Tgf β1

Macrophages were co-cultured with 1 × 10⁵ human primary dermal fibroblasts (ATCC, USA) within the matrix for 3 days in standard cell culture conditions. As negative and positive controls, 10⁵ human primary dermal fibroblasts were cultured onto 3D collagen matrices with and without the presence of 10 ng/mL of transforming growth factor beta 1 (TGF-β1; Biolegend, USA). Afterwards, RNA was extracted and gene expression of myofibroblast markers was quantified using quantitative polymerase chain reaction (qPCR, see gene expression analysis section). Furthermore, as TGF-β1 is involved in the myofibroblast differentiation, we quantified the free active TGF-β1 in the cell culture supernatant using the TGF-β1 ELISA (enzyme-linked immunosorbent assay) kit (Biolegend, USA) following the protocol by the manufacturer. All experiments were undertaken in 4 replicates.

For naïve T cell and DC co-cultures, naive CD4⁺ T cells (5 × 10⁵ cells/mL) and DCs (5 × 10⁵ cells/mL) were co-cultured in 96-well U-bottom plates and stimulated with soluble anti-CD3ε mAb (5 μg/mL) supplemented with 0.1 ng/mL TGF-β1 and 10 ng/mL IL-2 (BioLegend) for 3 days. For naïve T cell monocultures, naive CD4⁺ T cells (5 × 10⁵ cells/mL) were cultured in 96-well flat-bottom high-binding plates (Corning). The cells were stimulated with immobilized anti-CD3ε mAb (5 μg/mL) and soluble anti-CD28 mAb (5 μg/mL) supplemented with 0.1 ng/mL TGF-β1 and 10 ng/mL IL-2 (BioLegend) for 3 days. LA, 10,13(OH)18:0, 17,18-DiHETE, 9,10-EpOME, 9,10-DiHOME, and 9,10-EpOME were purchased from Cayman Chemical Company and stored at -30 °C in methyl acetate before use. For all experiments, the methyl acetate was removed under a nitrogen steam. The compound was resuspended in TexMACS medium (Miltenyi Biotec) containing 20 µM GlutaMAX (Thermo Fisher Scientific), 55 µM 2-mercaptoethanol, 10 mM HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin, or RPMI 1640 medium containing 10% FBS, 20 µM GlutaMAX (Thermo Fisher Scientific), 55 µM 2-mercaptoethanol (Thermo Fisher Scientific), 10 mM HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin. The methyl formate fraction dissolved in ethanol was diluted in the culture medium at the same ethanol concentration (0.5%) under all dilution conditions.

Differentiation of murine T cells was performed as previously described (40 (link)). Splenic T cells were isolated by magnetic activated cell sorting using the “pan T cell isolation kit II” according to the manufacturer’s instructions (Miltenyi Biotech). Cells were fluorescently stained with an antibody cocktail containing αCD4-FITC (RM4-5, eBioscience), αCD44-PE (IM7, Biolegend), αCD62L-APC (MEL-14, eBioscience), and αCD25-PE-Cy5 (PC61.5, eBioscience) and were subsequently isolated by fluorescence activated cell sorting on MoFlo (Beckman-Coulter, Brea, CA, USA) in the FACS-core unit of the University Hospital Erlangen. Sorted naive T cells (CD4⁺CD62L⁺CD44^lowCD25⁻) were stimulated by plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen, San Diego, CA, USA) and soluble anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and cultured for 4 days in the presence of IL-6 (40 ng/ml, R&D Systems, Minneapolis, MN, USA) and TGFβ1 (2 ng/ml, Biolegend) for Th17, IL-12p70 (20 ng/ml,R&D Systems) and anti-IL-4 (10 µg/ml, Biolegend) for Th1 or TGFβ1 (10 ng/ml, Biolegend) alone for Treg differentiation. T cell frequencies were analyzed by flow cytometry (FACSCantoII, BD Biosciences) using the antibodies listed in the section “Murine FACS analysis.”