Inf γ

Chemicals were of the purest analytical grade. Human recombinants IL-2, IL-4, IL-6, IL-10, INF-γ, and LIF, calpain substrate [N-Suc-Leu-Tyr-AMC (7-amido-4-methyl-coumarin)], AA861 (specific inhibitor of 5-LOX), and E64D (specific inhibitor of calpain) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse anti-cytochrome c antibody was from Cell Signalling Technology Inc. (Danvers, MA, USA); mouse anti-calpain-1 was from Calbiochem (Merck Darmstadt, Germany). Rabbit anti-LIF, anti-IL-2, anti-IL-4, anti-IL-6, anti-IL-10, anti-INF-γ, secondary antibodies conjugated to horseradish peroxidase (HRP), and enhanced chemiluminescence (ECL) kit were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Goat anti-rabbit conjugated to alkaline phosphatase (GAR-AP) was from Bio-Rad (Hercules, CA, USA).

Bone marrow-derived macrophages (BMDMs) were extracted from 12-week-old C57 BL/6J mice. BMDMs were cultured in DMEM medium containing 10% FBS and 10 ng/ml Recombinant Mouse M-CSF Protein (416-ML, R&D), followed by treatment with DMY (50, 100 μmol/L) or control vehicle for one hour, then stimulated with 100 ng/ml LPS (Cat# L2880, Sigma-Aldrich) and 20 ng/ml INF-γ (IF005, Sigma-Aldrich) for 24 hours. Lipofectamine 3000 transfection reagent (L3000008, Invitrogen) was used for miRNA (miR10000142-1-5, miR20000142-1-5, RiboBio) and siRNA (AM16708, Thermo Fisher Scientific) transfection according to the manufacturers' instructions.

Conditionally immortalized wild-type (WT) and mutant IFT88^orpk mouse chondrocyte cell lines were generated as described previously²⁷ (link). The IFT88^orpk chondrocytes harbour an insertional hypomorphic mutation to the gene tg737, encoding for the anterograde intraflagellar trafficking protein IFT88, resulting in strong inhibition of primary cilia assembly. Chondrocytes were maintained in DMEM supplemented with 10% FCS, 88 U/ml penicillin, 90 μg/ml streptomycin, 10 ng/ml INF-γ and 2.5 mM L-glutamine (Sigma–Aldrich, Poole, UK). Immortalized cells were maintained under permissive conditions at 33°C, 5% CO₂ in the presence of 10 nM IFN-γ. For experiments, cells were cultured in non-permissive conditions at 37°C (without IFN-γ) for 3 d, to switch off SV40 control and restore the primary chondrocyte phenotype, then seeded in 24 wells plate for another 24 h. Cell viability was assessed with trypan blue.

Based on all tests, 4 samples were selected for further cell culture characterization: CR-PLD-L, CR-PLD-P, CR-PLD-Cu-L, and CR-PLD-Cu-P. The samples were put into the wells of 24-multiwell plate and preincubated in the complete culture medium. Human THP-1 monocytes were seeded directly on the biomaterials at the density of 2.5 × 10⁵ cells/sample. Monocytes grown in a polystyrene well of the 24-multiwell plate without biomaterial served as negative control (CTRL−), showing a physiological level of cytokine release. Monocytes cultured in a polystyrene well of the 24-multiwell plate in the medium supplemented with 100 ng/mL of lipopolysaccharide (LPS) and 25 ng/mL of interferon-gamma (INF-γ, Sigma-Aldrich Chemicals) served as positive control (CTRL+), showing increased level of cytokine release due to stimulation with pro-inflammatory factors. After 48-h culture, cell culture supernatants were harvested to assess concentrations of interleukin (IL)-4, IL-6, Il-10, and IL-13 by human specific ELISA kits (EIAab). ELISAs were carried out according to the manufacturer’s protocol using three independent samples. Statistically significant differences between the samples were considered at p < 0.05 according to#]p098 One-way ANOVA with post-hoc Tukey test (GraphPad Prism 8.0.0 Software, San Diego, CA, USA).

Treatment of HBCs was performed in complete MaM medium, treatment of THP-1 macrophages in complete RPMI-1640 and performed in T25 nunclone flasks (Nunc, Waltham, MA, USA) at a cell density of 1 × 10⁶ cells/mL. For induction of polarization towards M1 phenotype, lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) and Interferon gamma (INF-γ, Sigma, St. Louis, MO, USA) were added to a final concentration of 50 and 20 ng/mL, respectively. For induction of the M2 phenotype, interleukin 4 and 13 (IL-4, IL-13, both Sigma, St. Louis, MO, USA) were added to a final concentration of 20 ng/mL each. Both cell types were cultivated for 72 h after addition of stimulating agents. Untreated controls were included in each individual experiment. THP-1 macrophages were harvested after 72 h treatment and used for subsequent analysis. HBCs received a second treatment with stimulating substances and were cultivated for an additional 24 h before harvesting and proceeding. Preliminary experiments on HBCs from our group and also others [56 (link)] have shown that the isolation protocol using extensive digestion steps can reduce presence of surface markers in FACS and that up to 72 h recovery period are needed before e.g., CD163 is fully present again. Therefore it was decided to offer HBCs more time in culture exposed to respective stimuli.

THP-1 (ATCC TIB202) cells, an in vitro model extensively used to study human monocytes and macrophages [48 (link)], were maintained in RPMI media (Cat# 11875093, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cat# 10437028, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C in a humidified incubator with 5% CO₂. The cells were subcultured every 2 to 3 days in order to maintain a cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL. THP-1 cells were seeded in assay plates in RMPI plus 10% FBS medium containing 25 ng of PMA (Cat# P8139, Sigma-Aldrich, St. Louis, MO, USA) per mL in order to induce differentiation of the THP-1 cells. After 24 h of cell plating, macrophages were activated with 5 pg/mL LPS (Cat# L8274, Sigma-Aldrich) and 10 ng/mL INFγ (Cat# SRP3058, Merck, Kenilworth, NJ, USA), and treated with 10 μM hemin (Cat# 51280, Sigma-Aldrich), 10 μM 3,4DHPAA (Cat# 850217, Sigma-Aldrich), or 10 μM 4HPAA (Cat# H50004, Sigma-Aldrich) for 72 h (Scheme 2).