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Deubiquitinase inhibitor compound C6 effect on cellular metabolism

ABSTRACT: 6.0 X 10^6 cells were plated in 10 cm Tissue Culture dishes and allowed to recover overnight at 37 degrees and 5% CO2. In the am, supernatant was removed, and 12 mL of fresh medium with either 2.5 micromolar Compound C6 or a v/v match of DMSO (vehicle control) was added to the cells. Cells were incubated at 37 degrees in 5% CO2 for 30 minutes. Cells were washed 3X with cold DPBS++ and 10 mL of medium was added to the cells with either 1.0 micromolar Compound C6 or v/v match of DMSO. Cells were incubated at 37 degrees and 5% CO2 for 7.0 hours. Cells were washed with 10 mL of 150 mM Ammonium Acetate, swirled, washed again with 5 mL, and immediately quenched with liquid nitrogen. Cells were then frozen at -80 degrees.

ORGANISM(S): Mouse Mus Musculus

TISSUE(S): Cultured Cells

SUBMITTER: Maureen Kachman

PROVIDER: ST000835 | MetabolomicsWorkbench | Wed Aug 02 00:00:00 BST 2017

REPOSITORIES: MetabolomicsWorkbench

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Project description:Purpose: To characterize the processes defining the end dendritic cell (DC) phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from human donors using Ficoll-Paque gradient centrifugation. CD14+ monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) and cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 µM 2-Mercaptoethanol, 1% Penicillin-Streptomycin at 37°C and 5% CO2. During the differentiation stage, complete medium was added 150 U/mL recombinant human IL-4 and 160 U/mL recombinant human GM-CSF to obtain monocyte-derived dendritic cells (moDCs). Medium was replaced after 3 days of culturing, and moDCs were used 6 days after the start of culture. The cells were on average >70% CD1a-positive, and with <3% CD14+CD16+ cells, based on flow cytometry, and responded with high levels of IL-12p70 production upon stimulation with LPS and IFN-y (mean IL-12p70 production of 4641.8 pg/mL; SD = 688.1 pg/mL). Cells were harvested and rested for 1 hour at 37°C and 5% CO2 before stimulation. At this point, moDCs were supplemented with complete medium containing TSLP (to final concentration of 25 ng/mL), IL-25 (25 ng/mL), TSLP+IL-25 (both 25 ng/mL), helminth PCF (100 μg dry matter/mL, tested in different doses in pre-study experiments), butyrate (2 mM), or succinate (0.5 mM and 2 mM, di-sodium succinate hexahydrate). Unstimulated cells were supplemented with complete medium alone. Immediately afterwards, medium containing Escherichia coli O26:B6 LPS (final concentration of 100 ng/mL) and IFN-γ (10 ng/mL) was added. Cells were stimulated for 4 or 20 h at 37°C, 5% CO2, and 95% humidity. Results: Our data show that helminth PCF and butyrate treatment suppress the Th1-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS+IFN-γ-matured DCs by downregulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in TLR4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, while transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were upregulated. Conclusion: Collectively, our results further our understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

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Deubiquitinase inhibitor compound C6 effect on cellular metabolism

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