Cholera toxin

4–6 week old female mice underwent weekly sensitization with 2 mg peanut extract and 10 μg Cholera toxin (List Biological laboratories, Campbell, CA) in 200 μL volume for three weeks by oral gavage followed by 1 week of 5 mg peanut extract and 10 μg Cholera toxin by oral gavage. One week after this final sensitization, mice were bled by submandibular bleed to collect serum for immunoglobulin quantification. The following day, mice undergoing an oral challenge were gavaged with 9 mg peanut extract while mice undergoing IP challenge received 200 μg peanut extract. Core body temperatures were monitored every fifteen minutes using a rectal thermometer (Physitemp, Clifton, NJ). For serum MMCP-1, histamine, and Ara h 1, 2, and 3 measurements, blood was collected 60 min after oral challenge. Serum levels of MMCP-1 (eBioscience, San Diego, CA), histamine (Beckman Coulter, Brea, CA) and Ara h 1, 2, and 3 (Indoor Biotechnologies, Charlottesville, VA) were measured by ELISA according to manufacturer’s instructions

For cholera toxin responses, mice were immunized with 10 ug of cholera toxin (List Biological Laboratories) in PBS by oral gavage. Animals received cholera toxin every 7 days for three consecutive weeks. Mice were analyzed 7 days after last immunization. For Salmonella infection mice were orally gavage three times, unless differently specified in the figure legend, on alternate days with 10⁹ CFUs of aroA-Salmonella in 200 ul 5% sodium bicarbonate. Serial dilutions of bacterial preparations were plated onto LB-agar plates to confirm administered dose. 10⁹ CFU (Wt) Salmonella were injected once orally, and mice were harvested 48 hours later. To prevent lymphocyte egress from Peyer’s patches, the S1PR1 agonist FTY-720 (Fingolimod-HCl., Selleck Chemicals ) was dissolved in saline solution and administered to mice daily i.p. at 1 mg/kg for 3–7 days. For antibiotics treatment mice were gavage with 200ml of antibiotic cocktail containing metronidazole 1mg/ml, ampicillin 0.5 mg/ml, neomycin 0.5 mg/ml, vancomycin 0.5 mg/ml or single antibiotic at the same concentration per day for 1 week. For LTbR-blocking experiments, animals were treated with 100 μg of LTβR-Fc or hIgG-FC on days −3 and −1 before sacrifice. For Diphtheria toxin treatment mice received 100 ng of DT (EMD Bioscience) i.p 18 h before sacrifice.