Abstract
BackgroundPlasmacytoid 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.ResultsGlobal gene expression was evaluated using the Affymetrix U133A GeneChip® and selected genes were confirmed using real time TaqMan® 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 IFNα subtypes, IFNα2, α5, α6, α8, α1/13, α10, α14, α16, α17, α21. 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.ConclusionThus 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.
Highlights
Plasmacytoid Dendritic Cells 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
We show that stimulation of Plasmacytoid Dendritic Cells (pDC) through TLR7 results in the induction of IFNB and the majority IFNA subtypes induced by viruses such as herpes simplex virus type 1(HSV-1) and Sendai virus (SV) [26] and H3N2 flu virus [27]
We have made the first comprehensive global gene expression analysis of high purity pDC stimulated with TLR7 agonists
Summary
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. 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 these cytokines in response to viral infections [5] and stimulation with TLR7 and TLR9 agonists [6,7]. Type I IFNs are clinically important cytokines and are used for anti-viral [8] and anti-cancer therapy [9] In addition to their direct anti-viral and anti-proliferative affects, type I interferons are important in bridging the innate and adaptive immune responses. Type 1 interferons have been shown to increase the expression of MHC class I and II, enhance co-stimulatory marker expression on DC, modulate immunoglobulin production, synergize with IL-12 to enhance IFN-γ production, and augmente NK and CTL responses [2,5,3]
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