Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: Role of macrophage scavenger receptor class A type I and II

Glycation structures in AGE-OVA produced by the Maillard reaction. A, AGE-OVA was prepared by means of incubation of 1 mmol/L OVA with 1 mol/L glucose at 50°C for 6 weeks. The formation of glycation structures was verified by means of ELISA. Native OVA and OVA thermally processed without glucose (thermally processed OVA) were analyzed as controls. The data represent means ± SEMs of 3 independent experiments. B, Structural formula of the glycation structures. Lys, Lysine residues of proteins.

AGE-OVA enhances the activation and proliferation of OVA-specific CD4⁺ T cells. A, CD4⁺ T cells isolated from OT-II mice were cocultured with mDCs and stimulated with native OVA, OVA thermally processed without glucose, or AGE-OVA for 24 hours. Concentration of IL-2 in the culture supernatant was measured by means of ELISA. ∗P < .001. B, CFSE-stained CD4⁺ T cells were cocultured with mDCs and stimulated with either form of OVA. After 72 hours, CFSE intensity of CD4⁺ T cells was measured by means of flow cytometry. The data are representative of 3 independent experiments.

AGE-OVA does not induce the maturation of mDCs. mDCs were stimulated with 20 μg/mL native OVA, OVA thermally processed without glucose or AGE-OVA or with 10 μg/mL LPS. A, Expression of CD40, CD80, CD86, and MHC class II molecules on mDCs was analyzed with flow cytometry. Gray areas represent mDCs cultured without stimulation. B, Levels of IL-10 and IL-12 p70 in the culture supernatants of mDCs were measured by means of ELISA. The data are representative of 3 independent experiments.

The uptake of OVA by mDCs is increased by means of glycation. RAGE and galectin-3 are not involved. A, Wild-type mDCs were incubated with 0.5 or 5.0 μg/mL FITC-conjugated native OVA, OVA thermally processed without glucose, or AGE-OVA for 15 minutes. The mDC uptake of the samples was analyzed by means of flow cytometry. B, The mDC uptake of FITC-conjugated AGE-OVA was verified by using confocal microscopy. C, Wild-type or RAGE-deficient mDCs were incubated with 5.0 μg/mL FITC-conjugated AGE-OVA for 15 minutes. The uptake of AGE-OVA was analyzed by using flow cytometry. D, Wild-type mDCs were treated with or without 150 mmol/L lactose for 30 minutes before incubation with 5.0 μg/mL FITC-conjugated AGE-OVA to prevent galectin-3–mediated uptake. The uptake of AGE-OVA by mDCs was analyzed by means of flow cytometry. Gray areas represent mDCs cultured with medium only. The data are representative of 3 independent experiments.

SR-AI/II is involved in the uptake of AGE-OVA by mDCs. A, Wild-type or SR-AI/II–deficient mDCs were incubated with 5.0 μg/mL FITC-conjugated recombinant OVA, native OVA, OVA thermally processed without glucose, or AGE-OVA for 15 minutes. The mDC uptake of the samples was analyzed by means of flow cytometry. Gray areas represent mDCs cultured with medium only. B, Wild-type or SR-AI/II–deficient mDCs were incubated for 3 hours with either form of OVA and then fixed with 0.008% glutaraldehyde before 21 hours of coculturing with CD4⁺ T cells isolated from OT-II mice. The concentration of IL-2 in the culture supernatant was measured by means of ELISA. ∗P < .01. ∗∗P < .001. The data are representative of 3 independent experiments.

AGE-BSA, but not native BSA, inhibits the uptake of AGE-OVA by mDCs. mDCs were incubated with or without 50 μg/mL native BSA and AGE-BSA together with 5.0 μg/mL FITC-conjugated, native OVA, OVA thermally processed without glucose, or AGE-OVA. After incubation for 15 minutes, the mDC uptake of either form of OVA was analyzed by means of flow cytometry. Gray areas represent mDCs cultured with cell culture medium only. The data are representative of 3 independent experiments.

Structural analysis of glycated proteins. A, SDS-PAGE profiles of OVA and BSA before and after thermal processing at 50°C for 6 weeks with or without glucose. Lane 1, Native protein; lane 2, protein thermally processed without glycose; lane 3, AGE product. B, The secondary structure of the OVAs was analyzed using circular dichroism spectroscopy.

AGE-OVA enhances cytokine-γ production of OVA-specific CD4⁺ T cells. CD4⁺ T cells isolated from DO11.10 mice were cocultured with mDCs and stimulated with AGE-OVA, native OVA, or OVA thermally processed without glucose for 72 hours. The concentration of IL-2, IL-4 and IFN-γ in the culture supernatant was measured by means of ELISA. ∗P < .001. The data are representative of 2 separate experiments.

The uptake of native OVA by mDCs is higher than that of recombinant OVA. Wild-type mDCs were incubated with 0.5 or 5.0 μg/mL FITC-conjugated native OVA or recombinant OVA for 15 minutes. The uptake of OVAs by the mDCs was analyzed by means of flow cytometry. Gray areas represent cells cultured with medium only. The data are representative of 2 separate experiments.

The uptake of native OVA and OVA thermally processed without glucose is not attenuated by the absence of RAGE or blockade of galectin-3. A, Wild-type or RAGE-deficient mDCs were incubated with 5.0 μg/mL FITC-conjugated native OVA or thermally processed OVA without glucose for 15 minutes. B, Eild-type mDCs were treated with or without 150 mmol/L lactose for 30 minutes before incubation with the FITC-conjugated OVAs to inhibit galectin-3–mediated uptake. The uptake of OVAs by the mDCs was analyzed by means of flow cytometry. Gray areas represent cells cultured with medium only. The data are representative of 3 separate experiments.

Mannan inhibits the uptake of AGE-OVA in SR-AI/II–deficient mDCs. SR-AI/II–deficient mDCs were incubated with or without 3 mg/mL mannan for 30 minutes before incubation with 5.0 μg/mL FITC-conjugated native OVA, OVA thermally processed without glucose, or AGE-OVA to inhibit the MR-mediated uptake. The uptake of OVA by the mDCs was measured by means of flow cytometry. Gray areas represent cells cultured with medium only. The data are representative of 3 separate experiments.

SR-AI/II is involved in the uptake of AGE-OVA by mDCs. A, Wild-type or SR-AI/II–deficient mDCs were incubated with 0.5 μg/mL FITC-conjugated recombinant OVA, native OVA, OVA thermally processed without glucose, or AGE-OVA for 15 minutes. The mDC uptake of the samples was analyzed by means of flow cytometry. Gray areas represent mDCs cultured with medium only. The data are representative of 3 separate experiments.

Uptake of AGE-BSA is attenuated in SR-AI/II–deficient mDCs. Wild-type or SR-AI/II–deficient mDCs were incubated with 0.5 or 5.0 μg/mL FITC-conjugated AGE-BSA for 15 minutes. The uptake of AGE-BSA by the mDCs was measured by means of flow cytometry. Gray areas represent cells cultured with medium only. The data are representative of 3 separate experiments.

SR-AI /II deficiency does not affect expression of costimulatory molecules and the uptake ability of mDCs. Wild-type or SR-AI/II–deficient mDCs were incubated with 10 mg/mL LPS for 18 hours. Expression of CD40, CD80, CD86, and MHC class II molecules on the mDCs was analyzed by means of flow cytometry. Gray areas represent mDCs cultured without LPS stimulation. The data are representative of 3 separate experiments.

AGE-OVA and native OVA induce comparable activation of OVA-specific CD8⁺ T cells. CD8⁺ T cells isolated from OT-I mice were cocultured with mDCs and stimulated with native OVA and AGE-OVA for 24 hours. The concentration of IL-2 in the culture supernatant was measured by means of ELISA. The data are representative of 3 separate experiments.

Expression of receptors for AGEs on mDCs. SR-AI, galectin-3, and RAGE expression on the surface of mDCs was analyzed by means of flow cytometry. A, SR-AI expression on wild-type and SRAI/II–deficient mDCs. B, Galectin-3 expression on wild-type mDCs. C, RAGE expression on wild-type mDCs. Gray areas represent mDCs stained with the isotype control for the respective antibody.