive arthritogenic T cells in vivo.

	Materials and Methods

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Mice

Female BALB/c retired breeder mice, aged between 16 and 26 wk, were purchased from Charles River Laboratories. PG-TCR Tg mice were bred and kept under standard conditions (31, 32). Animals were routinely housed and received water and chow ad libitum. Experiments were approved by the Animal Experimental Committee of the University of Utrecht.

Abs and Ag

Anti-CD8 (53-672), anti-CD11b (M1/70, MAC-1), anti-F4/80, anti-MHC class II (M5/114), and anti-CD45R (6B2) were used as culture supernatants at predetermined optimal concentrations. PE-conjugated anti-CD4 (GK1.5), anti-CD25 (PC61), anti-CD69 (H1.2F3), anti-CD103 (M290), and anti-CD27 (Lg.3A1) were purchased from BD Pharmingen.

Proteoglycan (PG) (aggrecan) was purified from human articular cartilage by 4 M guanidinium chloride extraction, and depleted of glycosaminoglycan side chains using endo-β-galactosidase (0.22 mU/mg dry weight) and testicular hyaluronidase (5 U/mg dry weight) as described before (13, 14). For intranasal treatment, potentially present contaminating endotoxins were removed from PG by a Triton X-114 gradient. After this, the PG was extensively washed and a Limulus amebocyte lysate assay was performed according to the manufacturer’s protocol to test for endotoxin contamination. Endotoxin levels were <50 EU/mg.

Tolerance induction and arthritis induction

Mice were either tolerized by 3x 100 µg endotoxin-low PG intranasally (i.n.) in 10 µl PBS or 3x 1 mg PG via gavage in 200 µl PBS on days –7, –5, and –3. Control mice received OVA grade V as a control. Subsequently, mice were immunized for the induction of arthritis on days 0 and 21. Mice were injected i.p. with 400 µg PG in the synthetic adjuvant dimethyl dioctadecyl ammonium bromide (DDA; 2 mg) in 200 µl solution with PBS.

Assessment of arthritis

Paws of all mice were examined three times per week to record abnormalities due to arthritic changes of the joints. The onset and severity of arthritis were determined using a visual scoring system based on swelling and redness of paws in a blinded setup as described previously (28, 29, 30). In brief, the degree of joint swelling for each paw (scored ranging from 0 to 4) was used to express a cumulative arthritis score, with a possible maximum severity index of 16 per animal. The first clinical appearance of swelling was recorded as the onset of arthritis.

Cytokine secretion and cytokine ELISA

The percentage of cytokine-secreting cells and the amount of cytokine secreted during a 24-h restimulation period were assessed in spleen and draining lymph node (DLN) cells. Single-cell suspensions were incubated at 5.10⁶ cells/ml with 50 µg/ml PG (or medium as control) for 24 h. Brefeldin A was added for the last 4 h of culture. Cells were subsequently stained with anti-CD4 (GK1.5), fixed, and permeabilized (Cytofix/Cytoperm, BD Biosciences) and stained with anti-IL-10 (JES5–16) and anti-IFN- (AN-18). Cells were analyzed on a FACSCalibur and data were analyzed with CellQuest software.

In separate 96-well plates, cells were incubated with either 50 µg/ml PG or medium as a control for 72 h and culture supernatants were analyzed using the Luminex 100 system (BD Biosciences) in combination with the LINCOplex assay performed according to the manufacturer’s instructions (Linco Research). In brief, Ab-coated microspheres were incubated with standards, controls, and samples (25 µl) in a 96-well microtiter filter plate overnight at 4°C. After incubation, beads were washed and a mixture containing IL-10-specific and IFN--specific detection Abs was added. After 30 min of incubation at room temperature, streptavidin-PE was added for an additional 30 min. After a final wash step, the beads were resuspended in buffer, read on the Luminex model 100 instrument to determine the concentration of the cytokines of interest, and results were analyzed using LMAT software (Luminex).

ELISA for PG-specific Abs

PG-specific Abs were measured by ELISA as described previously (28, 30, 33). ELISA 96-well plates (Corning) were coated overnight with hPG (0.1 µg protein/well) or native mPG (0.15 µg protein/well) and blocked with 1% fat-free milk in PBS. Sera were applied at increasing dilutions, and isotypes of PG-specific Abs were determined using peroxidase-conjugated mAbs to mouse IgG1 or IgG2a (BD Biosciences) as secondary Abs (33). Serum Ab levels were calculated relative to a corresponding mouse IgG isotype standards (all from BD Biosciences) or mouse serum Ig fractions (Sigma-Aldrich) (28, 30, 33).

Transfer to assess regulatory function of T cells

Single-cell suspensions from spleens of donor mice were depleted from erythrocytes in ACK lysis buffer (150 mM NH₄Cl, 1 mM NaHCO₃ (pH 7.4) and were stained with mAbs specific for CD45R, CD11b, F4/80, MHC class II, and CD8. Positive cells were removed with sheep anti-rat-conjugated Dynal beads (Dynal Biotech).

Negative cells, denoted as enriched CD4⁺ T cells (purity routinely between 85 and 95%), were resuspended in PBS. Per recipient, 1 x 10⁶ cells were transferred at day 20. Mice were immunized at days 0 and 21 for induction of arthritis. For tracking of CD4⁺ cells, we resuspended enriched CD4⁺ cells in PBS at 10⁷ cells/ml and incubated these for 10 min at 37°C with CFSE (Molecular Probes) at a final concentration of 5 µM to follow their division profiles in vivo. CFSE-labeled CD4⁺ T cells were washed in ice-cold PBS with 2% FCS and resuspended in saline. Each mouse received 1 x 10⁷ CD4⁺ CFSE-labeled cells in 100 µl saline by i.v. injection. For tracking of naive CD4 ⁺ T cells isolated from PG-TCR Tg mice, cells were labeled with PKH26. Cells were incubated with 2 x 10^–6 M PKH26 dye in diluent C for 5 min at 25°C at 2 x 10⁷ cells/ml. Labeled cells were washed in PBS with 2% FCS and resuspended in saline.

Quantitative PCR

For quantitative analysis of mRNA expression, 1 x 10⁶ cells or total joint-extracted cells were isolated and total RNA was isolated using the Qiagen RNeasy kit. Subsequently, RNA was transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s protocol. Real time quantitative PCR (Q-PCR) was performed using a Bio-Rad iCycler based on (specific primers and) general fluorescence detection by SYBR Green. HPRT and GAPDH were used as control for sample loading and to allow normalization between samples. cDNA obtained from lymphoid tissues from naive mice was used to allow normalization between experiments. Primers used were: HPRT: sense 5'-CTG GTG AAA AGG ACC TCT CG-3', antisense 5'-TGA AGT ACT CAT TAT AGT CAA GGG CA-3'; GAPDH: sense 5'-CAA CTC ACT CAA GAT TGT CAG CAA-3', antisense 5'-GGC ATG GAC TGT GGT CAT GA-3'; Foxp3: sense 5'-CCC AGG AAA GAC AGC AAC CTT-3', antisense 5'-TTC TCA CAA CCA GGC CAC TTG-3'; IL-10: sense 5'-GGT TGC CAA GCC TTA TCG GA-3', antisense 5'-ACC TGC TCC ACT GCC TTG CT-3'; CD4: sense 5'-GAC TGA CCC TGA AGC AGG AG-3', antisense 5'-CTG GTT CAC CCC TCT GGA TA-3'; myeloperoxidase (MPO): sense 5'-GCT ACC CGC TTC TCC TTC TT-3', antisense 5'-GGT TCT TGA TTC GAG GGT CA-3'.

Statistics

Data were analyzed with a one-way ANOVA with a Bonferonni posthoc test to determine significance for differences between individual groups. p < 0.05 was considered significant.

	Results

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Mucosal Ag application prevents PGIA

To explore whether mucosal tolerance suppressed disease in the chronic PGIA model, PG was applied on days –7, –5, and –3 either i.n. or intragastrically (i.g.). Control mice received OVA protein i.n. and/or i.g. All mice were subsequently immunized for the induction of arthritis on days 0 and 21.

Intranasal and oral application of PG before immunization significantly reduced the development of arthritis, as shown by the lower mean arthritis score when compared with control OVA-treated mice (Fig. 1). Also, the maximum arthritis score of the individual animals was reduced. OVA-treated controls showed an average maximum score of 7.4 ± 4.0, while tolerized animals after nasal treatment only reached a maximum score of 3.2 ± 2.6 and orally tolerized animals had a score of 2.7 ± 2.4 (Table I). Assessment of the mean day of arthritis onset revealed that mucosally treated mice not only developed less severe disease, but also that initial signs of disease occurred significantly later (Table I).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Mucosal application of human PG induces tolerance. All mice were immunized by two immunizations with human PG at day 0 and day 21 to induce arthritis. Before the first immunization mice were tolerized with either 100 µg human PG in 10 µl PBS intranasally () or 1 mg PG orally () at days –6, –3, and –1. Control mice received OVA mucosally. Arthritis scores were analyzed three times a week with a maximum score of 16 per mouse. Data are the mean values of at least 5 mice per group ± SD. Data are representative of four separate experiments. *, p < 0.05 compared to control.

Mucosal Ag enhances Foxp3, IL-10, and TGF-β expression by CD4⁺ splenocytes

Subsequently, we analyzed the effect of mucosally applied Ag on differentiation of CD4⁺ T cells within the paw DLNs and spleens of mucosally tolerized mice as based on cytokine profile and surface marker expression. DLN and spleen cells were isolated on day 40 after the first immunization, and single-cell suspensions were cultured for 24 h in the presence of 50 µg/ml PG. The last 4 h of culture brefeldin A was added to analyze the intracellular cytokine content. For expression of surface markers, cells were analyzed without prior in vitro reactivation.

Both intranasally and orally tolerized mice showed enhanced numbers of PG-specific cells producing IL-10 within the draining lymph nodes compared with OVA-treated controls. Fewer IL-10-producing cells in the OVA-treated control mice coincided with enhancement of the IFN--producing cell population in the DLNs (Fig. 2).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. CD4+ splenocytes of tolerized mice produce more IL-10. Mice were treated as described in Fig. 1. Spleen cells were isolated at day 40 and single-cell suspensions were (re)stimulated in vitro for 24 h with 10 µg PG or medium as control. Brefeldin A was added for the last 5 h. Subsequently, cells were fixed and stained for intracellular cytokines IFN- and IL1–10. Samples are represented as net producing cells compared with medium control samples. FACS plots are representative figures for IFN- and IL-10 producing populations. Data are the mean values of at least 5 mice per group ± SD. *, p < 0.05 compared to control. Data are representative of at least three separate experiments.

Within the spleen cell population of the protected mice, only few cells were found to produce IL-10 in an Ag-specific manner. Therefore, no differences as found in DLNs can be observed by flow cytometry. However, when CD4⁺ cells were isolated to assess in situ expression of IL-10, TGF-β, and Foxp3 (Fig. 3), in both nasally and orally tolerized mice the levels of IL-10, TGF-β, and Foxp3 mRNA were enhanced in the splenic CD4⁺ population compared with control-treated arthritic mice.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. CD4+ splenocytes of tolerized mice express higher levels of Foxp3, IL-10, and TGF-β mRNA. Mice were treated as described in Fig. 1. Spleen cells were isolated at day 40 and RNA was isolated directly. cDNA was used in Q-PCR/SYBR Green analysis for Foxp3, IL-10, TGF-β, and CD25. Data are expressed as relative expression to a calibrator ± SEM of at least 5 mice per group. Data are representative of at least three separate experiments.

Taken together, our data showed that PG-specific T cells in the DLNs of tolerized mice displayed a regulatory cytokine profile upon Ag recognition. Additionally, splenocytes of these mice showed enhanced transcription of IL-10, Foxp3, and TGF-β, all coding for proteins associated with immunomodulatory Tr cells.

Mucosal Ag induces CD4⁺ Tr cells in the spleen with the capacity to suppress arthritis

To determine whether the enhanced expression of regulatory molecules observed in spleen reflected functional differentiation of mucosal Tr cells, we isolated CD4⁺ splenocytes from intranasally tolerized, orally tolerized, and OVA-treated control mice. These CD4⁺ splenocytes, potentially containing Tr cells, were subsequently transferred i.v. to mice on day 20, before the second PG immunization on day 21.

As shown in Fig. 4, mice that received CD4⁺ splenocytes from nasally or orally tolerized mice only developed mild arthritis compared with control groups, indicating that CD4⁺ spleen cells from mucosally tolerized mice had obtained regulatory capacity. On the other hand, CD4⁺ splenocytes from OVA-tolerized control mice slightly enhanced the onset of arthritis, as several mice already showed signs of arthritis on day 26 whereas donor mice developed the first clinical arthritic scores on average on day 29 (Table I).

View larger version (13K): [in this window] [in a new window]   FIGURE 4. CD4+ splenocytes transfer tolerance to arthritogenic immunization in recipient mice. All mice were immunized with 400 µg human PG and 2 mg DDA in 200 µl PBS at day 0 and day 21. Before the second immunization, mice received 1 x 106 CD4+ splenocytes from mice that were tolerized as described in Fig. 1. Arthritis scores were analyzed three times a week (with a maximum score of 16 per mouse). Mean arthritis scores per group per day ± SD are shown. Data are the mean values of at least 5 mice per group ± SD. Data are representative of three separate experiments. *, p < 0.05 compared to control.

Thus, oral or nasal application of PG before induction of arthritis seemed to have induced functional Tr cells that suppressed arthritis in already immunized acceptor mice.

Transferred mucosal Tr cells suppress both T and B cell immunity in arthritis

Because PG-specific autoantibodies are essential for inducing severe arthritis in PGIA, the effect of Tr cell transfer on the B cell response was studied. Both nasally and orally induced Tr cells suppressed the B cell-dependent Ab response as measured by the levels of Ag-specific IgG1 and IgG2a (Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Reduced Ab response to human PG and murine PG after transfer of Tr cells. Mice were treated as described in Fig. 4, and at day 40 blood was drawn and serum was analyzed by ELISA for IgG1 and IgG2a to human and murine PG. Data are the mean values of at least 5 mice per group ± SD. Data are representative of three separate experiments. *, p < 0.05 compared to control.

To follow the effect of Tr cell transfer on infiltration of T cells and neutrophils into the joints of arthritic Tr cell recipients, we analyzed CD4 and MPO contents in joint infiltrates. Q-PCR analysis of cells isolated from the joints of Tr cell recipients showed a clear reduction of infiltrating CD4⁺ and MPO⁺ cells, indicating reduced influx of both CD4⁺ T cells and neutrophils (Fig. 6). This observation correlated with the reduced arthritis scores as shown in Fig. 4.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Reduced infiltration of CD4 T cells in joint tissue of Tr cell acceptor mice. Mice were treated as described in Fig. 4. At day 40 cells were isolated from the knees of arthritic mice. mRNA was isolated, subsequently treated with DNase, and reversely transcribed to cDNA. Q-PCR analysis using cDNA was performed for CD4 and MPO to assess infiltration of CD4+ T cells and neutrophils. mRNA is normalized to HPRT as housekeeping gene.

To analyze the immunomodulatory role of the transferred Tr cells in recipient mice, we analyzed the proteoglycan-specific cytokine response in the joint DLNs. Single-cell suspensions of the paw DLNs were restimulated at 5 x 10⁶ cells/ml with 50 µg/ml PG in vitro during 18 h, and brefeldin A was added for the last 6 h. For the analysis of cytokine secretion in the culture supernatant, cells were stimulated for 72 h in the absence of brefeldin A.

Transfer of nasally and orally induced Tr cells enhanced not only the percentage of IL-10-producing CD4⁺ T cells (Fig. 7A), but also significantly enhanced the concentration of IL-10 in response to PG in the culture supernatant (Fig. 7C). Additionally, PG-specific IFN- secretion was significantly reduced in Tr cell recipients when compared with mice receiving control cells (Fig. 7B). In contrast, no significant differences were detected in the concentration of TNF- (Fig. 7D) or the relative expression of IL-17 mRNA (Fig. 7G). Also, spleen cells from Tr cell recipients restimulated with PG in vitro produced significantly more IL-10 than did cells from control animals (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Enhanced numbers of IL-10 producing cells and IL-10 secretion upon transfer of Tr cells. Mice were treated as described in Fig. 4. A, Intracellular staining for IL-10. Draining lymph node cells were isolated at day 40 and single-cell suspensions were restimulated in vitro for 18 h with 10 µg/ml PG. Brefeldin A was added for the last 4 h. Samples are represented as net producing cells compared with medium control samples. B–D, Single-cell suspensions were restimulated with 50 µg/ml human PG and supernatants were analyzed after 72 h. E–I, Lymph node cells were isolated at day 40, and mRNA was isolated directly, DNase digested, and reversely transcribed. cDNA was used as template for Q-PCR analysis for IL-10, Foxp3, IL-17, TGF-β, and CD25. Data are expressed as relative expression to a calibrator ± SEM. Data are represented as average ± SEM. *, p < 0.05 compared to control. Data are the mean values of at least 5 mice per group ± SD. Data are representative of three separate experiments.

These data indicate that transfer of Tr cells from mucosally tolerized mice modulated the cytokine profile in PG-specific T cells in the paw DLNs as shown by the reduced IFN- production.

Tr cells proliferate in the DLNs and inhibit proliferation of naive T cells

Because it is unclear at present where transferred Tr cells exert their regulatory function, we sought to identify the location of Tr cell activation. Therefore, we labeled 5 x 10⁶ CD4⁺ mucosally induced Tr cells with CFSE before transfer into already immunized recipient mice. Subsequently, mice were immunized the next day and spleen and paw DLNs were isolated 4 days after the second PG immunization and were characterized for proliferation by flow cytometry of CFSE dilution.

Comparable numbers of CFSE-labeled cells were detected in spleens and all LNs analyzed of recipient mice irrespective of the tolerization route of the donor mice, indicating that cells migrated through the lymphoid tissues equally well. Intriguingly, we observed significantly enhanced proliferation of transferred Tr cells within the paw DLNs as compared with control cells (Fig. 8, top panel).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. Transferred Tr cells show enhanced proliferation in DLN. CD4+ splenocytes from mucosally tolerized donors were labeled with CFSE before transfer. Naive CD4+ splenocytes from PG-TCR Tg mice were labeled with PKH-26 and transferred as described in Materials and Methods. Five days after transfer into PG-immunized acceptor mice, spleen and DLN cells were isolated and analyzed for CFSE dilution. The percentage of CFSE+ cells that had divided is plotted. *, p < 0.05 compared to control. Data are the mean values of at least 5 mice per group ± SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mucosal administration of autoantigens has been shown to be a powerful way to induce tolerance in several models of autoimmune inflammation. However, trials of Ag feeding in patients with chronic autoimmune diseases have been disappointing. Therefore, unraveling the cellular basis of Ag-specific mucosal tolerance in more detail may help clinical application of mucosal tolerance induction. In this study we show the induction of mucosal tolerance in PGIA, a model for chronic and progressive arthritis that crucially depends on T cell- and B cell-mediated responses. The clinical relevance of this Ag is growing because literature shows that at least a subset of patients with RA exhibits Ag-specific T and B cell responses against cartilage matrix components (20, 21, 22).

This is the first study showing that both nasal and oral application of PG can suppress PGIA via the induction of mucosal Tr cells. Both routes of Ag delivery significantly reduced arthritis severity and incidence and altered the T cell response in the joint DLNs. Earlier studies in the laboratory showed that treatment of mice with either soy bean trypsin inhibitor or mouse serum albumin did not induce protection against arthritis (maximum score ± SEM of control group 3.3 ± 0.8 vs mouse serum albumin-treated mice 4.3 ± 1.8), underlining the Ag specificity of the response. Additionally, adoptive transfer of CD4⁺ T cells from mucosally tolerized mice showed that functional Tr cells had developed with the potential to suppress T cell- and B cell-mediated immunity in already immunized mice. These transfer studies also showed that Tr cells isolated from spleen migrated to the joint DLNs and suppressed proliferation of naive PG-specific T cells in vivo under disease-inducing conditions.

Most studies exploring the role of mucosal tolerance in autoimmune diseases have focused on oral tolerance induction. However, for practical reasons the nasal route might be more attractive as a therapeutic approach compared with oral application. Nasal application will most likely lead to reduced antigenic degradation, thereby lowering the dose of Ag needed to achieve tolerance. As we have demonstrated previously, the conversion of naive T cells into functional mucosal Tr cells occurs in the mucosa-draining lymphoid tissue within 48–72 h after Ag encounter, and the dose needed for the induction of nasally induced Tr cells was 175-fold lower than that needed for the induction of oral Tr cells (34, 35, 36).

Our data confirm earlier findings on nasal tolerance in arthritis (37, 38, 39). However, our study is the first showing that nasal application of PG was sufficient to induce functional Tr cells that are suppressive upon transfer without further treatment. This is in contrast with an earlier study in the PGIA model (33), which showed that continuous nasal treatment was needed to maintain tolerance after transfer of splenocytes of tolerized mice to SCID acceptor mice. This difference can be explained by a difference in Tr cell population, because we transferred CD4⁺ T cells to immune competent hosts instead of splenocytes to SCID mice. In the earlier study, transfer of unfractionated spleen populations may have led to cotransfer of potentially arthritogenic cells that contributed to disease induction.

Mucosal tolerance to Ags has been considered an effective means to prevent T cell-mediated immune responses to the same Ag. In humans, however, oral tolerance failed to suppress Ag-specific B cell responses to an exogenous Ag (12). B cells are known to play a crucial role in the pathogenesis of RA via the induction of autoantibodies, activation of autoreactive T cells, and formation of tertiary lymphoid structures (40). Modulation of the B cell response via mucosal tolerance induction could therefore strongly enhance therapeutic benefit. Herein we show that mucosal Tr cells are capable of suppressing the Th1-mediated Ag-specific IgG2a response irrespective of the site of their induction to both the tolerizing Ag and the murine PG. However, only orally induced Tr cells also suppress the Ag-specific IgG1 response, indicating a more general suppression of both Th1 and Th2 cell-mediated immune responses. This is in agreement with an earlier study showing that orally tolerized T cells can no longer provide cognate help to B cells (41). Given our finding that nasal tolerance was effectively suppressing disease, we may conclude that suppression of IgG1 is not essential for suppression of disease. These findings indicate that although both oral and nasal tolerance can induce tolerance via the induction of Tr cells, the suppressive mechanisms might differ.

Even though both oral and nasal Ag application resulted in suppression of disease, no obvious changes in cell surface marker expression of T cells in DLNs or the spleen of treated mice were detected. This is in agreement with recent studies exploring the phenotype of mucosally induced Tr cells rapidly after Ag application, as these studies showed that such Tr cells can hardly be distinguished from other recently activated T cells and that regulatory capacity resides in both CD25⁺ and the CD25^– populations (34, 35, 36). Additionally, phenotypic differences in a small population will not be reflected by differences in the entire CD4⁺ T cell population in spleen. However, the observation that differences in regulatory markers are present in spleen mRNA in combination with their ability to transfer tolerance to immunized recipients suggests that these Tr cells do reside in this tissue.

In this study we collected evidence that mucosally induced Tr cells were not only able to migrate into the joint DLN, but also that they were activated locally, as clearly shown by their in vivo proliferation. Transferred CD4⁺ cells from nontolerized mice proliferated significantly less in the DLN; however, we cannot exclude that this reduced proliferation compared with Tr cells is due to the activation that occurred in the severely diseased donor mice. The data indicate that mucosal Tr cells suppressed arthritis development by suppressing proliferation of pathogenic T cells in the joint DLNs, thereby reducing subsequent immune responses at both the T cell and B cell levels, in line with the findings of enhanced IL-10 expression in CD4⁺ T cells and reduced IgG2a levels.

Herein we show that both oral and nasal Ag application can induce functional Tr cells in the chronic and relapsing PGIA model. It is possible that effective translation into therapeutic application in humans will lead to combination therapy with other antiinflammatory approaches, such as anti-TNF-. Such combination of therapies will then broadly target autoaggressive T effector cells while inducing or expanding Ag-specific Tr cells, diverting the autoimmune response into a more regulatory type (42). Recently, the effectiveness of such a combined approach was demonstrated in RA patients (43, 44). The skewing of Ag-specific inflammatory responses toward more tolerogenic responses can become a major addition to available therapeutic options for autoimmune diseases.

	Acknowledgments

 
We thank Dr. Albert Marsman and colleagues of the hospital Hilversum (Hilversum, The Netherlands) for help in obtaining essential materials.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Femke Broere, Division of Immunology, Institute of Infectious Diseases and Immunology, University of Utrecht, Yalelaan 1, 3584 CL Utrecht, The Netherlands. E-mail address: f.broere{at}uu.nl

² Abbreviations used in this paper: Tr cell, regulatory T cell; DDA, dimethyl dioctadecyl ammonium bromide; DLN, draining lymph node; i.g., intragastric; i.n., intranasal; MPO, myeloperoxidase; PG, proteoglycan; PGIA, proteoglycan-induced arthritis; Q-PCR, quantitative PCR; RA, rheumatoid arthritis; Tg, transgenic.

Received for publication December 14, 2007. Accepted for publication May 1, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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