Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis

Construction of NPs Containing MOG_35–55 and ITE.

MS and EAE are caused by an autoimmune response directed against the CNS (1). To induce CNS-specific Tregs, we constructed NPs containing the AhR ligand ITE and MOG_35–55, which contains a CD4⁺ T-cell epitope targeted by effector and Tregs during the course of EAE (21). By using gold particles (60 nm in diameter), we constructed four types of NPs that were stabilized with a layer of thiol-polyethylene glycol (PEG) (16): (i) unloaded NPs, (ii) NPs loaded with ITE (NP_ITE), (iii) NPs loaded with MOG_35–55 (NP_MOG), and (iv) NPs loaded with ITE and MOG_35–55 (NP_ITE+MOG; Fig. 1A).

As a first step toward the characterization of the NPs we studied their UV–visible absorption spectra. We detected a prominent absorption at 530 nm in unloaded gold NPs, which results from the excitation of surface plasmon vibrations in the gold NPs (22) (Fig. 1B). Loading of ITE, MOG_35–55, or MOG_35–55 and ITE shifted the absorption peak to 560 nm, reflecting the binding of ITE and/or MOG_35–55 to the NPs (Fig. 1B). MOG_35–55 was detected in NP_MOG and NP_ITE+MOG, but not in NP or NP_ITE, by gel electrophoresis followed by silver staining (Fig. 1C). Further analysis by transmission EM showed that the NPs had a round morphology and a diameter of ∼60 nm (Fig. 1D).

We analyzed the ability of NP-delivered ITE to activate AhR by using a mammalian cell line stably transfected with a construct carrying the luciferase gene under the control of an AhR-responsive promoter. We found that treatment of the reporter cell line with ITE-containing NPs (NP_ITE and NP_ITE+MOG) led to the significant activation of the AhR-responsive promoter (Fig. 1E). The AhR-responsive promoter, however, was not activated by NPs that did not carry ITE (NP and NP_MOG; Fig. 1E). Thus, ITE loaded into NPs can be released to trigger AhR-dependent signaling.

Compounds loaded onto NPs are protected from enzymatic degradation (23), so we investigated the effect that the incorporation of ITE into NPs might have on its degradation by liver enzymes. Free ITE and NP_ITE were preincubated with a preparation of hepatic microsomes (24), and their ability to activate an AhR-responsive promoter was analyzed in aliquots taken at different time points. Incubation with hepatic microsomes for 1 h decreased the ability of free ITE to activate AhR to ∼50% of its initial value, whereas ITE in NPs maintained its ability to activate AhR under the same experimental conditions (Fig. 1D).

To investigate whether the stabilization of ITE by NPs might have any effect on its in vivo activity, we compared the suppressive activity of free and NP-bound ITE on the experimental autoimmune disease EAE. In agreement with our previous results, daily administration of ITE (100 μg per mouse, i.p.) led to a significant suppression of EAE, but no significant suppression was observed when ITE was administered once per week (Fig. S1). However, the weekly administration of NP-bound ITE (i.p.) led to a significant suppression of EAE development (Fig. S1). Taken together, these data suggest that NPs protect ITE from enzymatic degradation and boost its immunoregulatory activity in vivo.

NP_ITE+MOG Induces Tolerogenic DC.

DCs play a central role in the activation and polarization of T cells in vivo (25), so we investigated the effect of NPs on DC function. Splenic DCs were isolated from naive mice and incubated with NPs, and NP uptake was analyzed by transmission EM. NPs could be detected inside DCs 1 h after their addition to the cells, and were still detectable inside DCs 24 h later (Fig. 2A). To investigate whether the uptake of NPs carrying ITE activates AhR signaling in DCs, we analyzed the expression of cyp1a1, a gene that is directly transactivated by AhR (26). We found that cyp1a1 expression was up-regulated in DCs treated with NP_ITE and NP_ITE+MOG, but not by NP and NP_MOG (Fig. 2B). Thus, ITE-containing NPs are uptaken by DCs and activate AhR signaling.

We (11) and others (13–15) have shown that AhR activation induces tolerogenic DCs that have a decreased ability to polarize naive T cells into effector Th1 or Th17 cells and promote the differentiation of Tregs. Thus, we studied the effects of NPs on the response of DCs to stimulation with the Toll-like receptor agonist Escherichia coli lipopolysaccharide (LPS). Splenic DCs isolated from naive mice were incubated in vitro with NPs and activated with LPS. Incubation with empty NPs did not modify the expression of the class II MHC or costimulatory molecules in DCs, and did not affect their ability to activate naive T cells (Fig. S2 A and B). However, we found that NP_ITE+MOG-treated DCs showed a significant decrease in the expression of MHC-II, CD40, and CD86, but no significant change was seen in CD80 (Fig. S2C). Moreover, incubation with NP_ITE or NP_ITE+MOG led to a significant reduction in the production of the Th1 and Th17 polarizing cytokines il12 and il6, respectively (Fig. 2C).

To directly investigate the effects of NPs on the activation and polarization of T cells by DCs, NP-treated DCs were activated with LPS and used to stimulate CD4⁺ 2D2⁺ T cells, which express a transgenic T-cell receptor that recognizes MOG_35–55 (27). We found that incubation of naive CD4⁺ 2D2⁺ T cells with DCs and NP_MOG resulted in the proliferation of the 2D2⁺ T cells in the absence of exogenous MOG_35–55, demonstrating that NP-delivered MOG_35–55 is presented by the DCs (Fig. 2D). Activation of 2D2⁺ T cells with DCs and NP_ITE+MOG, however, triggered a significantly reduced proliferative T-cell response (Fig. 2D), which resembles the reduced response of T cells incubated with ITE-treated DCs (11). We also observed a significant decrease in the proliferative response of 2D2⁺ T cells activated with DCs and NP_ITE+MOG or NP_ITE in the presence of exogenously added MOG_35–55 (Fig. S2D).

The analysis of cytokine secretion showed that 2D2⁺ T-cell activation with DCs and NP_MOG triggered the production of significant amounts of IFN-γ and IL-17, indicative of the polarization of Th1 and Th17 cells (Fig. 2E). The production of IFN-γ and IL-17, however, was significantly reduced when 2D2⁺ T cells were activated with DCs and NP_ITE+MOG (Fig. 2E). Conversely, DCs incubated with NP_ITE+MOG showed an increased ability to promote the differentiation of FoxP3⁺ Tregs (Fig. 2F). Taken together, these results are in agreement with the reported effects of AhR activation on the ability of DCs to activate an polarize effector and Tregs (11, 13–15), and demonstrate that NP_ITE+MOG induces tolerogenic DCs that favor the generation of FoxP3⁺ Tregs.

NP_ITE+MOG Administration Suppresses EAE.

We then studied the effects of NPs in vivo. Following parenteral administration, pegylated gold NPs have been reported to home to the spleen (16). DCs control T-cell activation and polarization in vivo (25), so we studied the effect of NP administration on splenic DCs. NPs were parenterally administered to naive mice, and, 6 h later, AhR activation in splenic DCs was analyzed by quantitative PCR. We found a significant up-regulation of cyp1a1 expression in DCs isolated from NP_ITE- or NP_ITE+MOG-treated mice, but not in those isolated from NP- or NP_MOG-treated mice (Fig. 3A). We also found a significant activation of AhR, as indicated by cyp1a1 expression, in splenic macrophages and B cells isolated from NP_ITE+MOG-treated mice (Fig. S3A).

Given the central role of DCs in priming encephalitogenic Th1 and Th17 cells in vivo (25), we analyzed the effect of NP administration on the production of Th1 and Th17 polarizing cytokines. DCs from NP_ITE+MOG-treated mice showed a significant decrease in the production of IL-6 and IL-12 in response to activation with LPS ex vivo (Fig. S3 B and C). Taken together, these data demonstrate that ITE-containing NPs activate AhR signaling in splenic DCs in vivo and decrease the production of Th1 and Th17 polarizing cytokines.

Based on the effects of NP_ITE+MOG on the activation and antigen presenting cell (APC) function of DCs in vitro and in vivo, and the role played by DCs in the differentiation of encephalitogenic and Tregs (25), we studied the effects of NPs on EAE. EAE was induced by immunization with MOG_35–55 in naive B6 mice, NPs were administered weekly (6 μg per mouse) starting on the day of EAE induction, and the animals were monitored daily for the development of the disease. NP_ITE+MOG administration resulted in a significant suppression of EAE development (Fig. 3B). Treatment with NP_ITE also led to a significant amelioration in EAE symptoms, but the protective effects of NP_ITE were not as strong as those observed with NP_ITE+MOG, and this difference in the protection achieved by NP_ITE or NP_ITE+MOG treatment was found to be significant (Fig. 3B). Of note, the coadministration of free MOG_35–55 and ITE had no significant effect on the development of EAE (Fig. S4).

EAE in B6 mice is driven by MOG_35–55–specific Th1 and Th17 cells (21). Thus, to study the suppression of EAE by NP_ITE+MOG, we analyzed the effect of NP administration on the T-cell recall response to MOG_35–55. Treatment with NP_ITE and NP_ITE+MOG resulted in a significant decrease in the proliferation and the production of IFN-γ and IL-17 triggered by MOG_35–55 in recall experiments; this decrease was significantly stronger in the NP_ITE+MOG group (Fig. 3 C and D). No significant effect was detected when the response to anti-CD3 stimulation was investigated (Fig. S5 A and B), suggesting that NP-treated mice are not systemically immune-suppressed. Thus, NP_ITE+MOG suppress the encephalitogenic Th1 and Th17 T-cell response and the development of EAE.

To investigate the potential therapeutic value of the coadministration of antigen and ITE using NPs, we used the SJL model of EAE induced by immunization with the 139 to 151 region of the proteolipid protein (PLP). In this model, the chronic phase of EAE is characterized by the spreading of the T-cell response to the PLP epitope placed between residues 178 and 191 (PLP_178–191) (28). Thus, we tested the therapeutic effect on SJL EAE of treatment with NP_ITE, NPs loaded with ITE and PLP_139–151 (NP_ITE+139), and ITE and PLP_178–191 (NP_ITE+178). In addition, an experimental group was treated with both NP_ITE+139 and NP_ITE+178; empty NPs were used as controls. EAE was induced by immunization with PLP_139–151 in naive SJL mice, and, on day 17 after disease induction, the mice were assigned to the different experimental groups and treated with NPs (6 μg per mouse); the animals were treated weekly during the duration of the experiment. We observed that treatment with NP_ITE had no significant effect in the development of EAE (Fig. 3E). We also found that the administration of NP_ITE+139 had a transient beneficial effect on the development of EAE; however, the administration of NP_ITE+139 and NP_ITE+178 led to a significant reduction in the EAE score, maximal EAE score, and the number of relapses after the initiation of NP treatment (Fig. 3E and Table S1). Treatment with NP_ITE+139 alone had no significant effect on the chronic phase of EAE. Taken together, these data suggest that a combination of NPs targeting several relevant T-cell reactivities can be engineered to control epitope spreading and chronic inflammation in established CNS autoimmunity.

FoxP3⁺ Tregs Mediate Suppression of EAE by NP_ITE+MOG.

We (9–11) and others (13, 14, 29–31) have shown that AhR activation can expand the FoxP3⁺ T_reg compartment, so we investigated the effect of ITE on FoxP3⁺ T_reg. We found that the suppression of EAE development by AhR activation with NP_ITE+MOG was associated with a significant increase in the frequency of CD4⁺ Foxp3⁺ Tregs in the spleen (Fig. 4B) and the blood (Fig. 4B). Thus, NP_ITE+MOG administration suppresses the encephalitogenic Th1/Th17 (Fig. 3D) whereas it promotes the generation of FoxP3⁺ Tregs.

We then performed transfer experiments to study the role of CD4⁺ Foxp3⁺ Tregs in the suppression of EAE triggered by NP_ITE+MOG. EAE was induced in Foxp3^gpf mice carrying a GFP reporter in the foxp3 gene (32), the mice were treated with NP or NP_ITE+MOG, and CD4⁺ T cells were isolated and transferred into naive mice. We found that naive recipients could be protected from the development of EAE by the transfer of 5 × 10⁶ CD4⁺ T cells isolated from NP_ITE+MOG-treated mice, but not with cells isolated from NP-treated mice (Fig. 4C). Removal of the CD4⁺FoxP3:GFP⁺ Treg fraction abrogated the protective effect of the transferred cells (Fig. 4C), suggesting that NP_ITE+MOG-induced CD4⁺FoxP3:GFP⁺ Tregs are responsible for the control of the encephalitogenic T-cell response.

Mice and Reagents.

C57BL/6 mice were purchased from Jackson Laboratories and kept, together with 2D2⁺ (27) and FoxP3^gfp (32) mice, in a pathogen-free facility at the Harvard Institutes of Medicine. All experiments were carried out in accordance with the guidelines of the standing committee of animals at Harvard Medical School. ITE was purchased from Sigma-Aldrich and from Tocris Bioscience.

NP Preparation.

NPs were produced by using PBS solution, 60 nm tannic acid-stabilized gold particles at a concentration of 2.6 × 10¹⁰ particles per milliliter (Ted Pella), methoxy-PEG-SH (molecular weight, ∼5 kDa; Nektar Therapeutics), ITE (Tocris Bioscience), and MOG_35–55 (MEVGWYRSPFSRVVHLYRNGK). Freshly prepared solutions of ITE (3.5 mM), MOG_35–55 (600 μg/mL), or ITE and MOG_35–55 (3.5 mM and 600 μg/mL, respectively) were added drop by drop to a rapidly mixing gold colloid at a 1:6 ITE solution:colloid volume ratio, which facilitates even distributions of the molecules on the gold particle surface (16). After 30 min incubation at room temperature with shaking, methoxy-PEG-SH (10 mM) was added drop by drop to the colloids. This surface coverage has been shown to result in a complete PEG monolayer on the gold particle surface, and stabilizes gold colloids against aggregation under various conditions (16). Moreover, it has been reported that the addition of 10- to 20-fold excess PEG-SH does not result in any changes in colloid stability or in the thickness of the polymer coating layer (16). After an additional 30 min incubation at room temperature, the colloids were pelleted by centrifugation, resuspended in PBS solution, and characterized by UV–visible spectroscopy and transmission EM.

Transmission EM.

DC-incubated NPs were fixed in the dish for at least 1 h at room temperature with 2.5% (vol/vol) glutaraldehyde, 1.25% (vol/vol) paraformaldehyde, and 0.03% picric acid in 0.1M sodium cacodylate buffer (pH 7.4). The cells were then postfixed for 30 min in 1% OsO₄/1.5% (wt/vol) KFeCN₆, washed in water three times, and incubated in 1% aqueous uranyl acetate for 30 min followed by two washes in water and subsequent dehydration in grades of alcohol [5 min each; 50%, 70%, 95% (vol/vol), twice at 100%]. Cells were removed from the dish in propylene oxide, pelleted at 1,000 × g for 3 min, and infiltrated for 2 h in a 1:1 mixture of propylene oxide and TAAB Epon (Marivac). The samples were then embedded in TAAB Epon and polymerized at 60 °C for 48 h.

Ultrathin sections (approximately 60 nm) were cut on a Reichert Ultracut-S microtome, picked up onto copper grids stained with lead citrate, and examined in TecnaiG² Spirit BioTWIN, and images were recorded with an AMT 2k CCD camera.

Gel Electrophoresis.

NPs (3.5 μg per lane) and MOG_35–55 (1, 0.1, and 0.01 μg) were run by using NuPAGE 4% to 12% 1.0-mm Bis-Tris gels (Invitrogen), and MOG_35–55 was visualized by silver staining (SilverQuest staining kit; Invitrogen) and Coomassie brilliant blue staining.

Reporter Assays.

HEK293 cells were transfected by using FuGENE HD (Roche), and the cells were analyzed after 24 h with the dual luciferase assay kit (New England Biolabs). Tk-Renilla was used for standardization.

Microsomal Degradation.

ITE or NP_ITE was incubated with mouse hepatic microsomes (2 mg/mL; Sigma-Aldrich) in a reaction buffer containing NAPDH (1 mM), MgSO₄ (8 mM), KCl (45 mM), and 3.3 glucose 6-phosphate, pH 7.4, at 37 °C for different periods of time.

Purification of DCs.

DCs were purified from the spleens of naive B6 mice by using CD11c⁺ magnetic beads according to the manufacturer’s instructions (Miltenyi). To generate bone marrow-derived DCs, bone marrow cells were isolated from the femurs of naive mice and cultured for 5 d in the presence of IL-4 (10 ng/mL) and GM-CSF (10 ng/mL). On day 5, the cells were purified with CD11c⁺ magnetic beads (Miltenyi), incubated with NPs, and stimulated with LPS.

Real-Time PCR.

RNA was extracted from cells by using an RNA Easy Mini Kit (Qiagen), cDNA was prepared as recommended, and real-time PCR was performed by using an ABI7500 cycler (Applied Biosystems). All values are expressed as fold increase or decrease relative to the expression of GAPDH.

T-Cell Differentiation in Vitro.

CD4⁺ T cells were activated with bone-marrow-derived cells or DCs at a 3:1 (100,000:30,000) T-cell-to-DC ratio, and activated with MOG_35–55 (20 μg/mL) as described (11).

Cell Proliferation and Cytokine Production.

Cells were cultured in serum-free X-VIVO 20 media (BioWhittaker) for 72 h. During the last 16 h, cells were pulsed with 1 mCi of [³H]thymidine (PerkinElmer), followed by harvesting on glass fiber filters and analysis of incorporated [³H]thymidine in a β-counter (1450 Microbeta Trilux; PerkinElmer). Culture supernatants were collected after 48 h, and cytokine concentration was determined by ELISA by using antibodies to IFN-γ and IL-17 from BD Biosciences.

FACS.

For intracellular cytokine staining, cells were stimulated in culture medium containing phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1 μg/mL; Calbiochem), and GolgiStop (BD Biosciences) for 4 h. After staining of surface markers, cells were fixed and permeabilized as described and incubated with cytokine-specific antibodies (1:100) at 25 °C for 30 min.

NP Administration and EAE Induction.

NPs were administered i.v. or i.p. (6 μg per mouse). EAE was induced by s.c. immunization with 100 μg of the MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) in complete Freund adjuvant (CFA) and administration of 150 ng of pertussis toxin (Sigma-Aldrich) i.p. on days 0 and 2 as described (10). Clinical signs of EAE were assessed according to the following score: 0, no signs of disease; 1, loss of tone in the tail; 2, hind limb paresis; 3, hind limb paralysis; 4, tetraplegia; and 5, moribund.

Statistical Analysis.

Statistical analysis was performed by using Prism software (GraphPad). P values <0.05 were considered significant.