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| Status |
Public on Sep 11, 2019 |
| Title |
Body fluid from the parasitic worm Ascaris suum inhibits broad-acting pro-inflammatory programs in dendritic cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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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| Overall design |
The transcriptional profile of stimulated human moDCs was evaluated, in triplicates, after 4 hours of stimulation. After stimulation for 20 hours, the cell-free culture supernatants were harvested and frozen at -80°C until further analysis. IL-12p70 levels were determined using ELISA in triplicates.
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| Contributor(s) |
Arora P, Moll JM, Andersen D, Workman CT, Williams AR, Kristiansen K, Brix S |
| Citation(s) |
31705653 |
| Submission date |
Sep 10, 2019 |
| Last update date |
Dec 11, 2019 |
| Contact name |
Pankaj Arora |
| E-mail(s) |
sbrix@dtu.dk, sbp@bio.dtu.dk, panar@dtu.dk, panar.arora@sund.ku.dk
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| Phone |
+45 45252784
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| Organization name |
Technical University of Denmark
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| Department |
Department of Biotechnology and Biomedicine
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| Street address |
Søltofts Plads - blg. 224 room 218, DK-2800 Kgs. Lyngby
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| City |
Lyngby |
| ZIP/Postal code |
2800 |
| Country |
Denmark |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA564834 |
| SRA |
SRP221281 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE137229_FPKM.gene.matrix_moDCs.txt.gz |
485.2 Kb |
(ftp)(http) |
TXT |
| GSE137229_ReadCount.gene.matrix_moDCs.txt.gz |
382.8 Kb |
(ftp)(http) |
TXT |
| GSE137229_Supplementary_data_sheet1_moDCs_Arora_et_al.__2019.xlsx |
22.5 Kb |
(ftp)(http) |
XLSX |
| GSE137229_TPM.gene.matrix_moDCs.txt.gz |
484.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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