Clone mp5 20f3

Naïve CD4 T cells stained with APC-labeled anti-CD4 (clone RM4-5) and FITC-labeled anti-CD62L (clone MEL-14) were isolated, with purity of over 98%, from IL-6 deficient OT-II mice using a FACSAria II. CD49b(DX5)⁺ c-kit⁻ basophils were isolated, with purity higher than 95%, from the BM culture supplemented with IL-3 (10 ng/ml, Peprotech) for 7–9 days using a FACSAria II. Conventional CD11c⁺ DCs were isolated, with purity higher than 90%, from BM cells cultured with GM-CSF (20 ng/ml, Peprotech) using CD11c microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). 2.5 × 10⁴ CD4 T cells were cultured with 0.5 × 10⁴ basophils activated with IgE (10 μg/ml, BD biosciences) and anti-IgE (10 μg/ml, BioLegend) and mature CD11c⁺ DCs for 3 days. 10 μM of OVA_323–339 peptide (ISQAVHAAHAEINEAGR), anti-IL-4 (10 μg/ml, clone 11B11, BD biosciences), anti-IFN-γ (10 μg/ml, clone XMG1.2, BD biosciences), anti-IL-6 (5 μg/ml, clone MP5-20F3, BD biosciences), and TGF-β (5 ng/ml, Peprotech) were added as indicated. Cell culture was performed in 96-well round-bottomed plates in a total volume of 200 μl.

Neurons/microglia co-cultures or microglia cultures were stimulated for 48 h with SN from non-infected or B. abortus-infected astrocytes diluted in complete medium (1/2, unless otherwise stated). Untreated wells were used as a negative control. When indicated, co-cultures were treated with astrocytes SN in the presence of recombinant annexin V (200 nM; eBioscience), cyclic RGD peptide (cRGD, Cyclo(-Arg-Gly-Asp-D-Phe-Val)), cRAD (Cyclo(-Arg-Ala-Asp-D-Phe-Val)) peptides (100 μM; Bachem), or recombinant mouse IL-6 (5, 10 or 15 ng/mL; Peprotech). Also, co-cultures were pre-treated for 1 h with aminoguanidine (AG) (200 μM; Sigma Aldrich) or recombinant mouse gp130Fc chimera protein (100 ng/mL; R&D Systems), then they were treated with astrocytes SN for 48 h in the presence of the inhibitor. Neutralization experiments were performed using anti-mouse TNF-α (10 μg/mL; clone MP6-XT3; BD Pharmingen), anti-mouse IL-1β (10 μg/mL; clone B122; eBioscience), anti-mouse IL-6 (5 μg/mL; clone MP5-20F3; BD Pharmingen) monoclonal antibodies, or an isotype control (10 μg/mL; BioLegend). Culture supernatants from B. abortus-infected astrocytes were pre-incubated with the respective neutralizing antibodies for 1 h at 37°C before stimulating neurons/microglia co-cultures.