Grade 2 ovalbumin

FCS was from Hyclone (Logan, UT); culture media RPMI 1640 from RHyClone, Thermoscientific, (Waltham, MA); recombinant murine interleukin-5 (IL-5) from R&D Systems (Minneapolis, MN, USA); grade II ovalbumin (Cat. A5253), methylcellulose (Cat. M0387) from Sigma (St. Louis, MO, USA); aluminium ammonium sulfate [AlNH₄(SO₄)₂·12H₂O, alum] (Cat. 01S1048.01.AF) (Manufacturer, Synth, Brazil), grade V ovalbumin (Cat. 950 512), from ICN Biomedicals (USA); and MK886 (Cat. 10133), montelukast (Cat. 10008318), from Cayman Chemical (USA).

BALB/c mice aged 6–8 weeks were obtained from Chiyoda Kaihatsu Co. (Tokyo, Japan) and sensitized and challenged with grade II ovalbumin (Sigma‐Aldrich., St. Louis, MO) as previously described.¹², ¹³, ¹⁴ All animal experimentation procedures were approved by the Institutional Animal Care and Use Committee of Yamaguchi University (No. 29‐S01), and all methods were conducted in accordance with approved guidelines. Additionally, animal experiments conformed to the revised Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council “Guide for the Care and Use of Laboratory Animals” published by the National Academy Press, Washington, D.C. 1996.

The mice were divided into four allergic and nonallergic group pairs receiving antibiotic treatment in the drinking water: (1) amoxicillin (Amox) at 50 mg/kg; (2) trimethoprim/sulfamethoxazole (TMP/SMX) at 24/120 mg/kg; (3) metronidazole (Metro) at 40 mg/kg; and (4) water-only control. The antibiotic doses were chosen based on literature (28 (link)–30 (link)). Treatment consisted of 7 days with antibiotics in the drinking water followed by 7 days of washout (resting), repeated for 3 cycles (Figure 1(a)). Washout periods of 7 days were chosen to allow at least 5 half-lives of the drug with the higher elimination period (metronidazole, 4 hours) and to allow reconstitution of the microbiota. Ovalbumin (OVA) allergic airway inflammation model was performed as described (31 (link)), with minor modifications. Allergic groups received intraperitoneal injections with 10 μg of grade II ovalbumin (Sigma-Aldrich, Missouri, USA) with Alhydrogel® (Brenntag, Denmark) as adjuvant after the first cycle of antibiotics and 14 days later (Figure 1(a)). Nonallergic groups received injections with Alhydrogel® only. At the end of the third recovery period, the animals were challenged in a nebulization chamber with aerosolized 1% ovalbumin suspension for 4 times on alternate days for 20 minutes each session.