Ovalbumin (ova)

Polyvinylpyrrolidone (PVP, K30), ferricyanide (K₃[Fe(CN)₆]·3H₂O), iron (II) chloride (FeCl₂), manganese (II) chloride (MnCl₂) were purchased from Aladdin Reagent Company (Shanghai, China). Ovalbumin (OVA) was obtained from Sangon Biotech Company (Shanghai, China). Phosphate buffered saline (PBS) was procured from meilunbio company (Dalian, China). Dulbecco's Minimum Essential Medium (DMEM) and RPMI Medium Modified (RPMI-1640) were procured from Hyclone (Logan, UT, USA). Fluorescein diacetate (FDA) and Propidium iodide (PI) were acquired from Sigma (New York, NY, USA). Cell counting kit-8 (CCK-8) was obtained from Vazyme (Nanjing, China).

SPF C57BL/6 mice (Female, 5-week-old, 16–18g) were purchased from Anhui Laboratory Animal Center (Hefei, China). All mice were housed at a temperature of 20–26°C and on a 12 h light and dark alternating time. This study was conducted in accordance with the Basel Declaration and the ethical guidelines by the International Council for Laboratory Animal Science (ICLAS). The Animal Care and Use Committee of Anhui Medical University approved all experimental procedures (Permit Number: LLSC20170361).
The murine macrophage cell line RAW264.7 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Inc., St. Louis, MO), supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) and 1% penicillin/streptomycin solution (C0222, Beyotime Biotechnology, China) at 37°C in 5% CO₂. RAW264.7 cells were grown in 24-well plates to 70–80% confluence and then stimulated with OVA (500 μg/mL, Sigma). Some cells were treated with CpG-ODN 1668 (1.5 μM, 5 μM, Sangon, Shanghai, China) for 1 h prior to stimulation with OVA. Some cells were treated with SP600125 (10 μM, Sigma Inc., St. Louis, MO) for 2 h prior to stimulation with OVA.

A total of 20 BALB/C mice (gender, male; aged, 6–8 weeks; weight, 20–30 g) were purchased from Charles River (Beijing, China). All mice were housed in a suitable environment with 12 h dark/light cycle, temperature in 25°C, and enough food and water. One week after adaptation, the mice were randomly divided into two groups: control and OVA. All animal experiments were approved by the Animal Ethics Committee of Soochow University (SUDA20200510A02). Meantime, all experiments were carried out strictly according to the request of the Recommended Guideline for the Care and Use of Laboratory Animals formulated by the Chinese Council on Animal Research.
The mice in OVA group were intraperitoneally injected with 0.2 mL of normal saline containing 0.2 mg OVA (Sangon Biotech Co., Ltd, Shanghai, China) and 1 mg aluminum hydroxide (Beijing Solarbio Science & Technology Co., Ltd, China) at 1, 7, and 14 day. Next step, at 21, 22, 23, 24, and 25 day, the mice were challenged with 5% OVA for 30min daily. At 24 hours after the last challenge, peripheral bloods of the mice were collected, and then were used for qRT-PCR and Western blot analysis. The mice in control group were sensitization and challenge with normal saline.