Alternaria alternata

Mice were immunized weekly for 1, 6, or 12 weeks by oral gavage with ground blanched peanut (Western Mixers Produce & Nuts), roasted and unsalted peanut (Whole Foods Market), or wheat flour (Bob’s Red Mill Company) or OVA (Sigma) at 5 mg food antigen, with or without 10 μg of CT (List Biologicals, lot nos. 10165A1 and 10167A2) or 100 μg of Alternaria alternata and Alternaria tenuis extract (Greer Laboratories, lot no. 322776) in 200 μl of 0.2 M sodium bicarbonate buffer per mouse based on a protocol modified from (52 (link)). For daily peanut exposure, one-half of a peanut was left in the cage for each mouse daily for a period of 6 weeks after weaning for infants and 6 weeks after for 8-week-old adult mice.

Eight-week-old wild type C57BL/6 mice were purchased from Taconic (Hudson, NY). RAGE KO mice congenic with the C57BL/6 strain were provided by Dr. A. Bierhaus (University of Heidelberg), and a breeding colony was established (25 (link), 26 (link)). Animal studies were carried out under the National Research Council’s guidelines in the Guide for the Care and Use of Laboratory Animals, with oversight by the University of Pittsburgh Institutional Animal Care and Use Committee. HDM and Alternaria alternata extracts were purchased from Greer Laboratories. Recombinant IL-33 was purchased from BioLegend.

Alternaria alternata and house dust mite (HDM; Dermatophagoides farina) extracts were obtained from Greer Laboratories (Lenoir, NC). Hydrogen peroxide solution, l-glutathione (GSH), N-acetyl-l-cysteine (NAC), PAMAM dendrimer, and mouse genomic DNA were purchased from Sigma-Aldrich (St Louis, Mo). AZ 10417808 (AQZ-1), PD 150606, and decanoyl-RVKR-CMK were procured from Tocris Bioscience (Bristol, United Kingdom). Hanks balanced salt solution (HBSS), acetoxymethyl ester form of fura-2 (fura-2 AM), SYBR Gold, YoYo-1, SYTOX Green, 4′,6-diamidino-2-phenylindole (DAPI), and EDTA were obtained from Thermo Fisher Scientific (Waltham, Mass). Etoposide was acquired from Abcam (Cambridge, United Kingdom). Naphthofluroscein, caspase-3, and calpain small interfering RNA (siRNA), control siRNA (fluorescein conjugate), siRNA transfection reagent, and media were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Fluorescently labeled antibodies to CD3 (145-2C11), CD25 (PC51), CD44 (IM7), CD16/CD32 (2.4G2), CD14 (rmC5-3), CD45R/B220 (RA3-6B2), and IgG₂a isotype control were purchased from BD Biosciences (San Jose, Calif).