Mice
WT C57BL/6 and CD45.1 SJL mice, mice expressing Cre recombinase (LysM–Cre) under the control of the lysozyme (LysM) promoter, CX3CR1–ERT2 inducible Cre, Rosa26-eYFP (R26YFP) mice and RiboTag mice61 harbouring a modified allele of Rpl22 (Rpl22-HA) induced by the action of Cre recombinase were all purchased from Jackson Laboratories. Dhps floxed mice were a gift from S. Balabanov, Zurich. Mice were bred and maintained under specific-pathogen-free conditions, and experiments were performed under protocols approved by the Regierungspräsidium Freiburg, the Animal Welfare Committee of the Max Planck Institute of Immunobiology and Epigenetics, or the Institutional Animal Care and Use Committee of Johns Hopkins University School of Medicine, in accordance with the Guide for the Care and Use of Animals. Mice used for all experiments were littermates and matched for age and sex; both male and female mice were used. Mice of all strains were typically 6–12 weeks of age unless otherwise specified. Experiments were not performed blinded. Parabiosis experiments were performed in the laboratory of F.G., with Institutional Animal Care and Use Committee approval from the Biological Resource Center (A*STAR) in Singapore, as follows: the animals were monitored daily for evidence of fighting and were exposed to a gel food diet supplement at least two times, 2–6 weeks before performance of parabiotic attachment. Animals were randomly assigned a pair member, with the appropriate transgene or CD45 congenic allotype, within their cohoused cohort. Pairs established for parabiotic attachment were ‘cage paired’, meaning they were housed only with their future parabiotic pair member, 2 weeks before the parabiosis procedure, and the mice were observed again for aggressive behaviour. Aggressive and injured mice were removed from the study. With this husbandry protocol, aggression was highly uncommon.
Tissue processing for RTM isolation
Lungs, liver, kidney, brain and heart were harvested after perfusion with ice-cold phosphate-buffered saline (PBS) through the heart; organs were placed in 5-ml tubes containing 2 ml digestion solution with 2 mg ml−1 collagenase IV (Thermo Fisher) and 0.1 mg ml−1 DNAse I (Sigma) and cut into small pieces with scissors. The solution was incubated in a shaker at 37 °C for 1 h. Tissues were further homogenized using a 20 G × 1” needle and a 5-ml syringe, and homogenates were filtered through a 100-μm cell strainer. RBCs from lungs, liver, kidney and heart were lysed with ACK for 2 min, washed with PBS 1% fetal calf serum (FCS), 2 mM EDTA (FACS buffer) and kept on ice until flow cytometry staining. For splenic macrophages, no digestion solution was used. Cells were isolated by mechanical disaggregation of spleen on a 70-μm strainer, RBCs were lysed and cells were resuspended in FACS buffer. Microglia were isolated postdigestion using a 35–70% Percoll gradient with centrifugation for 20 min at 500g and room temperature without brakes. The leucocyte layer was transferred to tubes containing FACS buffer and kept on ice until flow cytometry staining. When indicated, alveolar macrophages were isolated by BAL and centrifuged to isolate lavage fluid for optical density measurement, and the pelleted cells were resuspended in FACS buffer for flow cytometry staining. Absolute numbers of macrophages were calculated using 123count eBeads Counting Beads (Thermo Fisher) following the manufacturer’s instructions. For solid organs, the weight of each tissue in milligrams was recorded after harvesting, and the number of macrophages per sample was normalized by this value to obtain the total cells per milligram tissue.
Peritoneal macrophages in vitro culture
Peritoneal macrophages were obtained by peritoneal lavage and either cultured in complete medium (RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 100 U ml−1 penicillin/streptomycin) with 20 ng ml−1 macrophage colony stimulating factor (PeproTech) on a six-well-plate, or transferred to a 96-well-plate and used directly for flow cytometry staining. For in vitro activation, peritoneal macrophages were cultured with 20 ng ml−1 IL-4 (PeproTech) overnight to generate alternatively activated macrophages. For IL-33 (R&D Systems) in vitro treatment, 20 ng ml−1 was used in all experiments unless otherwise specified, with culture for 72 h.
Parabiosis
Parabiotic mice were generated as reported62 from age- and weight-matched CD45.2+ (Dhpsflx/flx LysM–Cre negative (Dhps-WT) or Dhpsflx/flx LysM–Cre positive (Dhps-ΔM) and CD45.1+ (C57BL/6) mice (WT) between 6 and 8 weeks old.
Bone marrow chimera
CD45.1+ (C57BL/6) mice (WT) were irradiated (1,200 cGy, 2 split doses, 3 h apart) in a Cesium Mark 1 irradiator (JL Shepperd & Associates) and then infused with a mix of 5 × 106 bone marrow cells from Dhpsflx/flx LysM–Cre negative (Dhps-WT) or Dhpsflx/flx LysM–Cre positive (Dhps-ΔM) CD45.2+ cells mixed with 5 × 106 bone marrow cells from CD45.1+ mice (1:1 ratio). Mice were harvested, and the frequency of CD45.1+/CD45.2+ RTMs were analysed at 12 weeks after bone marrow transfer.
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