Immune Monitoring

The immune status of mice was evaluated at predefined time points, as described in the specific experimental set-ups. Mice were anesthetized with 80 μL ketamine [100 mg/mL; Nimatek (Eurovet, Bladel, Nederland)] and blood was collected from the retro-orbital plexus using glass capillaries. Blood was centrifuged at 8,000 rcf for 10 min. Serum was collected and stored at −80°C for further analysis. Next, the animals were euthanized by cervical dislocation. Peritoneal washing with 10 mL of PBS was performed to collect the circulating immune cells in ascites and from the peritoneal lining. Peritoneal washings were centrifuged for 5 min at 500 rcf and resuspended. Supernatant was collected and stored at −80°C for cytokine analysis. Using a Lymphoprep (Stemcell technologies, Vancouver, Canda) gradient, immune cells were isolated from the cell suspension and analyzed with flow cytometry (FACS).

Using flow cytometry, dead cells were excluded via eFluor780 fixable viability dye staining (Affymetrix Inc. San Diego, CA, USA). Immune cells were stained for myeloid cells, T cells and B cells using antibody panels, which are available as Supplementary Material (Supplementary Tables 1–3, respectively). For the myeloid panel, the cells were permeabilized using Leucoperm (Bio-Rad Laboratories Inc., Kidlington, UK) in accordance to manufacturers' protocol and stained for CD206. Permeabilization in the T cell panel was achieved using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher scientific, Waltham, Massachusetts, USA) and cells were then stained for FoxP3. Samples were acquired on the BD LSRFortessa (BD Biosciences, San Jose, CA, USA) and the analysis was performed using FlowJo Analysis software (Flow Jo, LLC, Ashland, Oregon, USA).

Cytokines in serum and ascites were determined using cytometric bead assay technique (BD Biosciences, San Jose, CA, USA). Both serum and peritoneal washings/ascites were used undiluted. The analysis was performed in accordance to the manufacturers' protocol using flex sets for IL-1 β, GM-CSF, IL-6, IL-10, MIP-1α, MIP-1β, TNFα, and IFNγ. Samples acquisition was performed on the BD LSRFortessa (BD Biosciences, San Jose, CA, USA) and the analysis was performed using FCAP Array Software v3.0 (BD Biosciences, San Jose, CA, USA).