Gentamycin

Raji DC-SIGN cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 100U/ml penicillin and 100U/ml streptomycin (100U/ml Pen/ Strep). Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (purchased Sanquin) obtained from healthy volunteers using ficoll-hypaque density centrifugation. PBMCs of 3 CCR5 wild-type homozygous donors were pooled and cultured in RPMI 1640 containing 10% FCS, 100U/ml Pen/ Strep, 100U/ml recombinant IL-2 (Chiron), and 2μg/ml phytohemagglutinin (PHA, Remel) was used to activate the cells. After 5 days of culture PBMCs were enriched for CD4⁺ T-cells by depleting the CD8⁺ T-cell population using CD8 dynabeads (Life Technologies) according to manufacturer’s protocol. Monocytes were isolated from PBMC as previously described [49 (link)]. Monocytes cultured in IMDM (Gibco) containing 5% FCS, 86μg/ml gentamycin (Duchefa), 500U/ml GM-CSF (Schering-Plough) and 10U/ml IL-4 (Miltenyi Biotec) for 6 days differentiated into immature DCs (iDCs). The CD4⁺ T-cell isolation MACS kit (Miltenyi Biotec, 130-091-155) was used according to manufacturer’s protocol to isolate CD4⁺ T-cells from the PBL fraction. Subsequently, the CD4⁺CD45RA⁺CD45RO^- naïve T-cells were isolated from the CD4⁺ T-cells using anti-CD45RO-PE (DAKO, R084301) and anti-PE beads (Miltenyi-Biotec, 130-048-801), described in detail [49 (link)].

Donor A iDCs were matured with 100ng/ml LPS (Sigma Aldrich) or 100ng/ml LPS and 10μM PgE₂ (Sigma Aldrich) in the absence or presence of 25μg/ml BmA or 2–4μg/ml ES-62. Matured DCs were washed 3 times, subsequently 5x10³ cells were co-cultured with 2x10⁴ CD4⁺CD45RA⁺CD45RO^- T-cells from donor B and 10pg/ml Staphylococcus enterotoxin B (Sigma-Aldrich) in IMDM, 10% FCS, 86μg/ml gentamycin (Duchefa). At day 5 and 7 the cells were split and the medium was supplemented with 20U/ml IL-2. At day 8, 5x10⁴ cells/well were plated and infected with SF162 (R5) (TCID₅₀/ml 1000) or LAI (X4) (TCID₅₀/ml 200). Cells were re-stimulated with 10ng/ml PMA (Sigma-Aldrich), 1μg/ml ionomycin (Sigma-Aldrich), 10μg/ml brefeldin A (Sigma-Aldrich) and 0.1μg/ml T1294 (Pepscan Therapeutics BV) for 6h day 5 and 7 post-infection. Subsequently, the cells were fixed in 3.7% formaldehyde and stored for no more than 1 week at 4°C in FACS buffer (PBS+ 2%FCS) for flow cytometry analysis. CD4⁺ T-cells are the major cell type being analysed here since the culture conditions used are non-permissive for iDC survival. HIV-1 infection is determined at the optimal time-point which varies between donor and virus and where cell viability is high (>50%).

Isolation and co-cultures of T cells, monocyte-derived DCs (moDCs) and neutrophils were performed as previously described [8 (link)]. In brief, following CD4⁺ T-cell isolation, CD45RA⁺ naive cells were separated from the CD45RO⁺ memory T cells by positive selection of memory T cells using CD45RO-Phycoerythrin antibody (DAKO) and magnetically-labeled anti-PE beads (Miltenyi Biotec). Naive CD4⁺ T cells were always more than 98% pure. Monocytes were differentiated into moDCs and harvested as immature DCs after 6 days. Co-cultures were initiated in 96-well Candida albicans hyphae-coated plates, with 50,000 DCs and 50,000 autologous CD4⁺ naive or memory T cells, with or without 100 ng/mL VSTM1-v2, and for the naive T cell cultures, with or without 100,000 autologous freshly isolated neutrophils. Co-cultures were done in IMDM supplemented with 5% HI-HS (Lonza) and gentamycin (86 μg/mL, Duchefa Biochemie). After 4 days, cells were transferred to 48-well plates (Costar) and refreshed every 2 days with IMDM/5% HS medium containing 10 U/mL IL-2. At 10–12 days of culture, when cells were resting, they were restimulated for 5h with PMA (100 ng/mL), ionomycin (1 μg/mL), and brefeldin A (10 μg/mL) (all Sigma-Aldrich).

A library consisting of 290 full-length Arabidopsis transporter cDNAs was screened for loganin uptake in Xenopus oocytes. The screen was performed using the transporter identification platform developed by Nour-Eldin et al. (2006 (link), 2012 (link)). In brief, the library was divided into 29 pools of each 10 cRNA species (50 ng µl^–1 cRNA) and was injected into Xenopus oocytes. Injected oocytes were kept for 2–4 d at 17°C in Kulori pH 7.4 (90 mM NaCl, 1 m M KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 5 mM HEPES), containing 100 µg ml^–1 gentamycin (Duchefa Biochemie). Loganin uptake assays were performed by incubating transporter-expressing oocytes for 1 h in Kulori pH 5.0 (90 mM NaCl, 1 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 5 mM MES) with 0.5–1 mM loganin {(1S,4aS,6S,7R,7aS)-6-hydroxy-7-methyl-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate; Sigma}. Methanol extracts were prepared from the assayed oocytes by disrupting them in 50% (v/v) methanol. After overnight incubation at –20°C, the extracts were centrifuged (>14,000 relative centrifugal force at 4°C for 10 min) and the supernatants were analyzed by liquid chromatography–mass spectrometry (LC-MS) (described below). Pools testing positive for loganin uptake were assayed as single transporters, to identify the membrane protein(s) responsible for transport.

Synovial fluid (SF) from inflamed knee joints was collected during active arthritis from 27 patients after obtaining informed consent. Patients’ characteristics are described in Table 1 and Supplementary Table S1. SF was centrifuged at 650× g for 20 min to pellet the cells. The SF was centrifuged at 3000× g for 30 min at RT to pellet all remaining cells and debris; the cell-free SF was collected and stored at −80 °C until further use. Meanwhile, the SF-derived cells collected after the first 650 g step were resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc., Waltham, Mass) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and passed through a 70 µm single-cell filter. Then, cells were resuspended in IMDM with 10% HI FBS at a concentration of 2 × 10⁶ cells/mL for flow cytometry analysis and culture experiments.

Blood was collected from healthy volunteer donors after obtaining informed consent into sodium heparin tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Neutrophils were isolated using density gradient followed by erythrocyte lysis, as previously described [23 (link)]. Neutrophils were then resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc., Waltham, MA, USA) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and used immediately. Neutrophil purity was analyzed by flow cytometry and was always >97%.

HEK293, HeLa, and B16F10 cells were obtained from the Korean Cell Line Bank and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS) (Merk, USA) and 1% penicillin- streptomycin (Corning, USA), ciprofloxacin (Fluka, USA), and gentamycin (Duchefa Biochemie, The Netherlands). All three cell lines were cultured in a 5% CO2 incubator at 37°C. HEK293, HeLa and B16F10 cells were plated in T75 flasks and transfected with 20 μg of control vector or inducible vector using 60 μg of polyethylenimine (PEI, molecular weight 4,000) (Poly Science, USA).