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


CONCLUSIONS
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.
Birmachu
Woubalem
W
Pharmacology, 3M Pharmaceuticals, St Paul, Minnesota, USA. wbirmachu@comcast.net
Gleason
Raymond M
RM
Bulbulian
Barbara J
BJ
Riter
Christie L
CL
Vasilakos
John P
JP
Lipson
Kenneth E
KE
Nikolsky
Yuri
Y
D017951:Antigen Presentation / Q000235:genetics; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D053263:Gene Regulatory Networks / Q000187:drug effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D051199:Toll-Like Receptor 7 / Q000819:agonists*; D051200:Toll-Like Receptor 8 / Q000819:agonists; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D051193:Toll-Like Receptors; D014157:Transcription Factors