Gene expression in primary human B cells sorted according to IL-10 secretion or not after 2 days of activation.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE49853 | GEO | 2014/03/24
SECONDARY ACCESSION(S): PRJNA215155
REPOSITORIES: GEO
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Project description:To determine characterize human B cells that express IL-10 on a molecular level, we compared the global gene expression of primary CD19pos B cells secreting IL-10 or not, upon activation with anti-CD40, IL-4 and CpG for 2 days. Human B cells from healthy donors were sorted according to CD19 expression by magnetic cell separation. The cells were stimulated at 2.5E06 cells/ml by activating anti-CD40 mAb (1 M-BM-5g/ml, clone 82111, RnD Systems), recombinant human IL-4 (10 ng/ml, Immunotools) and CpG2006 (3 M-BM-5g/ml) for 2 days following restimulation for 3 h with phorbol myristate acetate (PMA, 10 ng/ml) and ionomycin (1 M-BM-5g/ml). Staining of viable IL-10 secreting human CD19pos B cells was performed by the IL-10 capture assay according the manufacturers instructions (Miltenyi Biotec, n=2). Finally, the cells were sorted into IL-10posCD69pos and IL-10negCD69pos cells by flow cytometric cell sorting on a BD Aria II sorter (Becton Dickinson). Dead cells were excluded using propidium iodide. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to four HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.
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Project description:Transcriptional profiling of human peripheral B cell subsets sorted by flow cytometry based on the secretion of IL-10 in response to CpG2006 stimulation. Goal was to identify molecular markers to distinguish IL-10-producing B cells form non-IL-10-producing B cells. Two-condition experiment, non-IL-10-secreting B cells vs. IL-10-secreting B cells. Healthy donors. Biological replicates: 6
2013-03-04 | E-GEOD-35002 | biostudies-arrayexpress
Project description:Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10.
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Project description:Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10. Primary naM-CM-/ve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions. For activation, 0.25E06 naM-CM-/ve T cells were co-cultured with 0.75E06 MACSi Beads in 96-well flat bottom plates. MACSi Beads were coated with anti-CD3 and anti-CD28 (30 M-BM-5g of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to separate IL-10-secreting and non-secreting cells. Using a BD Aria or DIVA cell sorter (Becton Dickinson), living CD4+ IL-10-secreting and non-secreting cells, with (co-culture with Dll4; 'TH1Notch') or without ('TH1Control') activation of the Notch signaling pathway, were isolated. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The preparation for the hybridization to the chip was done using the GeneChip 3' IVT Express Kit. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 4 mouse genome 430 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.1.1., both Affymetrix. The data was analyzed using the original GCOS CHP-file Signals, Excel and AmiGO website.
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Project description:Mitochondrial electron transport chain (ETC) function modulates macrophage biology, however, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. Here we report that mutant mice with mitochondrial ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondrial CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Surprisingly, restoring mitochondrial respiration without generating superoxide using alternative oxidase (AOX) was not sufficient to reverse LPS-induced endotoxic shock susceptibility or restore IL-10 release. However, activation of protein kinase A (PKA) rescued IL-10 release in mitochondrial CIII-deficient BMDMs following LPS stimulation. Additionally, mitochondrial CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondrial CIII generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation
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Project description:Metabolism plays an essential role in shaping Th cell responses including the production of pro-inflammatory cytokines. However, its effect on the production of the anti-inflammatory cytokine IL-10 has not been investigated. We show here that the glucose analogue 2-deoxyglucose (2DG) specifically inhibited Th1 and Th2 cell differentiation and accompanying IL-10 production. In contrast, 2DG promoted IL-17A production by Th17 cells, even in the presence of IL-2 known to limit Th17 differentiation, whilst simultaneously also abrogating the production of IL-10. Rather than inhibiting glycolysis, 2DG was found to act through the inhibition of glycosylation, itself critical for IL-2Ra surface expression and downstream signaling, explaining the reciprocal effect of 2DG on the Th1 and Th2 versus Th17 differentiation. The essential role of IL-2 for IL-10 production by Th17 cells was confirmed by adding IL-2 or anti-IL-2, neither of which made the cells more pathogenic. The molecular mechanism of the IL-2 driven Th17 IL-10 production may be attributed to the transcription factor c-Maf, whose expression was significantly upregulated in the presence of IL-2 signaling. The overall RNA signature of sorted IL-10+ T cells driven by IL-2, be it IL-17A+ or IL-17A-, was dominated by protein synthesis whereas that of their IL-10- counterparts by cell cycle associated processes suggesting that IL-10 secreting Th17 cells trade off self-renewal for secretory capacity. Our study thus reveals a previously unappreciated, anti-inflammatory role for IL-2 in Th17 cell production of IL-10 and identifies a novel mechanism to limit Th17 pathogenicity.
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