Interleukin 4 (il 4)

Primary CD14+ monocytes were isolated from peripheral blood of healthy donors or from apheresis cones (NHS Blood & Transport Service) as described by Poole et al. [81 ]. Briefly, peripheral blood mononuclear cells (PBMC) were separated from whole blood by density-gradient centrifugation with Histopaque 1077 (Sigma, St. Louis, MO, USA), and from this, monocytes were isolated by magnetic-activated cell sorting (MACS) with CD14+ microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes were maintained in X-Vivo15 media (Lonza) at 37 °C in a 5% CO₂ atmosphere.
Monocytes were differentiated into monocyte-derive dendritic cells (moDCs) by the addition of 1000 U/mL IL-4 (Proteintech, Rosemont, IL, USA) and 1000 U/mL GM-CSF (granulocyte-macrophage colony-stimulating factor, Peprotech) for 5 days to generate immature DCs, followed by 2 days stimulation with 50 ng/mL LPS (lipopolysaccharide, Invivogen, Toulouse, France) to produce mature DCs. M2 macrophages were generated by the addition of 20 ng/mL IL-4 and 50 ng/mL M-CSF (macrophage colony-stimulating factor, Proteintech) for 6 days as described in Rostam et al. [82 (link)].
Cocultures to assess release of infectious virus were performed by addition of 3 × 10⁴ HFFs per well of a 96-well plate. Media was replaced with a mixture of 50% DMEM (supplemented with 10% FBS) and 50% X-Vivo 15.

Human monocyte-derived immature DCs were obtained from peripheral blood mononuclear cells (PBMC) extracted from healthy human ethylene diamine tetraacetic acid (EDTA) anticoagulated whole blood. PBMC were extracted using Ficoll (GE, Boston, MA, United States) and seeded at a density of 5 × 10⁵ cells/mL in 24-well plates. For the induction of cells, human granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL, Proteintech) and human interleukin-4 (IL-4, 50 ng/mL, Proteintech) were added to each well. On day 4, the cell culture supernatant of each group was co-cultured with immature DCs. On day 8, positive control groups were treated with 10 μg/mL LPS or 50 ng/mL TNF-α for 24 h.
On day 9, cell surface markers of DCs were evaluated by flow cytometry. DCs were stained on ice for 30 min with FITC-conjugated anti-CD11c and phycoerythrin (PE)-conjugated anti-CD83 (Biolegend, San Diego, CA, United States), and data were acquired and analyzed on a FACSCanto II flow cytometer (BD Biosciences).

RIPA lysis buffer was applied to extract total protein and nucleoprotein from rat testis tissue. According to the BCA Protein Determination Kit for protein quantification of each group, the protein was separated on a 10% SDS-PAGE gel and then transferred to the PVDF membranes. The membranes were sealed with a 5% skimmed milk solution at room temperature for 2 h, combined with the primary antibodies IL-1β (ProteinTech, 16806-1-AP), IL-6 (Abcam, ab229381), TNF-α (ProteinTech, 17590-1-AP), IL-10 (Abcam, ab271261), IL-4 (ProteinTech, 66142-1-Ig), MIS (Abcam, ab229212), ANXA2 (ProteinTech, 11256-1-AP), APP (ProteinTech, 25524-1-AP), SEMG1 (Abcam, ab139405), SEMG2 (ThermoFisher, PA5-88785), and β-actin (ProteinTech, 51067-2-AP/60008-1-Ig), and incubated overnight at 4°C. β-actin was used as internal reference l. TBST was used to wash the membranes three times. Then, the membranes were incubated with the secondary antibodies HRP goat anti-rat IgG (ProteinTech, SA00001-1) or HRP goat anti-rabbit IgG (ProteinTech, SA00001-2) 90 min at room temperature. After using ECL to develop color exposure, the Odyssey Infrared Imaging System was performed to detect protein bands.