Mice
For LD 50 and inhibitor experiments, C57BL/6 female mice from Jackson Laboratories (000664) were used. Young mice were used at 12 weeks of age or roughly 12 weeks of age (12–14 weeks). Aged mice were used at 72–76 weeks of age. Trim63 mutant mice were generated by Cyagen using CRISPR–Cas mediated genome engineering to delete exons 1–8. The mutant line was maintained as heterozygous and Trim63+/− and Trim63−/− were used as Trim63 knockout in our study. The Trim63+/+ generated from the heterozygous breeding scheme were used as wild-type controls. Foxo1 mckcre mice were a generous gift from M. Febbraio (Monash University) and were bred in our colony with the following breeding scheme: Foxo1 mckcre+ × Foxo1 mckcre−. cre+ and cre− littermates were used for experiments. Foxo1 Myh6cre mice were generated from a series of crosses using our floxed Foxo1 mice to B6/FVB-Tg(MyH6-cre_2182Mds/J) from Jackson Laboratories (011038). All experiments were performed in our AAALAC-certified vivarium, with approval from The Salk Institute Animal Care and Use Committee (IACUC). In accordance with our IACUC guidelines, in an effort to reduce the number of animals used for our experiments, where possible and appropriate, experiments were done in parallel and shared controls were used. This is indicated in the figure legends where relevant. Mice were housed with a 12-h light/dark cycle, humidity 30–70% and temperature of 18–26 °C (64–79 °F).
Bacteria
Bacteria used were E. coli O21:H+ (ref. 43) and S. aureus (American Type Culture Collection strain 12600). For a list of complete key resources used in this study, refer to Supplementary Table 1 in the Supplementary Information.
Method details
Culturing E. coli O21:H+ and S. aureus for mouse infection
E. coli O21:H+ was incubated overnight at 37 °C on an eosin methylene blue plate treated with ampicillin sodium salt (1 mg ml−1), vancomycin hydrochloride (0.5 mg ml−1), neomycin sulfate (1 mg ml−1) and metronidazole (1 mg ml−1) antibiotics to grow single colonies. S. aureus was incubated overnight on a Luria-Bertani (LB) plate at 37 °C without antibiotics for colony growth. The next day, a single colony of E. coli O21:H+ was inoculated into LB-AVNM media. A single colony of S. aureus was inoculated into LB without antibiotics. Both cultures were shaken overnight at 37 °C (250 rpm). The following morning, the optical density was measured and an inoculum with a 1:1 mixture of the bacteria was prepped with the appropriate amount of both bacteria in sterile 1× PBS that was used directly for mouse infections.
Mouse infection models
Mice were infected intraperitoneally with the appropriate dose of bacteria and put back into their home cage. Mice were group housed for the experiments. For LD 50 experiments the dose of total bacteria used was 1 × 108 CFU. This dose was titrated up or down for low dose and high dose models. Inoculums were serially diluted and plated to confirm the infectious doses. Immediately after infection, food was removed for the first 10–12 h postinfection to control for any potential variations in the sickness-induced anorexic response. Mice were clinically monitored as described below every 2 h postinfection. For some experiments, mice were clinically monitored every 2 h for the first 10–12 h postinfection and then again at 24 h. For experiments involving inhibitors, the details are provided below. Mice that reached clinical endpoints were euthanized according to our animal protocol.
Survival
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