Ciprofloxacin

NIH 3T3, a mouse embryonic fibroblast cell line, was purchased from ATCC (ATCC CRL-1648). Cells were maintained throughout the experiments in a 75-cm² flask (Falcon 250 mL 75 cm², #353135) in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, Saint-Quentin- Fallavier, France) containing 10% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% ciprofloxacin (Fresenius Kabi, France) and 1% fungizone (Gibco, USA).

Conventional IL-10^−/− mice (C57BL/6j background) were reared in the Forschungsinstitute für Experimentelle Medizin, Charité—University Medicine Berlin (Berlin, Germany), housed in cages equipped with filter tops under standard conditions (22–24 °C room temperature, 55 ± 15% humidity, 12 h light/12 h dark cycle) in an experimental semi-barrier facility and had free access to autoclaved standard food pellets (ssniff R/M-H, V1534-300, Sniff, Soest, Germany) and tap water. Female and male 3-week-old litter-mate mice were treated with broad-spectrum antibiotics, in order to deplete the commensal gut microbiota as reported earlier [41 (link),54 (link)]. In brief, mice were transferred to sterile cages (maximum of 4 animals per cage) and treated for eight weeks with an antibiotic cocktail consisting of ampicillin plus sulbactam (1 g/L; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany), imipenem (250 mg/L; Fresenius Kabi, Bad Homburg, Germany), vancomycin (500 mg/L; Hikma Pharmaceuticals, London, UK), metronidazole (1 g/L; B. Braun, Melsungen, Germany), and ciprofloxacin (200 mg/L; Fresenius Kabi, Bad Homburg, Germany) added to autoclaved drinking water (ad libitum). Microbiota-depleted mice were continuously kept and handled under strict aseptic conditions. Three days before infection, the antibiotic treatment was withdrawn, and mice received autoclaved tap water instead.

IL-10^−/− mice with a C57BL/6J background were bred and maintained under specific pathogen-free conditions in the identical unit of the Forschungseinrichtungen für Experimentelle Medizin (Charité-University Medicine Berlin). Under standard conditions (i.e., 22–24 °C temperature, 55% ± 15% humidity, 12 h light/12 h dark cycle), animals were kept in cages with filter tops within an experimental semi-barrier. Mice had access to autoclaved water and chow (food pellets: ssniff R/M-H, V1534-300, Sniff, Soest, Germany) ad libitum. To overcome physiological colonization resistance, mice were treated with broad-spectrum antibiotic compounds rendering them secondary abiotic, as described earlier [16 (link),36 (link)]. Briefly, immediately after weaning, 3-week-old mice were treated with an antibiotic cocktail consisting of ampicillin plus sulbactam (1 g/L; Dr. Friedrich Eberth Arzneimittel, Ursensollen, Germany), vancomycin (500 mg/L; Hikma Pharmaceuticals, London, UK), ciprofloxacin (200 mg/L; Fresenius Kabi, Bad Homburg, Germany), imipenem (250 mg/L; Fresenius Kabi), and metronidazole (1 g/L; B. Braun, Melsungen, Germany) dissolved in autoclaved drinking water (ad libitum) [36 (link)]. To ensure antimicrobial washout, the antibiotic cocktail was withdrawn and replaced by autoclaved water three days prior to the initial FMT.

Primary culture of MSCs was obtained from placental amnion by enzymatic method [25 (link)]. Amnion membranes were washed with PBS with 10% Ciprofloxacin (Fresenius Kabi, Bad Homburg, Germany), then dissected into small pieces and incubated in presence of 0.25% trypsin for 1 hour at 37°C. Following trypsin digestion, samples were filtered through 100 μm cell strainer (BD Biosciences, Durhan, USA), the cell suspension was centrifuged for 5 min at 1200 rpm (Heraeus Multifuge 1S-R, Thermo Fisher Scientific GmbH, Dreieich, Germany), the cell pellet was resuspended in MSC growth medium and plated into 10 cm cell dishes (Cellstar, Greiner BioOne, Frickenhausen, Germany).

Adrenal gland cells pooled from control rats (pooled from a total of n = 8–28 control animals depending on the size of the experiment) were seeded into 24 well plates as described, and on day 4 of culture, 0.75 × 10⁵ differentiated BMDCs from control or arthritic rats (not pooled) were added to the wells with co-culture inserts with 0.4 µm pore width (141002, Thermo Fisher scientific). After a co-culture period of 1 day, inserts including BMDCs were discarded, and adrenal gland cells were stimulated with 1 nM synthetic ACTH (Tetracosactid, Sigma-Tau) in DMEM-F12 supplemented with 2 mg/ml Ciprofloxacin (Fresenius Kabi), 1% P-S, 0.2% BSA and 1% glutamine (all from Sigma Aldrich) for 6 h. The level of corticosterone in supernatants was determined by ELISA (DEV9922, Demeditec, Germany).

Primary culture of MSCs was derived from the amnion of placenta via application of an enzymatic method [5 (link)]. Amnion membranes were washed in phosphate-buffered saline (PBS) supplemented with 10% ciprofloxacin (Fresenius Kabi, Bad Homburg, Germany) and then dissected into small pieces and incubated for 1 h at 37 °C in presence of 0.25% trypsin. After trypsin digestion the samples were filtered through a 100-μm cell strainer (BD Biosciences, Durhan, USA), followed by centrifugation of the obtained cell suspension for 5 min at 1200 rpm (Heraeus Multifuge 1S-R, Thermo Fisher Scientific GmbH, Dreieich, Germany). The supernatant was removed and the cell pellet was resuspended in MSC growth medium and plated on 10-cm cell culture dishes (Cellstar, Greiner BioOne, Frickenhausen, Germany).

Ficoll separation was performed to isolate mononuclear cells from bone marrow samples obtained from MDS patients or healthy donors following informed consent and under institutional review board guidelines. CD34⁺ cells were isolated from mononuclear cells using magnetic beads CD34⁺ separation protocol (CD34 MicroBead Kit human, MACS Miltenyi Biotec). Isolated CD34⁺ bone marrow cells were kept in cell culture at 37°C in serum-free medium containing 20% BIT9500 (Stemcell Technologies), 80% IMDM medium with Glutamax (Gibco), 10μM ß-Mercaptoethanol (Gibco), 8μg/mL ciprofloxacin (Fresenius Kabi) and 5 growth factors: 100ng/mL stem cell factor, 100ng/mL FLT3-Ligand, 25ng/mL thrombopoietin (TPO), 10ng/mL interleukin 3 and 10ng/mL interleukin 6 (R&D systems).