Interleukin 4 (il 4)

BM cells were stimulated with GM-CSF (5 ng/ml, Tonbo Biosciences, San Diego, CA) for 6 days to induce BMDC (12 (link)). On day 6, non-adherent cells were collected for gene analysis. BMDM were generated with MΦ colony-stimulating factor (10 ng/ml, Sigma-Aldrich, St. Louis, MO). For generation of M2, BMDM were treated with IL-4 (10 ng/ml, Tonbo Biosciences, San Diego, CA ) and IL-13 (10 ng/ml, Tonbo Biosciences, San Diego, CA) for additional 3 days (16 (link), 17 ). DNBS (0.05%, Sigma-Aldrich, St. Louis, MO) and 1µM dexamethasone (Sigma-Aldrich, St. Louis, MO) were used to stimulate cells for 16 hrs as outlined in figure legends and in text.

Primary BM progenitor cells were isolated and differentiated from wildtype or AhR knockout (AhR^−/−) C57BL/6 mice, as described earlier (Vogel et al., 2013 (link)). Briefly, femurs were isolated under sterile conditions, and BM cells were extracted via a Roswell Park Memorial Institute (RPMI) media-loaded syringe. Cells were passed through a 30 μm cell strainer, and the supernatant was centrifuged for 5 min at 1000 × g. The supernatant was decanted, and the pellet was resuspended and cultured in RPMI medium. Cells were plated in 100 mm cell culture dishes. Differentiation of BM-derived macrophages was performed in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/mL; Tonbo Biosciences, San Diego, CA) whereas BMDCs were differentiated in the presence of GM-CSF (20 ng/mL) and IL-4 (20 ng/mL; Tonbo Biosciences). Differentiation of both macrophages and DCs occurred over 6 days and nonadherent BMDCs were purified (≥85–90%) as described previously (Vogel et al., 2013 (link)), and the appropriate media was replenished every 2 days. On day 6, cells were transferred to a 24 well plate at 5 × 10⁴ cells/well and treated in triplicate with PBS, PM (50 μg/mL), OVA (10 μg/mL), or OVA+PM for 24 hours followed by RNA isolation to analyze gene expression.