Dextran sulfate sodium (dss)

BALB/c mice were purchased from Claire Japan, Inc. (Tokyo, Japan). SKG mice were obtained from Prof. Sakaguchi (Osaka University, Osaka, Japan). Both mice were bred under SPF conditions in the main facility of the Institute of Laboratory Animals, Kyoto University. All experiments were approved by the Kyoto University Animal Ethics Committee. In all experiments, only female mice 8 to12 weeks of age were used, since previous reports have shown that female SKG mice have higher clinical scores than male [10 (link), 13 (link)]. In the DSS administration cohorts, 1% DSS (MP Biomedicals, Santa Ana, CA, USA, molecular weight 35,000–50,000 kDa) in drinking water was administered for the first two consecutive weeks. In the set of cohorts with antibiotic or anti-fungal agents, 6% dimethyl sulfoxide (DMSO) (Nacalai Tesque Inc., Tokyo, Japan) as a control group, 6% DMSO and meropenem trihydrate (MEPM) (1 g/L) (FUJIFILM Wako Chemicals Inc., Tokyo, Japan) and vancomycin hydrochloride (VCM) (0.5 g/L) (Nacalai Tesque Inc.), and 6% DMSO and amphotericin B (AMPH-B) (0.3 g/L) (Nacalai Tesque Inc.) were administered for 1 week in drinking water, from the time point 2 weeks before the initiation of DSS administration. Ten mice were used per group to confirm disease phenotypes, and 5 mice were used per group in other cohorts for the FCM, qPCR, and NGS analyses.

To create colitis model mice (C57BL/6), wild-type (WT), Atg5^flox/+/villin-Cre, and Atg5^flox/flox/villin-Cre mice, all aged 8–10 weeks, were given 2.5% DSS (Nacalai Tesque, Kyoto, Japan) in drinking water for six days, and plain drinking water afterward. The body weights of the mice were measured every day, and, on day eight after DSS administration, the mice were sacrificed. The histological scores of the Hematoxylin and eosin (HE)-stained rectal specimens were determined as described previously[10 (link)]. Ethical permission for the experiments presented in this study was given by the Animal Ethical Committee of the Osaka Medical College, and all procedures were conducted according to the guidelines of the Institute for Laboratory Animal Research at Osaka Medical College (permission # 30049).

Acute colitis was induced by 3.5% (w/v) DSS (molecular weight, 5000; Fujifilm, Osaka, Japan) added to the drinking water for 7 days. Seven- to nine-week-old gender-matched mice were included in each group. Body weight was recorded daily. Mice were sacrificed on day 3 or 7, and the colons were collected. To inhibit the NLRP3 inflammasome in vivo, mice received an intraperitoneal injection of 50 mg/kg MCC950 (AdipoGen Life Sciences, San Diego, CA) (or PBS as a control) every other day from one day before to day 7 of DSS administration. To inhibit PGE₂ production in vivo, indomethacin (Nacalai Tesque, Kyoto, Japan) was dissolved in ethanol (10 mg/mL) and was added to the drinking water at 1 mg/kg per day, concurrent with DSS treatment.