Interferon ifn γ

3D co-cultures of PBMC with MSC (n = 6) were performed in 12-well plates (Corning). Cells were embedded in fibrin matrices in a ratio 1:100 (MSC:PBMC) using 2.5 × 10⁶ PBMC/cm² and cultured for up to 2 weeks in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences). Fibrin matrices were prepared as described above, but without addition of aprotinin. Control experiments were performed culturing PBMC separated from MSC by a 0.4 μm transwell insert (Corning), PBMC without support of stromal cells and MSC alone. To investigate the effect of paracrine inflammatory signals on stromal cells in the absence of immune cells, MSC (n = 4) were embedded in fibrin matrices in 24-well plates (Corning) at 2.5 × 10⁴ cells/cm² and cultured for 6 days in complete αMEM medium (Invitrogen) containing 10% fetal bovine serum (GE Healthcare Life Sciences) supplemented with 5 ng/ml tumor necrosis factor (TNF)α (PeproTech, Rocky Hill, NJ) and 10 ng/ml interferon (IFN)γ (PeproTech). Control experiments were performed in complete αMEM medium without cytokine supplementation. Cultures were maintained at 37°C (20% O₂ and 5% CO₂ humidified atmosphere), and medium was changed every 3 days. Cellular re-arrangement was monitored using a phase contrast microscope (Olympus) and documented using a digital camera (Olympus).

Cibinetide was provided by Araim Pharmaceuticals, Inc. (Tarrytown, NY, USA). Cibinetide stock solution (1.2 mg/mL, 1 mmol/L) was dissolved in phosphate buffered saline (PBS), aliquoted and stored at -20°C until use. The doses of cibinetide (100 nmol/L in in vitro study and 120 µg/kg in in vivo model) were selected based on our previous studies^{21 (link),22 (link)}. Recombinant human interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were purchased from PeproTech Inc., (Rocky Hill, NJ, USA).

PBMC or MDM were stimulated with MDP (10 µg/mL), lipidated L18-MDP (200 ng/mL), inactive D-D isomer of MDP (10 µg/mL), Flagellin from S. typhimurium (100 ng/mL), Pam3CSK4 (100 ng/mL, all Invivogen), Lipopolysaccharide (LPS) from Salmonella minnesota R595 (20–200 ng/mL, Enzo), interleukin (IL)-10 (20 ng/mL), interferon (IFN)-γ (50 ng/mL) or tumour necrosis factor (TNF) (10 ng/mL, all Peprotech). Lipidation of MDP (L18-MDP) allows reduction of MDP concentration in stimulation studies.

RAW264.7 cells were purchased from Procell Life Science&Technology (Wuhan, China) and authenticated by STR profiling. The cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Gibco, USA) with 10% fetal bovine serum (Australian origin; Gibco) and 1% penicillin-streptomycin (Gibco, USA) at 37 °C with 5% CO₂.
RAW264.7 macrophages were unstimulated to create M0 macrophages. For M1 macrophage creation, RAW264.7 cells were treated with LPS (10 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) and interferon (IFN)-γ (100 ng/ml; PeproTech) for 24 h. For the AAM, RAW264.7 cells were treated with IL-4 (20 ng/ml; PeproTech) for 24 h.
Apoptosis inducer (20 μM, 24 h), pyroptosis inducers (7 μg/ml, 24 h), necrosis inducer (4 x, 24 h), RSL3 inducer (5 μM, 5 h), and erastin inducer (60 μM, 24 h) were used to treat macrophages (M0, M1, and AAM). For 5 h, AAM was treated with RSL3 (5 μM) in the presence or absence of Fer-1 (400 nM). AAM was treated with the 1488 candidates combined with RSL3 (5 μM) for 5 h. AAM was treated with Lie (1, 5, and 10 μM) combined with RSL3 (5 μM) for 5 h. AAM was treated with RSL3 (5 μM) in the presence or absence of Lie (10 μM) for 5 h. AAM was treated with RSL3 (5 μM) in the presence or absence of Lie (10 μM) or bafilomycin A1 (25 nM) for 5 h.

To obtain human monocytes and T cells, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Chalfont St. Giles, UK) gradient centrifugation from buffy coats of healthy volunteers. Buffy coats were provided by the Portuguese Blood and Transplantation Institute (IPST) following an established protocol allowing access to buffy coats for scientific research with academic purposes only. The buffy coats were not specifically obtained for the present study and were provided without any personal detail from the donor.
Monocytes and T cells were isolated by positive selection using CD14 and CD3 antibody-coated magnetic beads (Miltenyi Biotec), respectively, as described by the manufacturer. T cells were maintained in RPMI medium until co-cultured with DCs. Monocytes were cultured at a density of 1 × 10⁶ cells/ml in the different culture media supplemented with 250 U/ml of IL-4 (Peprotech, London, UK) and 400 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech), for differentiation into immature DCs (iDCs). Each medium was refreshed every 2 days and DCs maturation was induced at day 6 of culture, by adding 50 ng/ml of tumor necrosis factor (TNF)-α (Biolegend), 25 ng/ml of IL-1β (Biolegend), 20 ng/ml of polyinosinic:polycytidylic acid (Poly-I:C) (Novus Biologicals, Abingdon, UK), and 100 ng/ml of interferon (IFN)-γ (Peprotech).