Animal studies.

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee at The University of Chicago (IACUC 71525). The transgenic mouse line RenTgMK (RenTg), which carries a single copy of mouse renin transgene driven by liver-specific albumin promoter/enhancer has been described previously (7). Colitis was induced in RenTg mice and wild-type (WT) littermates (2–3 mo old) by use of 2,4,6-trinitrobenzene sulfonic acid (TNBS), based on a published protocol (58) with some modifications. Briefly, mice were presensitized with 1% TNBS solution applied to the back skin for 8 days before they were treated with TNBS installation through the colon. Then, these presensitized mice, fasted overnight, were treated under anesthesia with 100 mg/kg TNBS dissolved in 50% alcohol via intrarectal injection, using a 1-ml syringe fitted with an 18-gauge stainless steel gavage needle, and control mice received 50% alcohol treatment. Mice were held upside down for 5 min after TNBS injection. Body weights, stool consistency, and rectal bleeding were monitored daily. Clinical scores and colonic damage scores were assessed as detailed previously (3, 4, 6, 10). Colons were fixed in 4% formaldehyde (pH 7.2), and colon histology was examined using the “Swiss roll” method (40). Histological scores were graded according to a previously published system (3, 23). Colonic mucosa was scraped and used for preparation of total RNAs or protein lysates. To assess the effect of JAK blockade on colitis, RenTg and WT littermates were orally gavaged with tofacitinib (provided by Pfizer) at 20 mg/kg per day dissolved in 0.5% methylcellulose and 0.025% Tween-20 (32) or vehicle 1 day before TNBS installation, and daily tofacitinib treatment continued for various days according to the experimental purpose.