Mice
All experiments were performed on 6-to-24-week-old C57BL/6 male and female mice. Young mice were defined as 8 to 12 weeks old, and old mice were defined as 22 to 24 months old at the time of analysis. Mice were maintained under specific pathogen-free conditions with chow and water provided ad libitum. mouse lines used were on the C57B1/6 J background and housed under specific pathogen-free conditions at the Centro Nacional de Investigaciones Cardiovasculares Carlos III, Singapore Immunology Network or Yale University. All mouse husbandry and experimentation was conducted using protocols approved by local animal ethics committees and authorities. Mice (Mus musculus) were maintained in racks with individual ventilation cages according to current Spanish, Singapore and US legislation (RD 53/2013 and EU Directive 63/2010, respectively). Mice have access to dust- and pathogen-free bedding, as well as sufficient nesting and environmental enrichment materials, to facilitate nesting. All mice were kept in environmental conditions of 45–65% of relative humidity, temperature of 21–24 °C, and a light:dark cycle of 12 h:12 h. Mice with neutrophil-specific deficiency in Tgfbr2 (TgfbrΔN) were generated by crossing MRP8CRE mice with Tgfbr2fl/fl mice41. Similarly, we generated neutrophil-specific mutants by crossing Junb-floxed42, Csf2r-floxed43 and Ifnar1-floxed mice44 with the MRP8CRE driver. Apoe–/– mice (B6.129P2-Apoetm1Unc; Taconic M&B). Ly6gcreERT2 mice were crossed with Rosa26Tdtomato mice as in ref. 1, resulting in the iLy6gtdTom mice used in our fate mapping experiments. Gavage administration of tamoxifen (2 mg per mouse) was performed to induce CRE recombinase activity in 6-to-12-week-old male iLy6gtdTom mice. JAXBoy (PtprcK302E) from Jackson laboratories and Tet2−/− mice19 were used for adoptive bone marrow cell transfer. Eight-week-old male Germ-free mice (C57Bl/6) were kindly provided by the laboratory of N. Palm. In brief, Germ-free C57BL/6 mice were bred and maintained under sterile conditions in flexible film isolators (Class Biologically Clean) in the Palm laboratory Gnotobiotic Facility at Yale School of Medicine. Mice were housed in a temperature- and humidity-controlled room under a dark:light cycle of 12 h:12 h. All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee (protocol 11513).
For the rewilding experiments, litters of mice were generated from multiple breeding pairs and randomly assigned to either remain in the institutional vivarium (laboratory mice) or be released into the outdoor enclosures (rewilded mice) to control for the microbiota at the onset of the experiment. Outdoor enclosures were previously described45 and the protocols for releasing laboratory mice into the outdoor enclosure facility and then returning them to vivaria were approved by Princeton University (protocol 1982) and Rutgers University (protocol PROTO999900794). All protocols were approved by the corresponding local authorities of Madrid, Singapore, Rutgers, Princeton and Yale University.
Mouse models of disease
Stroke
Thrombotic occlusion of the middle cerebral artery was induced by the ferric chloride (FeCl 3 ) stroke model. In brief, mice were anaesthetized and maintained at 2% sevoflurane in a mixture of 0.2 l min−1 O 2 :0.8 l min−1 air and temperature was kept at 36.5–37 °C using a heating blanket. The scalp was opened, and the middle cerebral artery was visualized with a stereomicroscope (PZMIV, World Precision Instruments). A piece of Whatman filter paper strip soaked in freshly prepared FeCl 3 (20%) was placed over the intact dura mater on the artery for 10 min and then removed to allow the formation of a thrombus. Following surgery, individual mice were returned to their cages with free access to water and food. Brains were collected 24 h after surgery to perform transcriptomics analysis.
Liver cholestasis
For the liver injury model, mice were fed a 0.1% of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 3 weeks before sample collection, housed with a 12 h:12 h light:dark cycle, and permitted ad libitum consumption of water as described46. An additional group of mice was fed a 0.1% DDC-supplemented diet for three weeks and afterward allowed to recover for three days under standard mouse diet to study the reversibility of the cholestatic phenotype.
Influenza A infection
A stock of the virus strain A/PR8/34 (H1N1) was diluted, and 100 plaque-forming units were administered intranasally to isoflurane-anaesthetized 8-to-12-week-old male mice in 50 μl of PBS. Mouse weight was monitored daily after infection and mice that presented weight loss of more than 20% of their initial body weight were euthanized and considered deceased. For transcriptomic studies, blood and lungs were collected on day 4 after infection.
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