Ifn β

LN, spleen, liver and epidydimal VAT were harvested 18 h after pDC transfer. LNs were smashed through a nylon net (pore size 70 μm). Spleen was digested with collagenase D (1 mg/ml) and DNase 1 (1 mg/ml) for 20 min at 37°C in RPMI 1640 media. Subsequently, the tissue was smashed through a nylon net and red blood cell lysis (Pharmlysis, BD) was performed before FACS staining. Liver was smashed through a nylon net by scraping it with a lid of the petri dish with PBS and 2 mM EDTA. The cell suspension was placed on a Lymphoprep® gradient (STEMCELL technologies) and centrifuged at 800 g without break for 20 min at RT. Cells were harvested form the interphase, washed with PBS and stained with antibodies for flow cytometry. VAT tissue was minced with a scalpel and digested with Collagenase 2 (1 mg/ml in RPMI 1640 media) for 25 min at 37°C under continuous rotation. The suspension was centrifuged and the cell pellet (stromal-vascular fraction) was taken up in red blood cell lysis buffer before staining for flow cytometry.
For the ELISA of IFN-α (PBL, InterferonSource) and IFN-β (Biolegend) one fat pad of epidydimal VAT was lysed in T-PER™ Tissue Protein Extraction Reagent (ThermoFisher) with protease inhibitor (Roche). Amount of cytokine was calculated per one total fat pad.

IFN-β^−/− BMDMs were infected as described previously and treated with 50 pg/mL IFN-β. An additional 50 pg/mL of IFN-β was added at 3 hpi, and cell culture supernatants were collected at 6 hpi. Concentrations of selected cytokines were determined using a custom ProcartaPlex magnetic bead-based multiplex assay (Thermo Fisher Scientific) on the Luminex MAGPIX^® platform according to manufacturer’s instructions. IFN-α (PBL Assay Science), IFN-β (BioLegend), CCL12 (R&D Systems) and CCL3 (R&D Systems) protein levels were measured using ELISAs.

Cytokines and chemokines were measured from sera, spleen, and liver taken from uninfected control mice as well as from infected mice on days 1 and 3 post-infection. Spleen and liver homogenates were centrifuged at 15,000 g for 15 min, and the supernatants were used for cytokine and chemokine analyses. The following ELISA kits were used: G-CSF (R&D Systems), IL-6, IFN-γ, and IL-12p70 (Invitrogen), MCP-1 and IFN-β (Biolegend). Data are expressed as mean pg/ml ± SEM.

Levels of cytokines from colon and sera samples were measured by ELISA according to the manufacturers’ instructions (IL-18 from eBioscience, IFN-β from BioLegend and all other cytokines from Millipore).