GoKawiilMice
Unless noted otherwise, young female (8 weeks old) and male (4 weeks old) C57BL/6 mice were obtained from The Jackson Laboratory and old mice (18 months old) were acquired from the National Institute on Aging. For co-housing, mice were housed either two young + three old, or three young + two old per cage. Males were co-housed before puberty to avoid fighting. Non-co-housed control young and old mice had their cages mixed so that two mice were swapped between each cage to control for the social effect of cage mixing. Where possible, mice were randomized to experimental groups, and experimenters were blinded to the experimental condition. Animals were housed in facilities at the University of Pennsylvania, the Arc Institute or Stanford University in 7 am to 7 pm light–dark cycles, 20–25 °C and 30–70% humidity. All experimental procedures were performed according to approval by the IACUC committees at the University of Pennsylvania, Stanford University and the Arc Institute.
Germ-free C57BL/6 mice were maintained in sterile isolators at the University of Pennsylvania Gnotobiotic Animal Facility. For FMT, one stool pellet per recipient mouse was homogenized in 1.5 ml of sterile phosphate-buffered saline (PBS) in an anaerobic hood and filtered through 70 μm filters. This homogenate (200 μl) was orally gavaged into germ-free mice housed in positive pressure isocages; in addition to oral gavage, 50 ml of used bedding from donor cages was added to the isocages.
The following mouse strains were used in manuscript and were purchased from The Jackson Laboratory: C57BL/6J (000664), CD45.1 (002014), DBA/2J (000671), Trpv1Cre (017769), Snap25-GCaMP6s (025111), DTA (009669), Phox2bCre (016223), Phox2bFlpO (022407), Il1rfl/fl (028398), Ccr2−/− (004999), Nlrp3−/− (021302), Tnfr−/− (003243), Cre-dependent hM4Di (026219), Cre/FlpO-dependent hM4Di (029040) and Ai65 (Cre/FlpO-dependent tdTomato) (021875). Gpr84−/− mice were a kind gift from I. Kimura60.
Antibiotic treatment, bacterial colonization and stool collection
Mice were given a combination of neomycin (1 g l–1; Research Products International); ampicillin (1 g l–1; Research Products International); vancomycin (0.5 g l–1; Mylan); metronidazole (0.5 g l–1; Research Products International); imipenem or cilastatin (0.5 g l–1; Fresenius Kabi); and ciprofloxacin (0.2 g l–1; Sigma-Aldrich) in their drinking water for two weeks. Parabacteroides goldsteinii (ATCC) and Alistipes shahii (ATCC) were grown under anaerobic conditions at 37 °C in Tryptic Soy Broth (BD Biosciences) supplemented with 5% defibrinated sheep’s blood (Hardy Diagnostics). Lachnospiraceae bacterium (DSMZ) was grown under anaerobic conditions at 37 °C in fastidious anaerobe broth (FAB) (Neogen). Lactobacillus acidophilus (ATCC) and Escherichia coli K12 (ATCC) were grown under aerobic conditions according to supplier instructions. Bacteria were gavaged into mice after two weeks of antibiotics treatment 24 h after the cessation of treatment. Mice were gavaged every other day the first week, and then once a week after. Control mice received PBS gavage. Stool samples for all experiments were collected fresh in 1.7 ml Eppendorf tubes and immediately snap frozen on dry ice before storage at −80 °C until DNA extraction.
Microbial metabolite isolation
P. goldsteinii and A. shahii were grown for two days in 50 ml tubes before centrifugation for 20 min at 3,220 × g at 4 °C. The supernatant was sterilized through 0.45 μm filters before 30 min of centrifugation through a 3 kDa size filter (Thermo Fisher Scientific) at 3,220 × g at 4 °C. The <3 kDa fraction was then treated with 25 μg ml–1 proteinase K (Thermo Fisher Scientific) for 1 h at 37 °C and then boiled for 10 min at 99 °C. This solution (300 μl) was gavaged into mice for five days before testing 1 h after the final gavage.
Bromodeoxyuridine labelling
Mice received 50 mg kg–1 bromodeoxyuridine (BrdU) (MedChem Express) dissolved in sterile PBS via intraperitoneal injection for five consecutive days, brains were collected four weeks after final injection.
... continue reading