Ain93g fomula diet

Six-week-old female C57BL/6JJmsSlc mice and 4-week-old female Syrian hamsters obtained from Japan SLC, Inc. were used as WT controls. For some experiments we used aged (52- to 122-week-old) female C57BL/6JJcl mice obtained from CLEA Japan, Inc. (Fig. 1e, f and Supplementary Figs. 7 and 10). B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice were purchased from The Jackson Laboratory and subsequently bred at The University of Tokyo. Mice were kept in an incubator (MIR-154, PHCbi, Japan) at 4 °C (80% relative humidity), 22 °C (53% relative humidity), or 36 °C (25% relative humidity). Cold, RT, or high-heat exposures were started 7 days before infection and continued for the entire duration of the experiments. These mice were allowed free access to food and drinking water and kept on a 12 h light/dark cycle. All animals used in this study were fed with a γ-ray-sterilized normal diet (CE-2, CLEA Japan, Inc.) or AIN93G-fomula diet (Oriental Yeast, Japan) as a LF diet. We confirmed that drinking water did not freeze at 4 °C. All animals were euthanized with deep anesthesia and cervical dislocation. All animal experiments were performed in accordance with The University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, The University of Tokyo (PA15-92, PA19-87, PA22-33).

Germ-free IQI mice were purchased from Sankyo Laboratories, Japan and maintained in germ-free vinyl isolators in the animal facility of RIKEN. Thirteen Treg-inducing Clostridia strains (strains 1, 3, 8, 18 were omitted from the previously reported VE202 cocktail consisting of 17 Treg-inducing strains [24 ]) were individually cultured in modified Eggerth Gagnon (EG) broth under strictly anaerobic conditions (80 % N₂, 10 % H₂, 10 % CO₂) at 37 °C in an anaerobic chamber (Coy Laboratory Products) to confluence. The cultured bacterial strains were then mixed at equal amounts of media volume and the mixture of 13 strains were orally inoculated into seven IQI germ-free adult mice. Five of the mice were fed with a gamma ray-sterilized high-fiber diet (CMF chow, Oriental Yeast Japan) for 5 weeks, then switched to a low-fiber diet (AIN93G-fomula diet, Oriental Yeast Japan) for 2 weeks, and returned to and maintained on the CMF high-fiber diet for 2 weeks. Two of the mice were maintained on the high-fiber diet for 5 weeks and were not switched to the low-fiber diet. Fecal samples were collected daily or every other day. Fecal pellets were collected at days 1–21 (daily), 23, 25, 27, 29, 31, 33, 35–60 (daily), 62, 63, and 65 for the five mice receiving the dietary perturbation and at days 1–21 (daily), 23, 25, 27, and 29 for the two mice not receiving the perturbation.