Induction of antigen-specific immune tolerance using biodegradable nanoparticles containing antigen and dexamethasone

Figures & data

Table 1 Characterization of PLGA-NPs

	Particle size (nm)	Polydispersity index	OVA loading capacity(μg/mg NP)	Dex loading capacity (μg/mg NP)	Zeta-potential (mV)
NP[OVA]	863.23±62.89	0.125±0.045	148.26±11.20	－	−1.85±0.13
NP[OVA+Dex]	864.80±20.50	0.269±0.022	148.37±10.92	0.978±0.022	−1.79±0.56

Notes: The average size, polydispersity index, and zeta-potential of the NPs were measured using a particle size analyzer. The OVA content was determined using a microbicinchoninic acid assay after lysing the NPs in a lysis buffer containing 0.1% SDS and 0.1 N NaOH. The concentration of Dex was determined by high performance liquid chromatography using a YMC J’sphere ODS-H80 column (150×4.6 mm) after lysing the NPs in a 1:2 mixture of dimethyl sulfoxide and methanol.

Abbreviations: Dex, dexamethasone; NPs, nanoparticles; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; SDS, sodium dodecyl sulfate.

Figure 1 Effects of NP[OVA+Dex] on the expression of cell surface molecules in DCs.

Notes: (A) DCs generated from the bone marrow cells of C57BL/6 mice were stimulated with IFN-γ (50 ng/mL) plus TNF-α (50 ng/mL), or treated with NP[OVA] or NP[OVA+Dex] (10 μg/mL as OVA) for 48 h. DCs were stained for CD11c, H-2K^b, I-A^b, CD80, CD86, and PD-L1. CD11c⁺ cells were gated and analyzed for the expression of cell surface molecules. The data shown are representative histograms of four independent experiments. (B) Mean fluorescence intensities of NP-treated, or untreated DCs. The data are presented as the mean ± SD of four independent experiments. (C) Expression of CD86 and PD-L1 in NP-treated, or untreated DCs. The data shown are representative histograms of four independent experiments. (D) The proportion of CD86^loPD-L1^hi cells in each experimental group is shown. The data are presented as the mean ± SD of four independent experiments. The significance of the data was evaluated using a Tukey test of one-way ANOVA test. *P<0.05, **P<0.01, ***P<0.001. “ns” indicates no significant difference.

Abbreviations: DCs, dendritic cells; Dex, dexamethasone; IFN-γ, interferon-γ; NP, nanoparticle; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; PD-L1, programmed death-ligand 1; SD, standard deviation; TNF-α, tumor necrosis factor-α.

Figure 2 Effects of NP[OVA+Dex] on cytokine production in DCs.

Notes: DCs generated from bone marrow cells of C57BL/6 mice were stimulated with IFN-γ (50 ng/mL) plus TNF-α (50 ng/mL), or treated with NP[OVA] or NP[OVA+Dex] (10 μg/mL as OVA) for 48 h. For TNF-α production, DCs were stimulated with IFN-γ (50 ng/mL) plus LPS (100 ng/mL). Cytokine secretion to culture supernatant was determined by ELISA. The data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. “ns” indicates no significant difference.

Abbreviations: DCs, dendritic cells; Dex, dexamethasone; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; LPS, lipopolysaccharide; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; SD, standard deviation; TNF-α, tumor necrosis factor-α.

Figure 3 DCs treated with NP[OVA+Dex] are impaired in both MHC class II-restricted exogenous antigen presentation and allogeneic T cell stimulatory capacity.

Notes: (A) DCs generated from bone marrow cells of BALB/c mice were treated with PBS, NP[OVA], or NP[OVA+Dex] (50 μg/mL as OVA) for 2 h. After washing and fixing, DCs were co-cultured with OVA_323-339-specific DOBW cells. The supernatants were harvested and IL-2 production was measured by ELISA. The data are presented as mean ± SD of three independent experiments. (B) DCs generated from C57BL/6 mouse bone marrow cells were stimulated with IFN-γ (50 ng/mL) plus TNF-α (50 ng/mL), or treated with NP[OVA] or NP[OVA+Dex] (10 μg/mL as OVA) for 48 h. DCs were then co-cultured with T cells isolated from the spleens of BALB/c mice at the indicated ratios for 96 h. T cell proliferation was measured by the incorporation¹H-thymidine added for the final 18 h of culture. **P<0.01, ***P<0.001.

Abbreviations: DCs, dendritic cells; Dex, dexamethasone; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; IL-2, interleukin-2; MHC, major histocompatibility complex; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; PBS, phosphate-buffered saline; SD, standard deviation; TNF-α, tumor necrosis factor-α.

Figure 4 DCs treated with NP[OVA+Dex] induce Foxp3⁺ Treg cells from naïve CD4⁺CD25⁻ T cells.

Notes: (A) Induction of Foxp3⁺ Treg cells from OVA-specific CD4⁺CD25⁻ T cells. DCs generated from C57BL/6 mouse bone marrow cells were stimulated with IFN-γ (50 ng/mL) plus TNF-α (50 ng/mL), or treated with NP[OVA] or NP[OVA+Dex] (10 μg/mL as OVA) for 48 h. DCs were co-cultured with CD4⁺CD25⁻ T cells isolated from the spleens of OT-II mice at a ratio of 1:10 in a medium containing recombinant human IL-2 (100 U/mL) for 4 days. Cells were gated on CD4⁺TCR Vα2⁺ cells and the expression of CD25 and Foxp3 was analyzed. The data show representative histograms of three independent experiments. (B) The proportion of CD25⁺Foxp3⁺ Treg cells in the CD4⁺TCR Vα2⁺ cell population of each experimental group is shown. (C) Induction of Foxp3⁺ Treg cells in allogeneic MLR. DCs were then co-cultured with CD4⁺CD25⁻ T cells isolated from the spleens of BALB/c mice at a ratio of 1:10 in a medium containing recombinant human IL-2 (100 U/mL) for 4 days. Cells were gated on CD4⁺ cells and the expression of CD25 and Foxp3 was analyzed. The data show representative histograms of three independent experiments. (D) The proportion of CD25⁺FoxP3⁺ Treg cells in the CD4⁺ cell population of each experimental group is shown. The data are presented as mean ± SD of three independent experiments. The data are presented as the mean ± SD of three independent experiments. **P<0.01, ***P<0.001. “ns” indicates no significant difference.

Abbreviations: DCs, dendritic cells; Dex, dexamethasone; Foxp3, forkhead box P3; IFN-γ, interferon-γ; IL-2, interleukin-2; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; MLR, Mixed lymphocyte reaction; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; SD, standard deviation; TCR, T cell receptor; TNF-α, tumor necrosis factor-α; Treg cells, regulatory T cells.

Figure 5 Effect of i.v. treatment of NP[OVA+Dex] on OVA-specific responses in vivo.

Notes: (A) Induction of OVA-specific CTLs. PBS, NP[OVA], or NP[OVA+Dex] (100 μg/mouse as OVA) was i.v. injected into C57BL/6 mice. After 7 days, OVA-specific cytotoxic activity was assessed using an in vivo CTL assay. The target cells were a 1:1 mixture of syngeneic cells pulsed with the OVA_257-264 peptide and then labeled with a high concentration of CFSE and syngeneic cells non-pulsed and labeled with a low concentration of CFSE. The target cells were i.v. injected into recipient mice and the specific cytotoxicity was determined 18 h later. (B) The proportion of killed target cells in each experimental group is shown. The data are presented as the mean ± SD of three independent experiments. (C-D) Production of OVA-specific IgG. PBS, NP[OVA], or NP[OVA+Dex] (100 μg/mouse as OVA) was i.v. injected into C57BL/6 (C) or OT-II mice (D). After 7 days, the sera were collected from the mice, and OVA-specific IgG levels were measured by ELISA. (E) The proportion of Foxp3⁺ Treg cells in the CD4⁺TCR Vα2⁺ cell population of each experimental group is shown. The data are presented as the mean ± SD of three independent experiments. **P<0.01, ***P<0.001. “ns” indicates no significant difference.

Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocyte; Dex, dexamethasone; ELISA, enzyme-linked immunosorbent assay; Foxp3, forkhead box P3; IgG, immunoglobulin G; i.v., intravenous; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; PBS, phosphate-buffered saline; SD, standard deviation; TCR, T cell receptor; Treg cells, regulatory T cells.

Figure 6 Effect of oral treatment of NP[OVA+Dex] on OVA-specific responses in vivo.

Notes: (A) Induction of OVA-specific CTLs. PBS, NP[OVA], or NP[OVA+Dex] (200 μg/mouse as OVA) was i.g. injected into C57BL/6 mice on day 0 and 2. On day 9, mice were i.v. injected with NP[OVA] (80 μg/mouse as OVA) or PBS. After 7 days, OVA-specific cytotoxic activity was assessed using an in vivo CTL assay, as described in Figure 5A. (B) The proportion of killed target cells in each experimental group is shown. The data are presented as the mean ± SD of three independent experiments. (C) Production of OVA-specific IgG. PBS, NP[OVA], or NP[OVA+Dex] (200 μg/mouse as OVA) was i.g. injected into C57BL/6 mice on day 0 and 2. On day 9, mice were i.v. injected with NP[OVA] (80 μg/mouse as OVA), or PBS. After 7 days, the sera were collected from the mice, and OVA-specific IgG levels were measured by ELISA. The data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. “ns” indicates no significant difference.

Abbreviations: CTL, cytotoxic T lymphocyte; Dex, dexamethasone; ELISA, enzyme-linked immunosorbent assay; i.g., Intragastric; IgG, immunoglobulin G; i.v., intravenous; NP[OVA+Dex], nanoparticles containing ovalbumin and dexamethasone; NP[OVA], nanoparticles containing only ovalbumin; OVA, ovalbumin; PBS, phosphate-buffered saline; SD, standard deviation.

Figure S1 Scanning electron microscopy showing the surface morphology of NP[OVA+Dex] with a mean size of 865 nm. The morphology of the NP[OVA+Dex] was visualized by scanning electron microscopy (LEO-1530, Carl Zeiss, Germany)

Figure S2 In vitro release kinetics. NP[OVA+Dex] was incubated in buffers with pH 3.0 and pH 7.0 at 37°C. At indicated time points, the NP[OVA+Dex] was collected by centrifuging at 3,000g for 10 min, and then lysed in a lysis buffer containing 0.5% SDS and 0.5 N NaOH for the determination of OVA content, or in a 1:1 mixture of DMSO and methanol for the determination of Dex. The OVA content was determined using a microbicinchoninic acid assay, and the Dex content was determined using HPLC, as described in detail in the methods section