Tlrl pms

The following ligands were used: Pam3CSK4 (TLR1/2; tlrl-pms; Invivogen, 200 ng/ml), FSL1 (TLR2/6; tlrl-fsl; Invivogen, 100 ng/ml), Poly (I:C) (TLR3; vac-pic; Invivogen, 10 μg /ml), LPS K12 (TLR4; tlrl-eklps; Invivogen, 200 ng/ml), Flagellin (TLR5; tlrl-stfla; Invivogen, 100 ng/ml), R837 (TLR7; tlrl-imqs; Invivogen, 10 μg/ml), R848 (TLR7/8; tlrl-r848; Invivogen, 100 ng/ml), CL075 (TLR8; tlrl-c75; Invivogen, 5 μg/ml), and CpG2006 (TLR9; tlrl-2006; Invivogen, 10 μM), and human interferon-beta 1a (100U/ml) was purchased from PeproTech GmbH (#300–02BC; Germany), 2’3’-cGAMP (InvivoGen, 1ug/ml), Lipofectamine 3000 (Invitrogen), diABZI STING agonist-1 (MedChemExpress, 1μM), Sendai Virus (Cantell Strain) (Charles River, 10 HA units/ml), and HSV-1 (MOI = 1), infected as previously described [28 (link)].

The following TLR agonists were used: TLR1/TLR2 agonist Pam3CSK4 (#tlrl-pms, InvivoGen, San Diego, CA, USA); TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] of high molecular weight type (#tlrl-pic, InvivoGen); TLR4 agonist LPS from E. coli (#L4391, Sigma-Aldrich, St. Louis, MO, USA); and TLR7 agonist CL264 (#tlrl-c264e-5, InvivoGen). The TLR agonists were used alone or in combination with mouse recombinant IFN-γ (#315-05, Peprotech, Rocky Hill, NJ, USA), mouse recombinant IFN-β (#8234-MB, R&D Systems Inc., Minneapolis, MN USA), or mouse recombinant IFN-α type A (#12100-1, PBL Assay Sciences, Piscataway, NJ, USA).

THP-1 cells and reporter cells were cultured according to a previously described protocol (Chen et al., 2020 (link)). Bone marrow-derived macrophages (BMDMs) were isolated from mice and differentiated for 5–6 days, following standard procedures (Hsu et al., 2015 (link)). THP-1 cells or BMDMs were treated with MDP fractions at the indicated concentrations for 6 h. The cells were then stimulated with 0.2 μg/ml Pam3CSK4 (tlrl-pms, InvivoGen) or 0.1 ng/ml LPS (SI-L8274, Sigma-Aldrich). After stimulation for 14–16 h, NF-κB activity was quantified by measuring the levels of alkaline phosphatase in the culture supernatant. Changes from baseline were calculated as -fold changes and normalized against unstimulated control cells.

Mice were administered 1 ml sterile thioglycolate (R064710, Remel, Fisher Scientific) by intraperitoneal injection. After 4 days, peritoneal cells were collected by lavage. Following red cell lysis, cells were plated in C-DMEM at 2x10⁵, 6x10⁵, or 1x10⁶ macrophages per well in 96-, 24-, or 12-well flat bottom tissue culture plates, respectively. For luminescence assays, macrophages were plated on white 96-well tissue culture plate (353296, Fisher Scientific) in 200 μL C-DMEM. C-DMEM and non-adherent cells were aspirated after overnight incubation, and fresh R-free C-DMEM was added with concentrations of L-arginine or L-citrulline noted in the text. Cells were infected and/or stimulated with IFN-γ (2 ng/ml, 14-8311-63, Invitrogen, Thermo Fischer Scientific – discontinued; PMC4034, Gibco), Pam3Cys (100 ng/ml, tlrl-pms, Invivogen), 1400w (100 μM, 214358-33-5, Cayman Chemical), 2-deoxyglucose (2-DG, D8375, Sigma-Aldrich), rapamycin (A8167, ApexBio), torin 1 (10997, Cayman Chemical) and/or dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, used as vehicle control for rapamycin and torin) where noted. DETA-NONOate (82120, Cayman Chemical) was added 24 hours post infection (40 μM) and then at 48 hours post-infection (80 μM) for the “high” dose, and only at 48 hours post-infection (80 μM) for the “low” dose.