Interleukin 4 (il 4)

After washing, adherent macrophages were immediately stimulated with IFNγ (40 UI ml⁻¹, Clinisciences), IL-6 (50 ng ml⁻¹, Clinisciences), LPS (1 ng ml⁻¹, Sigma), IL-4 (50 ng ml⁻¹, Miltenyi Biotech), IL-13 (50 ng ml⁻¹, Clinisciences), IL-10 (50 ng ml⁻¹, Clinisciences), 15-HETE (1 μM, Cayman) or DLPC (50 μM, Sigma) for 4 or 24 h. In indicated experiments, adherent macrophages were pre-incubated or not with a Jak-2/STAT6 inhibitor, AG490 (1 nM, Tebu-Bio).
The mRNA preparation was made using the EZ-10 Spin Column Total RNA Minipreps Super Kit (Bio Basic) using the manufacturer's protocol. Synthesis of cDNA was performed according to the manufacturer's recommendations (Thermo electron). RT–qPCR was performed on a LightCycler 480 system using LightCycler SYBR Green I Master (Roche Diagnostics). The primers (Eurogentec) were designed with the software Primer 3. Actb (Actin) mRNA was used as the invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in Supplementary Table 1.

This was done as we have previously described [25 (link)]. Briefly, buffy coats were obtained from healthy donors following ethics committee approval and informed written consent (National Blood Service, UK). Separation of peripheral blood monocytes (PBMCs) was done by means of density gradient centrifugation using Histopaque (Sigma-Aldrich, UK). CD14⁺ monocytes were isolated using magnetic-assisted cell sorting (Miltenyi Biotec, UK). The purity of isolated monocytes was confirmed by flow cytometry and it was consistently >95% (Supplementary Fig. 1). Purified CD14 monocytes were supplemented with RPMI media containing 10% heat-inactivated fetal bovine serum (FBS), 100 mg/mL streptomycin, 100 U/mL penicillin, and 2 mM l-glutamine (all from Sigma-Aldrich). To generate immature DCs, monocytes were then seeded (1 × 10⁶ cells per well) in a 24-well tissue culture plate with the presence of 50 ng/mL GM-CSF and 250 U/mL IL-4 (both from Miltenyi Biotec) and incubated at 37 °C in a 5% CO₂-humidified incubator for 6 days for differentiation, where fresh media was added on day 3. Immature DC phenotype and maturation were confirmed through flow cytometry (Supplementary Fig. 2).

PBMCs from healthy donors were purchased (Trima Residuals RE202, Vitalant) and purified by Ficoll-hypaque gradient centrifugation (Fisher Scientific, 45-001-749). Cryopreserved PBMCs were thawed using RPMI (Gibco-Invitrogen) complete media (1% Pen Strep, 1% L-Glutamine, 10% FBS Heat Inactivated Serum (Gibco-Invitrogen, 16000-044), and 0.5% DNase (Sigma, DN-25) and washed twice with PBS. CD14⁺ monocytes were selected using CD14 microbeads (Miltenyi Biotec, 130-050-201) and cultured for 5 days in CellGenix medium (0020801-0500) supplemented with 800 U/mL GM-CSF (Miltenyi Biotec, 130-095-372) and 500 U/mL IL4 (Miltenyi Biotec, 130-095-373) to generate iDC). At day 3, half of media was replaced and supplemented with fresh cytokines. iDC were matured on day 5 with 1000 U/mL IFN-γ (Peprotech, 300-02) and 250 ng/mL LPS (Sigma-Aldrich, L2630). Two types of tol-DC were generated. To obtain vitd3-tol-DC 100 nM of vitamin D₃ (Sigma, D1530) was added to cultures at d0 and day 3. And dexa-vitd3-tol-DC were generated by adding 100 nM of vitamin D₃ and 10 nM of dexamethasone (Sigma, D4902) at day 3 to cultures. Both tol-DC were matured as described above. Rapamycin (Selleckchem, S1039), Compound C (Selleckchem,7306) and BAY8002 (Selleckchem, S 8747) were added at iDC stage together with IFN-γ/LPS for 24 h.

B cells were isolated from PBMCs by immunomagnetic negative selection using EasySep Human B Cell Isolation Kit (Stemcell Technologies) in accordance with the manufacturer’s instructions. B cells were cultured in StemMACS HCS Expansion Media XF (Miltenyi Biotec) supplemented with 1% streptomycin and penicillin (Invitrogen), 5% Human AB Serum (Valley Biomedical), and 125 IU/mL of IL4 (Miltenyi Biotec) at a density of 5 × 10⁵ cells/ml. B cells were expanded by crosslinking CD40 using Human CD40-Ligand Multimer Kit (Miltenyi Biotec) at a concentration of 8 U/ml in accordance with the manufacturer’s instructions. Media, IL4, and multimeric CD40L were refreshed every 3–4 days throughout the duration of all experiments.

Bone marrow-derived macrophages were obtained by flushing from murine (OPA1^f/f or OPA1^M/M) femur and tibia and differentiated into macrophages in RPMI 1640, 10% FBS (Superior, Millipore), Glutamine (300 mg/L), sodium pyruvate (1 mM), 2-mercaptoethanol (0.01 mM), HEPES (25 Mm) in the presence of M-CSF (Miltenyi Biotec), 40 ng/ml for 5 days and then refilled with 2 mL more with M-CSF (20 ng/mL) for 2 more days. At day 7, cells were differentiated to: M1 with Lipopolysaccharide from E. coli O111:B4 (Sigma-Aldrich) (LPS) (500 ng/mL) and IFNγ, 25 ng/mL (Miltenyi Biotec) or M2 with IL-4 (Miltenyi Biotec) 25 ng/mL; for 24 h at 37 °C, 5% CO₂. Different treatments were performed blinded to the mouse genotypes.