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. 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.

Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo.

An etiological relationship between periodontitis, a significant oral health problem, and the anaerobe Porphyromonas gingivalis may be related to the expression of a variety of putative virulence factors. The objective of the experiments described here was to develop a quantitative reverse transcription polymerase chain reaction (QRT-PCR) method to examine P. gingivalis gene expression in human dental plaque from periodontitis subjects. PCR primers and probes for six target genes representing putative virulence factors were chosen and evaluated in vitro for specificity. A potential cross-reactivity level of only 10 copies/10(7) whole genomic equivalents was occasionally observed with non-P. gingivalis microbes. P. gingivalis cells stressed in vitro by a 5 degrees C temperature increase showed a rapid rise in the mRNA associated with the molecular chaperons (htpG, dnaK, groEL), SOD (sodA) and gingipain (rgp-1) genes. We examined the stability of bacterial RNA in plaque specimens and found no significant difference in the amount of RNA obtained before or after storage 3 months in a stabilizing buffer (p=0.786, t-test). Sixty-five percent of plaque samples obtained from two clinical locations contained P. gingivalis; there was a mean level of gene expression (fold increase) for all samples tested for groEL, dnaK, htpG, sodA, PG1431 and rgp-1 of 0.84+/-2.03 to 7.85+/-10.0. ANOVA showed that the levels of stress gene transcription for dnaK and htpG were significantly elevated (p<0.05) at diseased sites; groEL gene transcription approached statistically significant elevation (p=0.059). We found correlations between probing depth and increased transcription of groEL, htpG and rgp-1 and between attachment loss and htpG. When sorted by disease status, we detected correlations between disease status and elevated expression of dnaK and htpG.

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod.

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.