Impact of secondary structure of toll-like receptor 9 agonists on interferon alpha induction.

Oligodeoxynucleotides containing a CpG motif and double- or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-alpha) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-alpha-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonist's ability to induce immune responses, including IFN-alpha induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes. A third agonist showed a biphasic melting transition in the heating cycle and a monophasic melting transition with lower hyperchromicity during the cooling cycle, suggesting the formation of both intra- and intermolecular duplexes. All three agonists induced the production of Th1-type cytokines and chemokines, including high levels of IFN-alpha, in human peripheral blood mononuclear cell and plasmacytoid dendritic cell cultures. Subcutaneous administration of the two intermolecular duplex-forming agonists, but not the intramolecular duplex-forming agonist, induced cytokine secretion in mice. In nonhuman primates, the two agonists that formed intermolecular duplexes induced IFN-alpha and IP-10 secretion. On the contrary, the agonist that formed an intramolecular duplex induced only low levels of cytokines in nonhuman primates, suggesting that this type of structure formation is less immunostimulatory in vivo than the other structure. Taken together, the present results suggest that oligonucleotide-based agonists of TLR9 that form intermolecular duplexes induce potent immune responses in vivo.

Constitutive regulation of CYP1B1 by the aryl hydrocarbon receptor (AhR) in pre-malignant and malignant mammary tissue.

The aryl hydrocarbon receptor (AhR) is a receptor/transcription factor which regulates cytochrome P450 (CYP) gene transcription and which is activated by environmental carcinogens, some of which are associated with increased breast cancer risk. Here, we show that the AhR is over-expressed and constitutively active in human and rodent mammary tumors, suggesting its ongoing contribution to tumorigenesis regardless of tumor etiology. AhR regulation of CYP1A1 and CYP1B1 was studied to determine if constitutively active AhR effects the same transcriptional outcomes as environmental chemical-activated AhR. Elevated AhR and CYP1B1 but not CYP1A1 before tumor formation in a rat model of mammary tumorigenesis suggested differential CYP1B1 regulation by a constitutively active AhR. This hypothesis was tested with human mammary gland cell lines which hyper-express AhR and CYP1B1 but which express little or no CYP1A1. CYP1B1 expression was diminished by repression of AhR activity or by AhR knockdown, demonstrating AhR control of basal CYP1B1 levels. ChIP assays demonstrated constitutive AhR binding to both CYP1A1 and CYP1B1 promoters, demonstrating that differential CYP1A1 and CYP1B1 regulation by constitutively active AhR does not result from different amounts of promoter-bound AhR. While increasing AhR binding to both CYP1A1 and CYP1B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA in both a malignant and non-malignant line but increased only CYP1B1 mRNA in the malignant line, again demonstrating that the level of promoter binding does not necessarily correlate with gene mRNA levels. These studies suggest that constitutively active AhR mediates different molecular outcomes than environmental chemical-activated AhR, and further implicate the AhR in mammary tumorigenesis.

Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8.

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications. The present study describes one pool of RNA compounds, referred to as stabilized immune modulatory RNA (SIMRA) compounds, in which two RNA segments are attached through their 3' ends. SIMRA compounds showed greater stability in human serum compared with linear RNA and activated human TLR8, but not TLR7, in HEK293 cells without using lipid carriers. Interestingly, another set of SIMRA compounds containing 7-deazaguanosine substituted for natural guanosine activated human TLR7 and TLR8. Additionally, TLR7- and TLR8-activating compounds, but not the compounds that activated only TLR8, stimulated mouse immune cells in vitro and in vivo and produced dose-dependent T helper 1-type cytokines. Both types of compounds activated human peripheral blood mononuclear cells, but only TLR7- and TLR8-activating compounds activated plasmacytoid dendritic cells and produced high levels of IFN-alpha. In monkeys, s.c. administration of both types of SIMRA compounds induced transient changes in peripheral blood monocytes and neutrophils, and activated T lymphocytes, monocytes, and NK cells. Both types of compounds induced IFN-gamma-inducible protein 10, but only the 7-deazaguanosine-containing compound that activated both TLR7 and TLR8 induced IFN-alpha in monkeys. This is a comprehensive study of RNA-based compounds containing structures and synthetic stimulatory motifs in mouse, monkey, and human systems without using lipid carriers.