Aluminum hydroxide

A peroxidase-labeled mouse anti-human IgE Fc antibody and peroxidase-labeled goat anti-mouse IgE, IgG1 and IgG2a Fc antibodies were obtained from SouthernBiotech, USA (9160-05, 1110-05, 1070-05 and 1155-05); tetramethylbenzidine (TMB) was purchased from Solarbio, China (R1200); aluminum hydroxide was obtained from Thermo Fisher, USA (77161); and LPS was purchased from Sigma, USA (L3012). ELISA kits for IL-4, IFN-γ and TNF-α were obtained from Ebioscience, USA (88-7044, 88–7314 and 88–7324); ELISA kits for IL-5 and IL-13 were purchased from 4 A Biotech, China (CME0003, CME0009); an IRF4 antibody was obtained from Cell Signaling, USA (4964); an antibody against GAPDH was procured from Proteintech, China (10494-1-AP); a TLR4 signaling inhibitor was purchased from Invivogen, USA (CLI-095); an anti-mouse TLR2 Ab was obtained from Biolegend, USA (121802); the PE-CD80, PE-CD83, FITC-MHCII and FITC-CD40 antibodies were obtained from Ebioscience, USA (12–0801, 12–0831, 11–5321 and 11–0402); and mouse GM-CSF and IL-4 were purchased from Sino Biological, China (51048-M07H, 51084-M08B). Anti-CD3 and anti-CD28 antibodies were obtained from Ebioscience, USA (16-0031-82, 16-0281-82).

Recombinant dTCTP was cloned into the pRSET-A expression vector with a His-tag and was expressed in the Escherichia coli strain BL21 (DE3) pLysS (Merck, Germany). The resulting protein was purified using Ni-NTA agarose (QIAGEN, Germany) and ion exchange chromatography with HiTrap Q column (GE Healthcare Bio-Sciences Corp). dTBP2 (WYVYPSM) was synthesized by Peptron, Inc. (Daejeon, South Korea). The purity of the peptide was >90%. Methoxy PEG-propionaldehyde (mPEG-aldehyde; MW 10 kDa) and sodium cyanoborohydride (NaCNBH₃) were purchased from Sigma (St. Louis, MO). OVA and aluminum hydroxide were purchased from Thermo Fischer Scientific (Waltham, MA). IL-4, IL-5, IL-8, and OVA-specific IgE ELISA assay kit was purchased from Biolegend (San Diego, CA). IL-13 ELISA assay kit was purchased from R&D Systems (Minneapolis, MN). All the chemicals used in this study were of analytical grade.

WT and IL-22-KO mice were sensitized three times, at 7-day intervals, with 10 µg of OVA Grade VI (Sigma-Aldrich, St. Louis, MO, USA, A2512) and aluminum hydroxide (2 mg) (Thermo Scientific, Waltham, MA, USA, 77161) intraperitoneally. On days 21, 22 and 23 mice were challenged with 30 µg OVA Grade V (Sigma, A5503), intranasally, and samples were collected 24 h after the last challenge. TIGR4 strain of S. pneumoniae (ATCC BAA-334) frozen at −80 °C in tryptic soy broth (TSB) (BD, cat. 211825) containing 10% glycerol was thawed, plated on blood agar and incubated overnight at 37 °C, 5% CO₂. After growth, colonies were inoculated in TSB medium, when the growth was checked at OD 600 = 0.06, following incubation for 4 to 5 h. When OD 600 = 0.3, bacteria was counted, and the concentration was adjusted to 50 × 10⁸/mL of colony-forming unit (CFU) in sterile Phosphate Buffered Saline (PBS). Mice were infected with 1 × 10⁸ CFU of S. pneumoniae by intranasal route (20 µL/animal) before the second challenge with OVA. The euthanasia of the animals was performed 48 h after the infection.

Mice were sensitized by intraperitoneal injections of 50 μg of OVA emulsified in 2 mg aluminum hydroxide (77,161; Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 200 μl on days 1, 8, 15, and challenged with an aerosol instillation of 1% OVA in PBS for 40 min on day 22, 24, 26. Control animals received phosphate-buffered saline (PBS) (P1020; Solarbio, Beijing, China) only. 24 h after the final challenge, the lung, serum and bronchoalveolar lavage fluid (BALF) were collected for further analysis. BALF was collected after flushing with 2 × 500 μl PBS and analyzed for the presence of inflammatory cells. For histology, lung tissues were fixed in 4% paraformaldehyde (P1110; Solarbio) and stained following standard protocols for HE and PAS.
In some models, WT mice were sensitized by OVA, then intranasally administered with different reagents in 20 μl, such as PGE₂ (300 μg/kg), PGE₁-alcohol (500 μg/kg), ONO-AE3-208 (1,000 μg/kg) or T0070907 (500 μg/kg), before an aerosol instillation of 1% OVA on day 22, 24, 26.

Loratadine tablets [Bayer Healthcare (Shanghai) Co., Ltd., National Drug ApprOVAl Number H10970410]. OVA (Sigma, United States, A5503-5G). Aluminum Hydroxide (Thermo Fisher Scientific, United States, 77161). Mouse OVAsIgE enzyme-linked immunosorbent assay (ELISA) Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20466). Mouse IL4 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20266). Mouse IL5 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20267). Mouse IL13 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20247). DNA extraction kit (Omega Soil DNA Kit, D5625-01). High-fidelity DNA polymerase (NEB, M0491L). Gel recovery kit (Axygen, AP-GX-250). Fluorescence quantitative polymerase chain reaction (PCR) reagent kit (BioTek, United States, FLx800). High-fidelity DNA polymerase (NEB, M0491L). Gel recovery kit (Axygen, AP-GX-250). Fluorescence quantitative PCR reagent kit (BioTek, United States, FLx800).
Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-2000A); Circulating water vacuum pump (Zhengzhou Changcheng Science and Technology Trade Co., Ltd., SHB-III); Fc-type ELISA reader (Thermo, United States); High-speed refrigerated centrifuge (Beckman Coulter, Inc., United States); PCR instrument (Bio-Rad, Germany, C1000).