M csf

Bone marrow–derived macrophages (BMDM) were generated by culturing bone marrow cells from P47 and S47 mice with MCSF (BioLegend; 576406), as described previously (33 (link)). Bone marrow cells were cultured in DMEM with 10% FBS (Corning; 35-026-CV) and 100 U penicillin/streptomycin antibiotics in the presence of 40 ng/mL MCSF (BioLegend, 576406) at a density of 2–2.5 × 10⁷ cells/15 cm dish. After 5 days of differentiation, BMDMs were harvested using ice-cold detachment buffer containing: 1X PBS, 2% FBS, and 2 mmol/L etheylenediaminetetraacetic acid (EDTA) (Invitrogen; 15575-038) and used in polarization experiments. BMDMs were polarized to M1 phenotype by lipopolysaccharide (LPS) alone (100 ng/mL) or LPS + IFNγ (100 ng/mL; BioLegend, 575306) for 8 hours. BMDMs were polarized to M2 phenotype by IL4 (10 ng/mL; BioLegend, 574304) for 8 hours.

For BMDMs, bone marrow was differentiated into macrophages in DMEM containing 10% FBS (Sigma), 5% M-CSF conditioned medium or 50 ng ml⁻¹ M-CSF (Biolegend) (results did not differ), 1% penicillin-streptomycin (Gibco), 1% glutamine (Invitrogen) 0.5% sodium pyruvate (Invitrogen) for 7–9 days prior to experimental use. Peritoneal macrophages were collected after 96 h thioglycallate treatment. Macrophages were washed, counted, plated in the medium described above, activated by TLR agonists and maintained in culture for 24–48 h.

Bone marrow was collected from femurs and tibias of 8-week-old, male C3H/He mice (Japan SLC) and cultured for 3 days at a density of 5 × 10⁶ cells/100-mm dish in MEMα (FUJIFILM Wako, Osaka, Japan) supplemented with 10% FBS and 100 ng/mL of macrophage colony-stimulating factor (M-CSF; BioLegend, San Diego, CA, USA). To induce osteoclast precursor cells (OPCs), the cells were cultured for additional 3 days at 5 × 10⁵ cells/100-mm dish. For osteoclastogenesis, OPCs were cultured for 5 days at 1 × 10⁵ cells/12-well plate in MEMα with 10% FBS, 20 ng/mL M-CSF, and 50 ng/mL of receptor activator of NF-kappaB ligand (RANKL; Biolegend). To determine effects of SEVs, 1 × 10¹⁰ particles/mL or indicated doses of SEVs were added to the medium at the beginning of osteoclastogenesis. To determine the effects of miRNA, 60 pmol of miRNA mimic transfection control with Dy547 (miR-Ctrl) or hsa-miR-146a-5p mimic (Horizon Discovery, Cambridgeshire, United Kingdom) was transfected into OPCs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) at the beginning of osteoclastogenesis. Cellular growth was evaluated using WST-8 assay reagent (Nacalai Tesque, Kyoto, Japan). TRAP staining was performed after 5 days of osteoclastogenesis by using an Acid Phosphatase Leukocyte Kit (Merck), and was evaluated using microscopy BZ-X710 and BZ-X Analyzer.

Peripheral blood mononuclear cells (PBMCs) were isolated from human donor whole blood. Primary human monocytes and peripheral blood leukocytes were separated via elutriation by the UNMC Elutriation core. Monocytes, and PBLs were used immediately after separation or were cryopreserved in liquid nitrogen before use. PBMCs and monocytes were maintained at 37˚C in 5% CO₂ in RPMI media with glutamine, 10% fetal bovine serum, penicillin and streptomycin added. Polarization of macrophages was induced as we previously described (). Specifically, monocytes were differentiated and polarized to M2 macrophages with M-CSF (100 ng/mL, BioLegend #574,806) for 7 days to promote monocyte differentiation and growth. Then, the M-CSF stimulated macrophages were polarized to M2 by addition of IL-4 (20 ng/mL, BioLegend #574,002) for 24 h. 20uM of GKT137831 was added to the monocytes during IL-4 treatment.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/mL; PanBiotech, Aidenbach, Germany) and washed twice with DPBS (Gibco).
Cells were then resuspended in alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies, Darmstadt, Germany), 10 μg/mL streptomycin (Life technologies), and 25 ng/mL MCSF (BioLegend, Amsterdam The Netherlands). The cells were seeded at specific concentrations, depending on the culture plate used (1.5 × 10⁶ cells/well in 6 well plates, 0.15 × 10⁶ cells/well in 48 well plates, and 0.05 × 10⁶ cells/well in 96 well plates) and cultured at 37 °C, 5% CO₂. After 24 h, the medium was replaced with a differentiation medium containing alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies), 10 μg/mL streptomycin (Life technologies, Darmstadt, Germany), 25 ng/mL MCSF, and 50 ng/mL RANKL (BioLegend, Amsterdam The Netherlands). As an osteoclast differentiation control, cells were cultured without RANKL. The cells were cultured for 14 days, and the medium was replaced twice a week.

Plasma samples, collected after centrifugation from the top of the ficoll gradient separation of portal blood, were frozen at -80°C for later use in cytokine/growth factor analyses. GM-CSF, M-CSF, IL-34, IL-8, interleukin 1alpha (IL1α), interleukin 1beta (IL1β), prostaglandin E2 (PGE2), and IFNγ levels were measured in portal and peripheral blood plasma and in media supernatants of PortalBMC cells grown in culture for cluster formation (‘conditioned media’) using BioLegend Legendplex multi-analyte assay flow cytometry or conventional ELISA (Boster Scientific, Cayman Chemical). Cell-free culture supernatants were collected by plate centrifugation of 7 day cultures at 600xg, 5min at 25°C and frozen at -80°C until analysis in duplicate by ELISA. Since portal blood from healthy individuals is not accessible without invasive intervention, portal blood control samples could not be ethically obtained for comparison. Therefore, portal and peripheral blood samples were compared to manufacturer/literature cited mean or median values for healthy human peripheral blood plasma (mean 33.7pg/ml GM-CSF, BioLegend, mean 83.7pg/ml M-CSF, BioLegend, mean 7.13pg/ml IL-34 [23 (link)], median 83pg/ml IL-8 [24 (link)], mean 1.91pg/ml IFNγ [25 (link)], mean 0.74pg/ml IL1α [26 (link)], mean 1.85pg/ml IL1β [27 (link)], mean 312pg/ml PGE2, ThermoFisher).