Rtnf α

The Amaxa nucleofector system and the Amaxa Human Chondrocyte Nucleofector Kit were used for electroporation following the protocols from the manufacturer (Lonza, Walkersville, MD, USA). Briefly, each reaction contained 1.0 × 10⁶ cells, 5μM of pre-miR mimics (Table S2) in a total volume of 100 μL nucleofection solution. The cells were seeded in 20% PLP without antibiotics and left to recover overnight. The following day (day 1), the medium was changed to 10% PLP with 1% penicillin/streptomycin. On day 4, ACs were stimulated with 0.1 ng/mL recombinant IL-1β (rIL-1β) or 10 ng/mL rTNF-α (R&D Systems, Minneapolis, MN) for 24 h before harvesting for analysis.

Dendritic cells were derived from monocytes selected for CD14 by magnetic selection column (Myltenyi Biotec, Bergisch Gladbach, Germany). The CD14⁺ cell population was dispensed into six-well plates containing X-vivo 15 medium (Cambrex) supplemented with antibiotic-antimycotics (Gibco). To generate immature dendritic cells (DCs), the cells were cultured in the presence of 20 ng/mL recombinant IL-4 (rIL-4) and 50 ng/ml rGM-CSF (both from R&D Systems, Minneapolis, MN) for six days (indicated as D6 cells). Mature DCs were obtained after 24 h (D7) after stimulation of the immature DC culture with 10 ng/mL rTNF-α (R&D System) plus 0.01 mmol/L of PGE2 (Sigma) [23] (link).
In order to set up the MLR, matured DCs were irradiated at 1,500 rads and co-cultured with allogeneic lymphocytes at the concentration of 1 DC:100 Lymphocytes (1×10⁴:1×10⁶ respectively) in presence or absence of 1×10⁵ allogeneic hMSCs. The proliferation rates were determined after four days of co-culture. Lymphocyte proliferation was measured by KI-67 nuclear staining on CD3+ gated population. The lymphocytes were gated based on SSC vs FSC, followed by CD3 gating; the selected events were analyzed in a second plot for KI-67 expression. The proliferation negative control was lymphocytes without allogeneic DCs stimulation.

Animals were inoculated in the hind footpad with 1x10⁶ CFUs of BCG in 30 μl of PBS. Control animals received an equal volume of PBS. For assessment of cell migration from the footpad skin, animals previously injected with BCG or PBS where injected 24hrs before sacrifice in the same footpad with 20 μl of 0.5 mM 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). In select experiments animals were injected with 50 ng or 300 ng rTNF-α or 2 μg rIL-12p40 homodimer (R&D Systems) in the same footpad that 2hrs earlier was inoculated with BCG or PBS. For assessment of lymphatic drainage to LNs, 20 μl 5% Evan’s blue (Sigma) was injected in the footpad and LNs isolated at different time points after injection. For assessment of CD4⁺ antigen-specific T-cell responses, 1x10⁵ LN cells from P25 TCRTg RAG–1^-/- mice were first labeled with 1 μM CFSE and then injected i.v. in the tail vein of congenic CD45.1⁺ recipients in a final volume of 200 μl. For DC adoptive transfer experiments, 2x10⁶ naïve BMDCs generated from WT or transgenic mice were labeled with 3 μM CFSE and then injected into the footpad in a final volume of 40 μl. Two hrs after DC transfer, the same footpads were inoculated with 30 μl PBS or 1x10⁶ CFUs of BCG.