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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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Cluster analysis of 680 genes regulated in expression by 3M-852A and 3M-011. Gene expression analysis was performed at 4 hr post-stimulation of flow-purified pDC with 3M-852A and 3M-011 using the Affymetrix GeneChip U133A as described in Methods. D1 and D2, designate two different donors for pDC preparation in which the pDC purity was > 99%. Global gene expression was determined using the Affymetrix U133A GeneChip. Two-way hierarchical clustering was performed as described in the Methods section, using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. The log2 fold change values were used for the analysis. Insert bar chart shows the expression change scale with red, green, and white signifying increased, decreased, and unchanged expression, respectively. Expression changes were evaluated with respect to vehicle stimulated pDC from the same donor. Expression changes for the 680 genes are documented in Additional File 1.
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Figure 4: Cluster analysis of 680 genes regulated in expression by 3M-852A and 3M-011. Gene expression analysis was performed at 4 hr post-stimulation of flow-purified pDC with 3M-852A and 3M-011 using the Affymetrix GeneChip U133A as described in Methods. D1 and D2, designate two different donors for pDC preparation in which the pDC purity was > 99%. Global gene expression was determined using the Affymetrix U133A GeneChip. Two-way hierarchical clustering was performed as described in the Methods section, using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. The log2 fold change values were used for the analysis. Insert bar chart shows the expression change scale with red, green, and white signifying increased, decreased, and unchanged expression, respectively. Expression changes were evaluated with respect to vehicle stimulated pDC from the same donor. Expression changes for the 680 genes are documented in Additional File 1.

Mentions: In order to determine TLR7-mediated global gene expression changes, Affymetrix GeneChip analysis was performed after stimulation of pDC with TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. Stimulation of pDC for 4 hours resulted in changes in expression of 680 genes with consistent expression changes across replicates for at least one of the compounds tested (see Additional File 1). Out of the 680 genes, 430 genes were increased in expression and 250 genes were decreased in expression due to stimulation with at least one TLR agonist in all donors tested. Figure 4 shows a two way hierarchical clustering of the 680 genes altered in expression. Samples clustered primarily according to donor rather than compound indicating that the compounds exhibit an overall similar gene expression profile, consistent with gene expression changes regulated through activation of TLR7. Subtle differences are apparent in the response of the same donor to the different agonists, primarily in the magnitude of change in expression.


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)

Cluster analysis of 680 genes regulated in expression by 3M-852A and 3M-011. Gene expression analysis was performed at 4 hr post-stimulation of flow-purified pDC with 3M-852A and 3M-011 using the Affymetrix GeneChip U133A as described in Methods. D1 and D2, designate two different donors for pDC preparation in which the pDC purity was > 99%. Global gene expression was determined using the Affymetrix U133A GeneChip. Two-way hierarchical clustering was performed as described in the Methods section, using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. The log2 fold change values were used for the analysis. Insert bar chart shows the expression change scale with red, green, and white signifying increased, decreased, and unchanged expression, respectively. Expression changes were evaluated with respect to vehicle stimulated pDC from the same donor. Expression changes for the 680 genes are documented in Additional File 1.
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Related In: Results  -  Collection

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Figure 4: Cluster analysis of 680 genes regulated in expression by 3M-852A and 3M-011. Gene expression analysis was performed at 4 hr post-stimulation of flow-purified pDC with 3M-852A and 3M-011 using the Affymetrix GeneChip U133A as described in Methods. D1 and D2, designate two different donors for pDC preparation in which the pDC purity was > 99%. Global gene expression was determined using the Affymetrix U133A GeneChip. Two-way hierarchical clustering was performed as described in the Methods section, using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. The log2 fold change values were used for the analysis. Insert bar chart shows the expression change scale with red, green, and white signifying increased, decreased, and unchanged expression, respectively. Expression changes were evaluated with respect to vehicle stimulated pDC from the same donor. Expression changes for the 680 genes are documented in Additional File 1.
Mentions: In order to determine TLR7-mediated global gene expression changes, Affymetrix GeneChip analysis was performed after stimulation of pDC with TLR7 selective agonists 3M-852A, and the TLR7/8 agonist 3M-011. Stimulation of pDC for 4 hours resulted in changes in expression of 680 genes with consistent expression changes across replicates for at least one of the compounds tested (see Additional File 1). Out of the 680 genes, 430 genes were increased in expression and 250 genes were decreased in expression due to stimulation with at least one TLR agonist in all donors tested. Figure 4 shows a two way hierarchical clustering of the 680 genes altered in expression. Samples clustered primarily according to donor rather than compound indicating that the compounds exhibit an overall similar gene expression profile, consistent with gene expression changes regulated through activation of TLR7. Subtle differences are apparent in the response of the same donor to the different agonists, primarily in the magnitude of change in expression.

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