Laboratory rodent diet 5001

The allergic upper airway model was designed to reflect AR and was a modification of a previously published protocol ^{37 (link)} (Figure 1). Briefly, BALB/c female mice (Harlan Laboratories), aged 7–8 weeks (20–23gr) were sensitized on day 0 and day 7 by intraperitoneal injection (i.p.) of 200µl chicken ovalbumin (OVA; 1mg/ml) (Amresco, Ohio, USA) and aluminum hydroxide (alum; 50 mg/ml) (Acros Organics, New Jersey, USA) in PBS. Starting on day 14, awake mice were challenged twice a day by intranasal (i.n.) instillation of 20µl OVA (25mg/ml) daily until day 27. For the allergic lower airway model, a previously published protocol with OVA was used to reflect atopic asthma^{38 (link)}. Briefly, FVB male mice aged 5–7 weeks (Charles River Laboratories) were housed in isolation cages under viral antibody-free conditions. Mice were fed a standard diet (Laboratory Rodent Diet 5001, PMI Nutrition International). Mice were sensitized with i.p. injections of OVA (10µg) and 1mg alum in 0.2ml saline on days 0 and 7. Mice were then challenged on days 14–17 with an aerosol of 6% OVA (25min).

C57BL/6 (18–22 week old, male:female ratio 1:1, body weights 20–25 g; Charles River Laboratories, Wilmington, MA), Pld1^−/− and Pld2^−/− were housed in isolation cages in pathogen-free conditions on a light-dark cycle with light from 7:00 to 20:00 at 25°C. Pld1^−/− and Pld2^−/− were obtained from Dr. Gilbert Di Paolo (Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York)^{28 (link)} and Dr. Yasunori Kanaho (Department of Physiological Chemistry, University of Tsukuba, Tsukuba, Japan).^{29 (link)} The Pld1^−/− and Pld2^−/− mice were backcrossed with C57BL/6 mice for > 7 generations.^{29 (link),30 (link)} Mice were fed a standard diet (Laboratory Rodent Diet 5001; PMI Nutrition International, St. Louis, MO) containing 4.5% total fat with 0.3% ω-3 fatty acids and <0.02% C20:4 and were provided water ad libitum.

Alcohol-naïve male rats from generation 70 of selective breeding for alcohol preference were provided by the Alcohol Research Resource Center of the Indiana Alcohol Research Center. These alcohol-preferring (P) rats were 50 ± 1 days of age and 237 ± 3 g at the start of the study (n = 46). All rats were individually housed in stainless steel hanging cages in an isolated vivarium with controlled temperature (21 ± 1°C) and reverse 12-hour light/dark cycle (lights off at 1000 hour; procedures during the dark period were performed under dim red illumination). All rats were acclimated to individual housing and the light/dark cycle for 10 days before the study. Chow (Laboratory Rodent Diet 5001, PMI Nutrition International, Brentwood, MO) and water were available ad libitum except during the first 3 days of the alcohol drinking induction phase when 10% (v/v) alcohol was the only source of fluid. All experiments were approved by the Veterans Administration Puget Sound Health Care System Institutional Animal Care and Use Committee and conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals.