Lipopolysaccharide lps from escherichia coli o127 b8

Lipopolysaccharide (LPS) from Escherichia coli (O127:B8) (Sigma-Aldrich) was aliquoted to 5 mg/ml. The aliquots were frozen at − 20 °C and defrosted to be prepared fresh with PBS to a concentration of 100 μg in 200 μl intraperitoneally (IP). Prior to surgery, buprecare (Axience, Centravet, France) was injected at 0.2 mg/kg IP, and the following day, mice were injected with an additional 50 μl subcutaneously. Dolethal (Vétoquinol, Centravet, France) was used to induce lethal unconsciousness prior to intracardial blood extraction. Atropine, hexamethonium, propranolol and mifepristone (all from Sigma-Aldrich) were used at a concentration of 1 mg/kg, 10 mg/kg, 2.5 mg/kg and 80 mg/kg, respectively. All were diluted in PBS except for mifepristone which was diluted in a PBS/DMSO mix (10%) (Sigma-Aldrich). All were injected IP in 200 μl.

Monocytes were cultured in complete media consisting of Dulbecco’s Modified Eagle Medium containing high glucose and sodium pyruvate (ThermoFisher Scientific) with 10% heat-inactivated fetal bovine serum (Wisent BioProducts) and 100U/mL penicillin/streptomycin (Corning). NGS datasets produced by Novakovic et al. were produced using 5ug/mL of (1→3)-β-D-glucan from heat killed Candida albicans for 24 hours, as previously described (12 (link)). Cells in our in vitro experiments were stimulated with 5μg/mL of (1→3)-B-D-glucan from Alcaligenes faecalis (Sigma-Aldrich), 100ng/mL of lipopolysaccharide (LPS) from Escherichia coli O127:B8 (Sigma-Aldrich), 120μM L-buthionine sulfoximine (Sigma-Aldrich), and/or 1mg/mL of reduced glutathione (Sigma-Aldrich) for 24 hours unless otherwise stated in figure legends.

We used RPMI 1640 medium (Lonza) supplemented (cRPMI) with 10% heat‐inactivated fetal bovine serum (FBS, Hyclone) 100 μg/mL normocin (InvivoGen), 50 μg/mL penicillin‐streptomycin, 1% nonessential amino acids, 1% MEM vitamins and 1 mmol/L sodium pyruvate (all from Life Technologies). Lipopolysaccharide (LPS) from Escherichia coli O127:B8 and O155:B5 (Sigma–Adrich) were used for cell cultures and animal models, respectively. Pam3CSK4 (InvivoGen) as TLR2 ligand and a combination of TNFα and IL-1β (both from PeproTech) as a maturing factors (MFs) were used. CBR agonists WIN55212-2 (Sigma Aldrich) and HU210, selective CB2 agonist HU308, selective antagonists for CB1 (Rimonabant), CB2 (AM630), PPARα (GW6471), PPARγ (GW9662) (all from Tocris) and autophagy inhibitor 3-methyladenine (3-MA; InvivoGen) were used.