ABSTRACT
Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.
Subject(s)
Cytokines/metabolism , Gene Silencing , Immunization/methods , Oligoribonucleotides/chemical synthesis , RNA, Small Interfering/genetics , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/metabolism , Amino Acid Motifs , Animals , Blood Buffy Coat , Cytokines/genetics , Humans , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Oligoribonucleotides/genetics , Phosphorothioate Oligonucleotides/chemistry , RNA, Small Interfering/metabolism , Uridine/chemistryABSTRACT
Toll-like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen-associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R-848) activates both TLR7 and TLR8. We demonstrate that co-incubation of R-848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R-848 on TLR8-expressing HEK 293 cells, but abolished TLR7-mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLR-transfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN-alpha secretion by TLR7-expressing plasmacytoid DC and toward IL-12, TNF-alpha and IFN-gamma secretion by TLR8-expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence-selective interaction between these receptors and synthetic phosphorothioate ODN.
Subject(s)
Oligodeoxyribonucleotides/pharmacology , Thionucleotides/pharmacology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Cell Line , Cells, Cultured , Guanosine/analogs & derivatives , Guanosine/pharmacology , Humans , Imidazoles/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Ligands , Models, Immunological , Toll-Like Receptor 7/antagonists & inhibitorsABSTRACT
Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODN are recognized by Toll-like receptor 9 (TLR9). Three classes of TLR9 agonists have been described: B-Class CpG ODN that induce strong B- and NK-cell activation and A-Class ODN that induce very high levels of IFN-alpha by plasmacytoid dendritic cells. The recently described C-Class ODN combine most efficiently properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN-alpha secretion comparable to A-Class ODN. Here, we investigate sequence requirements of C-Class ODN regarding optimal IFN-alpha secretion. Sequence as well as backbone modifications like 2'-O-methyl modifications especially in the 5' part of the ODN influence IFN-alpha-producing capacity. Kinetic studies on mRNA level for CD69, IFN-gamma, IP-10 and IL-18 by semi-quantitative PCR demonstrated differences in mRNA transcription for some cytokines suggesting different regulatory mechanisms for different ODN classes. High amounts of IP-10 mRNA and protein as well as up-regulation of IL-18 mRNA were observed especially for the A- and C-Classes. According to these data, C-Class ODN can be described as strong Th1 inducers with the stimulation of type I and II interferon as well as IP-10 production and strong NK activation. These characteristics can be availed to induce potent anti-tumor or anti-viral effects. Consequently, C-Class CpG ODN represent ideal drug candidates for anti-viral and/or anti-tumor therapy.
