Ultra pure LPS from E. coli 0111:B4, Pam3CSK4 and IFN-γ were purchased from InvivoGen (San Diego, USA), pertussis toxin, polymixin B and 8Br-cAMP (B7880) from Sigma Dorset, UK and QCL-1000® Endpoint Chromogenic LAL Assay from Lonza Group, Basel, Switzerland. Mouse
CD40L was kindly provided by Dr. David Gray (University of Edinburgh). hBD3 (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK) and hBD2 (GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP) were purchased from Peptides International Louisville, USA and are oxidised so the disulfide connectivities are of the canonical β-defensin arrangement 32. Defb14 (FLPKTLRKFFCRIRGGRCAVLNCLGKEEQIGIRCSNSGRKCCRKKK) and LL37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) anti-PD-1 antibody were synthesized as previously described Erlotinib ic50 20, 33. RAW264.7 cells were maintained in DMEM (GIBCO Paisley, UK) and THP-1 cells in RPMI containing 10% FBS, essential amino acids and antibiotics. Balb/c, CBA and C57 Black/6 mice were obtained from
Charles River (UK) and Mc1r e/e and Mc3r KO mutants were bred in-house. C3H/HeJ OlaHsd-Tlr4 mutants and C3H/HeN controls were obtained from Harlan Laboratories, UK. Primary Mϕ were generated from femur BM and grown in DMEM containing 10% FBS and 20 ng/mL M-CSF (R&D Systems, Abingdon, UK) for 7 days. Cells were seeded at 1.25×105 into 48-well plates and grown without growth factor for 24 h prior to treatment. Replicate experiments were done with separate Mϕ preparations from at least three mice for each experiment. Human venous blood was collected according to Lothian Research Ethics Committee approvals ♯08/S1103/38, using sodium citrate anticoagulant (Phoenix
Pharma, Gloucester, UK), and cells were separated by Dextran sedimentation, followed by discontinuous, isotonic Percoll gradient centrifugation as previously described 33. PBMC were incubated at 4×106/mL in IMDM (PAA Laboratories, Somerset, UK) at 37°C, 5% CO2, for 1 h. Non-adherent L-gulonolactone oxidase cells were removed and adherent monocytes cultured for 6 days in IMDM with 10% autologous serum to generate monocyte-derived Mϕ. Cells were treated with LPS (50 ng/mL), Pam3CSK4 (100 ng/mL), CD40L (3 μg/mL) IFN-γ (5 ng/mL), hBD3, Defb14, LL-37, 8Br-cAMP (at concentrations shown) or combinations of these as described, in serum free media then incubated at 37°C, 5% CO2 for 18 h. Supernatants were collected and centrifuged to remove particulate debris. Levels of TNF-α, IL-6 and IL-10 in the supernatants were measured using human or mouse DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions. Cell viability was measured using TACS™ MTT assay (R&D Systems). Balb/c male mice (5–8 wk) were injected with 16 mg/kg of LPS (approx. 200 μg/mouse) with or without 10 μg of hBD3 in 200 μL of PBS. After 1 h mice were killed by cervical dislocation, exsanguinated and serum TNF-α levels measured by ELISA.
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