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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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Stimulation of pDC with 3M-852A and 3M-011 results in increased expression of a large number of chemokines. Protein-interaction network was generated for expression changes in pDC 4 hr post stimulation with 3M-852A and 3M-011. The network was generated using a list of genes in the GO function classification of chemokines and the shortest path algorithm. The network summarizes interactions between chemokines increased in expression and their respective receptors. Green arrows indicate binding interactions or covalent modifications that result in activation or increased transcription. Red arrows indicate binding interactions or covalent modifications that result in inhibition of activity or suppression of transcription.
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Figure 6: Stimulation of pDC with 3M-852A and 3M-011 results in increased expression of a large number of chemokines. Protein-interaction network was generated for expression changes in pDC 4 hr post stimulation with 3M-852A and 3M-011. The network was generated using a list of genes in the GO function classification of chemokines and the shortest path algorithm. The network summarizes interactions between chemokines increased in expression and their respective receptors. Green arrows indicate binding interactions or covalent modifications that result in activation or increased transcription. Red arrows indicate binding interactions or covalent modifications that result in inhibition of activity or suppression of transcription.

Mentions: In addition to the genes regulated by the transcription factors illustrated in Figure 5, analysis in MetaCore showed that a large number of genes that are increased in expression are known to be regulated by the transcription factors CREB1, c-Myc and p53. Four hr. post-stimulation with TLR7 agonists, one would expect transcriptional regulation through other secondary pathways activated by the genes induced in the primary TLR7-mediated pathways. In addition to type 1 interferons, a large number of other cytokine and chemokine genes were increased in expression including the chemokines, CCL5, IL6, IL8, MIP1A, MIP1B, and the cytokines TNFA and TNFB. In fact, at 4 hr post-stimulation, significant amounts the proteins for IL6, IL8, TNFα, MIP-1α and, MIP-1β were produced. These cytokines can act in an autocrine fashion to activate the IL8 receptors, CCR1 and CCR3, which are linked to G proteins that can in turn activate various transcription factors. Figure 6 shows a protein interaction network illustrating the various chemokines induced in pDC and their respective G-protein-coupled receptors. Potential activation of the transcription factors CREB1, c-Myc and NFkB via the chemokine receptors CCR1, CCR3 and the IL8 receptors is depicted in Figure 7. Chemokine receptors activate various guanine nucleotide proteins (G proteins) including the beta/gamma subunits which result in the activation of the phospholipase beta (PLCB) and Phosphatidyl inositol kinase (PI3K) pathways [43,44]. These signaling pathways culminate in the activation of various transcription factors including CREB, cMyc, and NFkB. In addition to the chemokines for these receptors, one of the G-proteins, GNG11 (guanine nucleotide binding protein gamma 11) is also increased 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)

Stimulation of pDC with 3M-852A and 3M-011 results in increased expression of a large number of chemokines. Protein-interaction network was generated for expression changes in pDC 4 hr post stimulation with 3M-852A and 3M-011. The network was generated using a list of genes in the GO function classification of chemokines and the shortest path algorithm. The network summarizes interactions between chemokines increased in expression and their respective receptors. Green arrows indicate binding interactions or covalent modifications that result in activation or increased transcription. Red arrows indicate binding interactions or covalent modifications that result in inhibition of activity or suppression of transcription.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Stimulation of pDC with 3M-852A and 3M-011 results in increased expression of a large number of chemokines. Protein-interaction network was generated for expression changes in pDC 4 hr post stimulation with 3M-852A and 3M-011. The network was generated using a list of genes in the GO function classification of chemokines and the shortest path algorithm. The network summarizes interactions between chemokines increased in expression and their respective receptors. Green arrows indicate binding interactions or covalent modifications that result in activation or increased transcription. Red arrows indicate binding interactions or covalent modifications that result in inhibition of activity or suppression of transcription.
Mentions: In addition to the genes regulated by the transcription factors illustrated in Figure 5, analysis in MetaCore showed that a large number of genes that are increased in expression are known to be regulated by the transcription factors CREB1, c-Myc and p53. Four hr. post-stimulation with TLR7 agonists, one would expect transcriptional regulation through other secondary pathways activated by the genes induced in the primary TLR7-mediated pathways. In addition to type 1 interferons, a large number of other cytokine and chemokine genes were increased in expression including the chemokines, CCL5, IL6, IL8, MIP1A, MIP1B, and the cytokines TNFA and TNFB. In fact, at 4 hr post-stimulation, significant amounts the proteins for IL6, IL8, TNFα, MIP-1α and, MIP-1β were produced. These cytokines can act in an autocrine fashion to activate the IL8 receptors, CCR1 and CCR3, which are linked to G proteins that can in turn activate various transcription factors. Figure 6 shows a protein interaction network illustrating the various chemokines induced in pDC and their respective G-protein-coupled receptors. Potential activation of the transcription factors CREB1, c-Myc and NFkB via the chemokine receptors CCR1, CCR3 and the IL8 receptors is depicted in Figure 7. Chemokine receptors activate various guanine nucleotide proteins (G proteins) including the beta/gamma subunits which result in the activation of the phospholipase beta (PLCB) and Phosphatidyl inositol kinase (PI3K) pathways [43,44]. These signaling pathways culminate in the activation of various transcription factors including CREB, cMyc, and NFkB. In addition to the chemokines for these receptors, one of the G-proteins, GNG11 (guanine nucleotide binding protein gamma 11) is also increased 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