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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

Cytokines secreted by flow purified pDC 4 hr post-stimulation with TLR7 agonists. Cell culture supernatants collected after stimulation were evaluated for cytokine protein expression using a Luminex 25-Plex assay. (A) Secreted TNFα, IL8, IFNα, MIP-1α, MIP-1β and IL6. (B) secreted IL1β, IL2R, IL12P70, Rantes, GM-CSF, MCP-1, and IP-10. Dotted bars, vehicle stimulated samples; hatched bars, 3M-852A-stimulated samples and solid bars, 3M-011-stimulated samples. The results are expressed as mean + SD, n = 2 donors.
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Figure 3: Cytokines secreted by flow purified pDC 4 hr post-stimulation with TLR7 agonists. Cell culture supernatants collected after stimulation were evaluated for cytokine protein expression using a Luminex 25-Plex assay. (A) Secreted TNFα, IL8, IFNα, MIP-1α, MIP-1β and IL6. (B) secreted IL1β, IL2R, IL12P70, Rantes, GM-CSF, MCP-1, and IP-10. Dotted bars, vehicle stimulated samples; hatched bars, 3M-852A-stimulated samples and solid bars, 3M-011-stimulated samples. The results are expressed as mean + SD, n = 2 donors.

Mentions: In order to confirm the expression of some of the genes at the protein level, ELISA and Luminex multiplex protein assays were used to determine the expression of several secreted cytokines and chemokines. Supernatants were collected after stimulation of pDC for 4 hr. Of the 25 cytokines and chemokines tested, IFNα, TNFα, IL8, IL6, MIP-1α (CCL3), and MIP-1β (CCL4) proteins were induced at high levels > 1000 pg/ml. This is consistent with the high level of expression of these genes observed by Affymetrix and real time RTPCR analysis (Table 1). Figure 3A shows the induction of these cytokines for 3M-852A and 3M-011. In contrast, low levels of IL-1β, IL12p70, GM-CSF, Rantes (CCL5), MCP-1 (CCL2), and IP10 (CXCL10) proteins were detected (Figure 3B). The genes for IL1β, IL12, GM-CSF, and MCP1 (CCL2) were not increased in expression in the Affymetrix or real time RTPCR data. In contrast, high levels of gene expression (up to a fold change of 900) were observed for CXCL10 and CCL5. The seeming disparity may be due to the fact that data was collected at 4 hr post stimulation and the proteins for these genes may not have reached peak expression. Consistent with the gene expression data, the proteins IL-1RA, IL-2, IL-4, IL-5, IL-7, IL-10, IL-13, IL-15, IL17, IFNγ, MIG (CXCL9), and Eotaxin (CCL11) were not induced by TLR7 agonists in pDC 4 hr post stimulation. However, induction of these genes and proteins at a different time than 4 hr post stimulation can not be ruled out.


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)

Cytokines secreted by flow purified pDC 4 hr post-stimulation with TLR7 agonists. Cell culture supernatants collected after stimulation were evaluated for cytokine protein expression using a Luminex 25-Plex assay. (A) Secreted TNFα, IL8, IFNα, MIP-1α, MIP-1β and IL6. (B) secreted IL1β, IL2R, IL12P70, Rantes, GM-CSF, MCP-1, and IP-10. Dotted bars, vehicle stimulated samples; hatched bars, 3M-852A-stimulated samples and solid bars, 3M-011-stimulated samples. The results are expressed as mean + SD, n = 2 donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Cytokines secreted by flow purified pDC 4 hr post-stimulation with TLR7 agonists. Cell culture supernatants collected after stimulation were evaluated for cytokine protein expression using a Luminex 25-Plex assay. (A) Secreted TNFα, IL8, IFNα, MIP-1α, MIP-1β and IL6. (B) secreted IL1β, IL2R, IL12P70, Rantes, GM-CSF, MCP-1, and IP-10. Dotted bars, vehicle stimulated samples; hatched bars, 3M-852A-stimulated samples and solid bars, 3M-011-stimulated samples. The results are expressed as mean + SD, n = 2 donors.
Mentions: In order to confirm the expression of some of the genes at the protein level, ELISA and Luminex multiplex protein assays were used to determine the expression of several secreted cytokines and chemokines. Supernatants were collected after stimulation of pDC for 4 hr. Of the 25 cytokines and chemokines tested, IFNα, TNFα, IL8, IL6, MIP-1α (CCL3), and MIP-1β (CCL4) proteins were induced at high levels > 1000 pg/ml. This is consistent with the high level of expression of these genes observed by Affymetrix and real time RTPCR analysis (Table 1). Figure 3A shows the induction of these cytokines for 3M-852A and 3M-011. In contrast, low levels of IL-1β, IL12p70, GM-CSF, Rantes (CCL5), MCP-1 (CCL2), and IP10 (CXCL10) proteins were detected (Figure 3B). The genes for IL1β, IL12, GM-CSF, and MCP1 (CCL2) were not increased in expression in the Affymetrix or real time RTPCR data. In contrast, high levels of gene expression (up to a fold change of 900) were observed for CXCL10 and CCL5. The seeming disparity may be due to the fact that data was collected at 4 hr post stimulation and the proteins for these genes may not have reached peak expression. Consistent with the gene expression data, the proteins IL-1RA, IL-2, IL-4, IL-5, IL-7, IL-10, IL-13, IL-15, IL17, IFNγ, MIG (CXCL9), and Eotaxin (CCL11) were not induced by TLR7 agonists in pDC 4 hr post stimulation. However, induction of these genes and proteins at a different time than 4 hr post stimulation can not be ruled out.

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