Oral gene application using chitosan-DNA nanoparticles induces transferable tolerance

Abstract

Oral tolerance is a promising approach to induce unresponsiveness to various antigens. The development of tolerogenic vaccines could be exploited in modulating the immune response in autoimmune disease and allograft rejection. In this study, we investigated a nonviral gene transfer strategy for inducing oral tolerance via antigen-encoding chitosan-DNA nanoparticles (NP). Oral application of ovalbumin (OVA)-encoding chitosan-DNA NP (OVA-NP) suppressed the OVA-specific delayed-type hypersensitivity (DTH) response and anti-OVA antibody formation, as well as spleen cell proliferation following OVA stimulation. Cytokine expression patterns following OVA stimulation in vitro showed a shift from a Th1 toward a Th2/Th3 response. The OVA-NP-induced tolerance was transferable from donor to naïve recipient mice via adoptive spleen cell transfer and was mediated by CD4(+)CD25(+) T cells. These findings indicate that nonviral oral gene transfer can induce regulatory T cells for antigen-specific immune modulation.

Fig 1

Gene expression kinetics after oral application of chitosan-DNA NP. Mice were fed a single dose of antigen-encoding chitosan-DNA NP containing 50 μg of plasmid DNA. At 0 h, 3 h, 6 h, 12 h, 24 h, or 48 h, Peyer's patches (A) and mesenteric lymph nodes (B) were removed and analyzed for gene expression by qRT-PCR. The results were normalized against GAPDH and are shown in relative expression units, representing the means of three independent experiments ± SDs.

Fig 2

OVA-encoding chitosan-DNA nanoparticles suppress delayed hypersensitivity and OVA-specific antibody formation. (A) BALB/c mice were fed five doses of OVA-NP containing 50 μg plasmid DNA (days 0 to 9). On day 12, mice were immunized by subcutaneous injection of 100 μg OVA in 100 μl of a 1:1 mixture of PBS and CFA. On day 19, mice were challenged by subcutaneous injection of 50 μg OVA in 30 μl PBS into the right ear pinna, while 30 μl PBS was injected into the left ear pinna as a control. (B) Twenty-four hours later, ear swelling was measured and the DTH response was calculated. (C) On day 21, blood samples were collected and the OVA-specific IgG concentrations were analyzed by endpoint dilution ELISA. Mice of the control groups were fed five times with PBS, OVA-plasmid (50 μg), pure chitosan solution, or EGFP-NP. The OVA protein-fed group received two doses of 25 mg OVA protein on days 0 and 2. Data shown were obtained for 4 to 8 mice per group (*, P < 0.05; n.s., P ≥ 0.05 for OVA-NP compared to controls).

Fig 3

OVA-NP suppress antigen-specific proliferation. OVA-NP-, OVA protein-, or PBS-fed mice were immunized against OVA on day 12, as described for Fig. 1. On day 26, mice received a second immunization. Thirteen days later, spleen MNCs were isolated and cultured with different OVA concentrations in R10+ medium at 37°C with 5% CO₂ and humidified atmosphere. After 72 h of incubation, [H3]thymidine (1 μCi/well) was added. Eighteen hours later, cells were harvested and thymidine incorporation was measured. Data shown represent duplicates and were obtained for 3 or 4 mice per group (*, P < 0.05 for OVA-NP or OVA protein compared to PBS).

Fig 4

Cytokine production patterns after OVA-NP application. OVA-NP-, OVA protein-, and PBS-fed mice were immunized against OVA on day 12. Eight days later, splenic MNCs were isolated and cultured in the presence of 20 μg/ml OVA. Forty-eight hours later, supernatants were analyzed for cytokine production by cytometric bead assay (CBA) or ELISA. Data shown were obtained for 4 or 5 mice per group (*, P < 0.05 for OVA-NP compared to PBS or OVA protein).

Fig 5

The tolerance induced by OVA-NP is transferable. (A) Mice were fed five doses of OVA-NP containing 50 μg plasmid DNA (days 0 to 9). The control group received PBS instead of nanoparticles. On day 11, mice were sacrificed and their spleen cells were isolated. (B) Naïve recipient mice were injected with 2 × 10⁷ splenocytes in the tail vein and immunized the next day. Seven days after immunization, the OVA-specific DTH response was analyzed. To identify the phenotype of regulatory cells mediating tolerance, isolated CD4⁺ T cells or the remaining CD4⁺-depleted fraction (C) and isolated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells (D) from OVA-NP- or PBS-treated mice were used for the adoptive transfer. Data shown were obtained for 4 to 6 mice per group (*, P < 0.05; n.s., P ≥ 0.05).