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Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists.

Birmachu W, Gleason RM, Bulbulian BJ, Riter CL, Vasilakos JP, Lipson KE, Nikolsky Y - BMC Immunol. (2007)

Bottom Line: A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC.Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted.The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology, 3M Pharmaceuticals, St Paul, Minnesota, USA. wbirmachu@comcast.net

ABSTRACT

Background: Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011.

Results: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNalpha subtypes, IFNalpha2, alpha5, alpha6, alpha8, alpha1/13, alpha10, alpha14, alpha16, alpha17, alpha21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.

Conclusion: Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.

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Purity of pDC preparations as determined by flow cytometry. Representative histograms for partially purified pDC (A) and flow purified pDC (B). pDC were separated from PBMC using anti BDCA4 microbeads. The partially purified cells were then labeled with CD123-APC, BDCA2-FITC, and CD11c-PE antibodies and sorted on a FACSAria flow cytometer to a final purity of > 99%. Numbers in the top, left hand corner of the dot plots indicate the percentage of CD123+, BDCA-2+, CD11c- cells.
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Figure 1: Purity of pDC preparations as determined by flow cytometry. Representative histograms for partially purified pDC (A) and flow purified pDC (B). pDC were separated from PBMC using anti BDCA4 microbeads. The partially purified cells were then labeled with CD123-APC, BDCA2-FITC, and CD11c-PE antibodies and sorted on a FACSAria flow cytometer to a final purity of > 99%. Numbers in the top, left hand corner of the dot plots indicate the percentage of CD123+, BDCA-2+, CD11c- cells.

Mentions: Even though pDC are reported to express high levels of TLR7, other cells in PBMCs, such as monocytes also express TLR7 [10] and can contribute to TLR7-mediated events. Therefore, in order to determine TLR7-mediated gene expression specific to pDC, high purity pDC preparations are necessary. Positive cell selection using BDCA4 antibody resulted in pDC enriched preparations with variable purity ranging from 40% to 80%. Further purification using flow cytometry after staining cells with APC-labeled anti-CD123 antibody and FITC-labeled BDCA2 antibody resulted in pDC preparations that were greater than 99% pure. Figure 1A and 1B show representative histograms from flow cytometer analysis of pDC-enriched (67%) preparation (Figure 1A) and a 99% pure pDC preparation (Figure 1B) after flow purification, as evaluated by the percentage of CD123+, BDCA2+, CD11c- cells.


Transcriptional networks in plasmacytoid dendritic cells stimulated with synthetic TLR 7 agonists.

Birmachu W, Gleason RM, Bulbulian BJ, Riter CL, Vasilakos JP, Lipson KE, Nikolsky Y - BMC Immunol. (2007)

Purity of pDC preparations as determined by flow cytometry. Representative histograms for partially purified pDC (A) and flow purified pDC (B). pDC were separated from PBMC using anti BDCA4 microbeads. The partially purified cells were then labeled with CD123-APC, BDCA2-FITC, and CD11c-PE antibodies and sorted on a FACSAria flow cytometer to a final purity of > 99%. Numbers in the top, left hand corner of the dot plots indicate the percentage of CD123+, BDCA-2+, CD11c- cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175514&req=5

Figure 1: Purity of pDC preparations as determined by flow cytometry. Representative histograms for partially purified pDC (A) and flow purified pDC (B). pDC were separated from PBMC using anti BDCA4 microbeads. The partially purified cells were then labeled with CD123-APC, BDCA2-FITC, and CD11c-PE antibodies and sorted on a FACSAria flow cytometer to a final purity of > 99%. Numbers in the top, left hand corner of the dot plots indicate the percentage of CD123+, BDCA-2+, CD11c- cells.
Mentions: Even though pDC are reported to express high levels of TLR7, other cells in PBMCs, such as monocytes also express TLR7 [10] and can contribute to TLR7-mediated events. Therefore, in order to determine TLR7-mediated gene expression specific to pDC, high purity pDC preparations are necessary. Positive cell selection using BDCA4 antibody resulted in pDC enriched preparations with variable purity ranging from 40% to 80%. Further purification using flow cytometry after staining cells with APC-labeled anti-CD123 antibody and FITC-labeled BDCA2 antibody resulted in pDC preparations that were greater than 99% pure. Figure 1A and 1B show representative histograms from flow cytometer analysis of pDC-enriched (67%) preparation (Figure 1A) and a 99% pure pDC preparation (Figure 1B) after flow purification, as evaluated by the percentage of CD123+, BDCA2+, CD11c- cells.

Bottom Line: A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC.Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted.The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology, 3M Pharmaceuticals, St Paul, Minnesota, USA. wbirmachu@comcast.net

ABSTRACT

Background: Plasmacytoid Dendritic Cells (pDC) comprise approximately 0.2 to 0.8% of the blood mononuclear cells and are the primary type 1 interferon (IFN), producing cells, secreting high levels of IFN in response to viral infections. Plasmacytoid dendritic cells express predominantly TLRs 7 & 9, making them responsive to ssRNA and CpG DNA. The objective of this study was to evaluate the molecular and cellular processes altered upon stimulation of pDC with synthetic TLR 7 and TLR 7/8 agonists. To this end, we evaluated changes in global gene expression upon stimulation of 99.9% pure human pDC with the TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011.

Results: Global gene expression was evaluated using the Affymetrix U133A GeneChip(R) and selected genes were confirmed using real time TaqMan(R) RTPCR. The gene expression profiles of the two agonists were similar indicating that changes in gene expression were solely due to stimulation through TLR7. Type 1 interferons were among the highest induced genes and included IFNB and multiple IFNalpha subtypes, IFNalpha2, alpha5, alpha6, alpha8, alpha1/13, alpha10, alpha14, alpha16, alpha17, alpha21. A large number of chemokines and co-stimulatory molecules as well as the chemokine receptor CCR7 were increased in expression indicating maturation and change in the migratory ability of pDC. Induction of an antiviral state was shown by the expression of several IFN-inducible genes with known anti-viral activity. Further analysis of the data using the pathway analysis tool MetaCore gave insight into molecular and cellular processes impacted. The analysis revealed transcription networks that show increased expression of signaling components in TLR7 and TLR3 pathways, and the cytosolic anti-viral pathway regulated by RIG1 and MDA5, suggestive of optimization of an antiviral state targeted towards RNA viruses. The analysis also revealed increased expression of a network of genes important for protein ISGylation as well as an anti-apoptotic and pro-survival gene expression program.

Conclusion: Thus this study demonstrates that as early as 4 hr post stimulation, synthetic TLR7 agonists induce a complex transcription network responsible for activating pDC for innate anti-viral immune responses with optimized responses towards RNA viruses, increased co-stimulatory capacity, and increased survival.

Show MeSH
Related in: MedlinePlus