Macrophage colony stimulating factor 1

Bone marrow cells collected from the femurs of 10–12-week-old C57BL/6 male mice were cultured at 37 °C in the presence of 5% CO₂ in ultra-low attachment flasks (Corning Inc., Corning, NY, USA) in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher Scientific, Waltham, MA, USA) containing 20 ng/mL macrophage colony stimulating factor-1 (Peprotech Inc., Rocky Hill, NJ, USA); the medium was changed twice weekly, as described previously [22 (link), 23 (link)]. After 7 days, the collected macrophages were harvested and seeded in 6-well Nunc™ Cell-Culture Treated Multidishes (Thermo Fisher Scientific) at a density of 3 × 10⁵ cells/well. Then, 25 ng/mL lipopolysaccharide (LPS from Escherichia coli O111:B4; catalog number L2630; Sigma-Aldrich, Tokyo, Japan) and 0.01 μM ONO-1301 or DMSO (control group) were added to the cultured macrophages. After 18 h, the macrophages were harvested, and the mRNA expression levels of genes encoding pro-inflammatory factors (e.g., interleukin-6 [Il6], tumor necrosis factor [Tnf-a], monocyte chemotactic protein-1 [Mcp-1], and inducible nitric oxide synthase [Inos]) and anti-inflammatory factors (e.g., interleukin-10 [Il10], chitinase 3-like 3 [Ym-1], and macrophage mannose receptor [Cd206]) were evaluated using real-time polymerase chain reaction (PCR) (Supplementary Table 1).

The B-cell Leukaemia C/EBPα (CCAAT-enhancer-binding protein α, where CCAAT stands for a Cytosine-Cytosine-Adenosine-Adenosine-Thymidine motif) Estrogen Receptor clone 1 cell line (BLaER1) [41 (link)] was kindly provided by Prof. Thomas Graf (Centre for Genomic Regulation, Barcelona). BLaER1 cells were maintained in RPMI medium supplemented with 1% L-Glutamine, 1% penicillin-streptomycin (P/S, Sigma-Aldrich, Darmstadt, Germany), 10% FBS and 1% sodium pyruvate (Thermofisher scientific, Darmstadt, Germany). BLaER1 cells were transdifferentiated into monocyte-like cells for 7 days in six-well plates by seeding 1 × 10⁶ cells/well in 3 mL of medium supplemented with 10 ng/mL of human recombinant IL-3, 10 ng/mL of human recombinant Macrophage Colony-stimulating Factor 1 (both from PeproTech, Hamburg, Germany) and 100 nM of β-Estradiol (Sigma-Aldrich, Darmstadt, Germany). Half of the medium was exchanged at days 2 and 5 for fresh medium containing identical supplements. On day 7 after differentiation, cells were harvested, counted with a CASY cell counter and cells were spun down (500× g, 5 min, RT). Cell pellets were resuspended in an adjusted volume of fresh medium without cytokines or P/S at a final density of 1 × 10⁶ cells/mL. Finally, 10⁶ cells were seeded per well in a 24-well plate for infection assays.

Bone-marrow cells collected from the femurs of 10-week-old male mice were cultured at 37 °C in the presence of 5% CO₂ in ultra-low attachment flasks (Corning, Corning, NY, USA) and medium (Dulbecco’s modified Eagle’s medium/F12; Thermo Fisher Scientific) containing 20 ng/mL macrophage colony-stimulating factor-1 (Peprotech Inc., Rocky Hill, NJ, USA); the medium was changed twice weekly, as described previously [6 (link)]. After 7 days, the macrophages were harvested. The harvested macrophages were seeded in 6-well ultra-low attachment dishes (Corning) at a density of 5 × 10⁶ cells/well. Then, 0.1 mg HMGB1 peptide (concentration: 1 mg/mL), 0.01 mg HMGB1 peptide (concentration: 0.1 mg/mL, low-dose group), or NS (control group) was added to the cultured macrophages. After 48 h, macrophages were harvested, and mRNA expression levels of genes encoding pro-inflammatory factors (e.g., interleukin-6 [Il6], tumor necrosis factor [Tnfa], monocyte chemotactic protein-1 [Mcp1], and inducible nitric oxide synthase [Inos]) and anti-inflammatory factors (e.g., Il-10, chitinase 3-like 3 [Ym1], found in inflammatory zone protein [Fizz1], and Cd206) were evaluated using real-time polymerase chain reaction (PCR).