Glucocorticoid transcriptome in macrophages upon activation [RNA-seq]

Glucocorticoids (GCs) are lifesaving medicines prescribed to treat inflammatory diseases. GCs act on numerous cell types and tissue of the body, however, one of the most important effects are their ability to suppress the immune system. Synthetic glucocorticoids, exemplified by dexamethasone (Dex) and prednisone, rank among the most widely prescribed anti-inflammatory and immunomodulatory drugs globally. The therapeutic efficacy of glucocorticoids extents across a broad spectrum of immune diseases, including conditions like acute and chronic inflammation. The action of the glucocorticoids in immune cells is mediated by the glucocorticoid receptor (here after GR; encoded by Nr3c1 gene) that is a member of the nuclear receptor superfamily of ligand dependent transcription factors. Macrophages emerge as central targets for the anti-inflammatory actions of glucocorticoids during periods of inflammation. Macrophages play a pivotal role during hyperinflammation and cytokine storm, two processes intricately linked to NLRP3 inflammasome activation in human diseases. Upon activation macrophages undergo a process of transcriptional reprogramming to resolve inflammation and restore homeostasis. To elucidate how glucocorticoids impact the transcriptome of macrophages upon activation, RNA-seq was performed on RNA isolated from macrophages unpolarized (M0) and polarized to M1 and M2 profile from WT and GRKO treated with vehicle and Dex. In addition, M0 and LPS M1-like macrophages were treated with vehicle and Dex and then analyzed by Cut&Tag. Overall design: Bone marrow derived macrophages (BMDMs) were obtained from 8- to 12-wk-old WT (GR-Flox) and GRKO mice. For polarization into M1-like and M2-like, BMDMs unpolarized (M0) were stimulated for 24 hours with 100 ng/mL of LPS for M1-like, and 10 ng/mL of murine recombinant IL-4 for M2-like, all in 10% charcoal-stripped serum medium. M0, M1-like and M2-like macrophages from WT and GRKO were stimulated with vehicle or 100nM Dex for 4 hours for gene expression. Total RNA was isolated from macrophages using Qiagen RNeasy Mini kit. A TruSeq RNA kit (Illumina, San Diego, CA) was utilized to prepare the poly(A)-enriched RNA-seq libraries that were sequenced on the Illumina NovaSeq 6000 in a 75-base paired-end mode according to the manufacturer's protocol. For Cut&Tag experiments, BMDM from vehicle, LPS treated for 4 hours; 4 hours Dex after LPS-primed and 4 hours LPS and Dex as a co-treatment were harvested. Cells were treated, harvested, and subjected to CUT&TAG analysis using a modified published protocol [Kaya-Okur et al., 2019]. Trypsinized cells were washed once with PBS and incubated with NE nuclear extraction buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitors (Roche cOmplete) for 10 min on ice. Samples were centrifuged for 3 min at 600g, nuclei pellet resuspended in NE buffer supplemented with 0.1 % formaldehyde and incubated at room temperature for 2 minutes followed by quench with 0.125 M glycine. Following formaldehyde cross-link, nuclei were re-centrifuged, resuspended in fresh NE buffer and counted with trypan blue dye using Bio-Rad TC20 Automated Cell Counter. 100,000 total nuclei [90,000 nuclei from experimental sample with 10,000 human spike-in nuclei] were incubated with activated concanavalin A-coated (ConA) magnetic beads (EpiCypher) in 0.2 ml thin-walled tubes for 10 min at room temperature. Nuclei-bound ConA beads were resuspended in antibody incubation buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Digitonin, 2 mM EDTA, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor), 0.5 ug primary antibody added and samples incubated overnight at 4C with gently rotation. Samples were incubated with 0.5 ug species specific secondary antibody for 30 min at room temperature with gentle rotation followed by two washes with Digitonin150 wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Digitonin, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor) then incubation with pAG-Tn5 in Digitonin300-wash buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.01% Digitonin, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor) for 1h at room temperature. After pAG-Tn5 incubation, bound ConA beads were washed twice with Digitonin300-wash buffer then resuspended in Tagmentation buffer (Digitonin300 wash buffer containing 10 mM MgCl2) and incubated for 1h at 37C. Beads were then pelleted, resuspended in TAPS buffer (10 mM TAPS pH 8.5, 0.2 mM EDTA). TAPS buffer removed and beads carefully resuspended in SDS Release buffer (10 mM TAPS pH 8.5, 0.1% SDS) and incubated for 1 h at 58 C. Following incubation, SDS Quench buffer (0.67% Triton X-100) was added to each sample along with 2 µl of a universal i5 and a uniquely barcoded i7 primers (from 10 µM stocks). Equal volume (25 ul) NEBNext High-Fidelity 2x PCR Master mix (M0541, New England Biolabs) was added to each sample and mixed by gentle pipetting. Samples subjected to DNA amplification using a thermocycler and the CUT&TAG-specific PCR cycling parameters: 58C for 5 min; 72C for 5 min; 98C for 45 sec; with 19 cycles of 98C for 15 sec and 60C for 10 sec; final extension at 72 °C for 1 min; and hold at 4C. DNA clean-up performed by adding 1.3x AMPure XP beads (A63880, Beckman Coulter) to each sample, allowing libraries to incubated with beads for 10 min at room temperature, followed by two washes with 80% ethanol, then elution in 15 µl of 10 mM Tris pH 8.0. While on magnetic stand, supernatant was carefully taken and transferred to a fresh tube. Library size distribution determined by Agilent TapeStation 4200 (Agilent Technologies) and libraries mixed to yield equal representation prior to paired-end (2 x 50 bp) Illumina sequencing by NIEHS The Epigenomics and DNA Sequencing Core Facility.