R848 resiquimod

For the in vitro infection assays, 1x10⁵ or 1x10⁶ HaCaT cells were seeded into a 24-well plate or a 12-well plate, respectively, and cultured to confluence at 37°C with 5% CO₂ for 24 hours. The following stimulating agents were used: lipopolysaccharide (LPS) 0.5 µg/ml (Invivogen), Pam3Cys (P3C) 0.5 µg/ml (EMC microcollections GmbH), resiquimod (R848) 5 µg/ml (Enzo Life Sciences GmbH), CpG oligodeoxynucleotides (CpG) 1 µM (MWG-Biotech AG), polycytidylic acid (Poly(I:C)) 1 μg/ml (Invivogen), peptidoglycan (PGN) 10 μg/ml (Invivogen) and muramyldipeptide (MDP) 5 μg/ml.

Whole venous blood was collected into tubes containing 1.8 mg/ml (K2) EDTA (Becton Dickinson, Oxford, UK) and stored at room temperature for up to 2 hours prior to cell separation. Leucocyte cones from blood donors were purchased from NHS Blood and Transplant (Tooting, UK) to determine SARM expression upon TLR activation . Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque gradients (Cedarlane, Burlington, Canada) as previously described [18 (link)]. Monocytes from PBMCs derived from whole venous blood were isolated using CD14⁺ selection beads (Miltenyi Biotec, Cologne, Germany) as per the manufacturer’s instructions. Peripheral blood monocytes from PBMCs derived from leucocyte cones were isolated by iso-osmotic Percoll gradient centrifugation, as previously described [19 ], before being cultured in RPMI1640 media supplemented with 5% (v/v) foetal calf serum and 1% (v/v) penicillin/streptomycin solution (PAA, Pasching, Austria) with or without 100 ng/ml PAM₃CSK₄ (Axxora, Nottingham, UK), 10 ng/ml lipopolysaccharide (LPS) (Axxora, Nottingham, UK) or 2 μg/ml resiquimod (R-848) (Enzo Life Sciences, Lausen, Switzerland) at 37°C, 5% CO₂. All TLR ligands were used at predetermined concentrations that induce maximal cytokine induction.

CD1c⁺ mDCs were cultured in RPMI 1640 Medium (Lonza) containing 5% Human Serum (Sigma-Aldrich) without antibiotics and challenged with BCG on the same day of isolation. Unless otherwise stated, cells were seeded at a density of 7.5 x 10⁵ cells/well in 200μl and infected for 20h with BCG at a ratio of 10 bacilli:cell. Alternatively, mDCs were stimulated with a combination of the TLR ligands Resiquimod (R-848, 5μg/ml, Enzo Life Sciences) and Polyinosinic-Polycytidylic acid (poly(I:C), 20μg/ml, Sigma-Aldrich) for the same period. Where indicated CD1c⁺ mDCs were treated for 1h prior to, and for the duration of infection with 1mM 2-Deoxyglucose (2-DG, Sigma-Aldrich).
For analysis by confocal microscopy CD1c⁺ mDCs were challenged with GFP-BCG as outlined above, washed and fixed with 2% paraformaldehyde and then mounted onto Polysine slides (ThermoFisher Scientific). Cells were imaged with a 63X magnification oil immersion objective on a Leica SP8 confocal microscope using LAS X software (Leica).