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Similarities and differences between human activated macrophages I

ABSTRACT: The human AA dataset 5 includes the gene expression profile of monocyte-derived macrophages isolated from blood donor buffy coats and plated on TCP in medium containing 10% autologous serum. Cells were stimulated for 120 hours with 50 ng/ml of IL-4, 50 U/ml of IFN-γ plus 12.5 ng/ml of TNF-α, 50 ng/ml of M-CSF or 50 ng/ml of IL-10. RNA samples were isolated using TriPure isolation reagent (Roche) and the RNeasy Mini Kit-Qiagen Clean up method. The transcriptional profile was evaluated in two independent cell preparations, each derived from a different single donor using the HT12 V4 R2 bead chip arrays from Illumina.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49240 | GEO | 2013/07/26

SECONDARY ACCESSION(S): PRJNA213374

REPOSITORIES: GEO

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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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Similarities and differences between human activated macrophages I

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