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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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Related in: MedlinePlus

Increasing purity of pDC preparations correlate with decrease in the expression of TLR4, TLR5, and TLR8. (A) Change in expression of TLR4 (circles), TLR5 (squares), and TLR8 (triangles), and (B) TLR1 (diamonds), TLR6 (squares), TLR7 (triangles), TLR9 (circles), and TLR10 (stars) with increasing purity of pDC preparation. TLR expression was determined by real time RTPCR analysis. Relative copy number was calculated with respect to 2 ng cDNA after normalization to expression levels of GAPDH in each sample.
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Figure 2: Increasing purity of pDC preparations correlate with decrease in the expression of TLR4, TLR5, and TLR8. (A) Change in expression of TLR4 (circles), TLR5 (squares), and TLR8 (triangles), and (B) TLR1 (diamonds), TLR6 (squares), TLR7 (triangles), TLR9 (circles), and TLR10 (stars) with increasing purity of pDC preparation. TLR expression was determined by real time RTPCR analysis. Relative copy number was calculated with respect to 2 ng cDNA after normalization to expression levels of GAPDH in each sample.

Mentions: Purity of pDC preparations were also followed by monitoring the expression of various TLRs using real time RTPCR. Figure 2A and 2B show the expression of various TLRs in preparations of varying pDC purity. Figure 2A shows that as the purity of the pDC preparation increased, the expression of TLR4, TLR5 and TLR8 decreased. Conversely, the expression of TLR7, TLR9, and TLR10 increased up to a purity of 82% then decreased somewhat (Figure 2B), potentially indicating loss of a cell type which may be enriched in expression of these TLRs. Alternatively the variability in the expression of these TLRs for preparation > 82% pDC purity may be explained by donor-to-donor variability in expression. The expression of TLR1 and TLR6 remained somewhat constant.


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)

Increasing purity of pDC preparations correlate with decrease in the expression of TLR4, TLR5, and TLR8. (A) Change in expression of TLR4 (circles), TLR5 (squares), and TLR8 (triangles), and (B) TLR1 (diamonds), TLR6 (squares), TLR7 (triangles), TLR9 (circles), and TLR10 (stars) with increasing purity of pDC preparation. TLR expression was determined by real time RTPCR analysis. Relative copy number was calculated with respect to 2 ng cDNA after normalization to expression levels of GAPDH in each sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Increasing purity of pDC preparations correlate with decrease in the expression of TLR4, TLR5, and TLR8. (A) Change in expression of TLR4 (circles), TLR5 (squares), and TLR8 (triangles), and (B) TLR1 (diamonds), TLR6 (squares), TLR7 (triangles), TLR9 (circles), and TLR10 (stars) with increasing purity of pDC preparation. TLR expression was determined by real time RTPCR analysis. Relative copy number was calculated with respect to 2 ng cDNA after normalization to expression levels of GAPDH in each sample.
Mentions: Purity of pDC preparations were also followed by monitoring the expression of various TLRs using real time RTPCR. Figure 2A and 2B show the expression of various TLRs in preparations of varying pDC purity. Figure 2A shows that as the purity of the pDC preparation increased, the expression of TLR4, TLR5 and TLR8 decreased. Conversely, the expression of TLR7, TLR9, and TLR10 increased up to a purity of 82% then decreased somewhat (Figure 2B), potentially indicating loss of a cell type which may be enriched in expression of these TLRs. Alternatively the variability in the expression of these TLRs for preparation > 82% pDC purity may be explained by donor-to-donor variability in expression. The expression of TLR1 and TLR6 remained somewhat constant.

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