Dexamethasone (dex)

S49 (Neo) are S49.1 mouse lymphoma cells stably transfected with a recombinant amphitropic retrovirus carrying a G418 (Neomycin) antibiotic resistance gene [20 (link)]. S49 Bcl-2 cells were additionally transfected with the Bcl-2 proto-oncogene [20 (link)]. Both cell lines were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum, 4.8 mM glutamine, 100 ug/ml streptomycin and 100 U/ml penicillin at 37 °C, 7% CO₂ atmosphere. Dexamethasone was purchased through Steraloids (Wilton, NH). Valinomycin, cereulide, and salinomycin were purchased from Thermo Fisher Scientific (Invitrogen; Hillsboro, OR), Focus Biomolecules (Plymouth Meeting, PA), and EMD Millipore Corporation (Seattle, WA), respectively.

Intestinal tissue explants (1 mm³ in size) were placed in 12-well culture plates (one explant per well) and cultured at 37 °C and 5% CO₂ in 1 ml serum-free DMEM medium supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, as previously described. For corticosteroid effect experiments, dexamethasone, prednisone or prednisolone (Steraloids, Inc. Newport, RI, USA) was added to the culture medium at a concentration of 100 nM as previously described. For recombinant human IL-33 (rhIL-33, R&D Systems, Minneapolis, MN, USA) effect experiments, 50 ng/ml was added to the culture medium as reported in in vitro and ex vivo model^{51 (link), 52 (link)}. After 24 hours ex vivo culture, supernatants were collected and stored at −80 °C until used for ST2 and cytokine measurement by ELISA (DuoSet, R&D Systems, Minneapolis, MN, USA) as previously above.

The A549 cell line (American Type Culture Collection; ATCC) was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 2 mM L-glutamine. The BEAS-2B cell line (ATCC) was grown in DMEM/F12 (Invitrogen) supplemented with 14 mM NaHCO₃, 2 mM L-glutamine and 10% FBS. Primary human bronchial epithelial (HBE) cells were isolated from non-transplantable normal human lungs obtained through a tissue retrieval service at the International Institute for the Advancement of Medicine (Edison, NJ), as previously described [25 (link), 26 (link)]. HBE cells were grown in submersion culture in bronchial epithelial cell growth medium (BEGM) (Lonza) containing all SingleQuots supplements (Lonza) except for hydrocortisone. All cells were incubated at 37 °C in 5% CO₂. Cell lines were passaged when 90–95% confluent and then cultured either in 6 or 12-well plates, as appropriate. Prior to experiments, all cells were incubated overnight in basal media that were serum- and additive-free. Budesonide (AstraZeneca, Sweden) and dexamethasone (Steraloids, Newport, RI) were dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich) as stocks of 10 mM. Final DMSO concentrations on cells were ≤ 0.1%.

Recombinant human IL1B, TNF and IFNG (R&D Systems, Minneapolis, MN) were dissolved in PBS containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO). Budesonide (AstraZeneca, Mölndal, Sweden), dexamethasone (Steraloids, Newport, RI), MG-132 (Millipore Sigma, St. Louis, MO), MG-262 (APExBIO, Houston, TX), PR-171 (Adooq BioScience, Irvine, CA), E-64 (APExBIO, Houston, TX), PS-1145, ML-120B (both Sigma-Aldrich) and Org34517 (gift from Chiesi Farmaceutici, Parma, Italy) were dissolved in dimethyl sulfoxide as stocks of 10 mM. Cycloheximide (Sigma-Aldrich) was dissolved in water at 10 mg/ml.

Female C57BL/6J mice randomized for experiments and male C57BL/6J mice were used as breeders for experiments (Jackson Laboratory; Bar Harbor, ME) and used between 12 and 14 weeks of age. All experiments of mice had ad libitum access to standard mouse chow and drinking water. Mice were exposed to a 12:12-h light/dark cycle. For pregnancy studies, male and female mice were set up for timed mating and the presence of a vaginal plug was marked at 0.5 dpc. Male mice were removed from the cage at 0.5 dpc and female mice were euthanized at either 14.5 dpc or PND1. Female mice not pregnant at 14.5 dpc were removed from the study. Glucocorticoid sensitivity was assessed in the livers of female mice at 14.5 dpc via injection of dexamethasone (10 μg/kg; I.P.) (Steraloids; Newport, RI). Four hours post I.P. injection, mice were killed and the livers were harvested. To reinstall GR during pregnancy, plugged mice were injected with 1 × 10¹¹vg in 25 μl via retro-orbital venous sinus injection. All animal experiments were performed in at least two independent cohorts of animals to ensure reproducibility. All animal experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee at the National Institute of Environmental Health Sciences, NIH.