ABSTRACT
TLR7 and TLR8 recognize RNA from pathogens and lead to subsequent immune stimulation. Here we demonstrate that a single naturally occurring 2'-O-methylation within a synthetic 18s rRNA derived RNA sequence prevents IFN-α production, however secretion of proinflammatory cytokines such as IL-6 is not impaired. By analysing TLR-deficient plasmacytoid dendritic cells and performing HEK293 genetic complementation assays we could demonstrate that the single 2'-O-methylation containing RNA still activated TLR8 but not TLR7. Therefore this specific 2'-O-ribose methylation in rRNA converts a TLR7/TLR8 ligand to an exclusively TLR8-specific ligand. Interestingly, other modifications at this position such as 2'-O-deoxy or 2'-fluoro had no strong modulating effect on TLR7 or TLR8 activation suggesting an important role of 2'-O-methylation for shaping differential TLR7 or TLR8 activation.
Subject(s)
RNA/chemistry , RNA/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Animals , HEK293 Cells , Humans , Interferon-alpha/biosynthesis , Interleukin-6/metabolism , Ligands , Methylation , Mice , RNA, Ribosomal, 18S/metabolism , Substrate SpecificityABSTRACT
Small interfering RNA (siRNA) is widely used to modulate gene expression, but its potential induction of cytokines via Toll-like receptors (TLR) strongly impairs its use. Selective 2'-O-ribose methylation of sense or antisense strand can abolish the immunostimulatory potential, however, no universal approach is available and the mechanism of action is unknown. Here, we demonstrate that alternating 2'-O-ribose methylation of the sense strand within a siRNA duplex specific for eGFP or beta(2)-microglobulin destroyed its immunostimulatory function in primary immune cells, while reduction in target gene expression was functional. Furthermore, addition of siRNA containing a 2'-O-ribose-methylated sense strand to immunostimulatory siRNA abolished its stimulatory activity and binding studies revealed that 2'-O-ribose-methylated RNA bound stronger to TLR7 than unmodified RNA. We conclude that 2'-O-ribose methylation acts as inhibitor for RNA-driven immune stimulation via TLR7 and recommend alternating 2'-O-ribose methylation of the sense strand as a universal approach for the generation of non-immunostimulatory siRNA.
Subject(s)
Dendritic Cells/metabolism , RNA, Small Interfering/immunology , Toll-Like Receptor 7/immunology , Animals , Cell Line, Transformed , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/pathology , Gene Expression Regulation , Humans , Immunization , Methylation , Mice , Mice, Knockout , Mice, Transgenic , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Ribose/analogs & derivatives , Ribose/chemistry , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolismABSTRACT
The mammalian immune system senses pathogens through pattern recognition receptors (PRR) and responds with activation. Toll-like receptors (TLRs) that are expressed on immune and non-immune cells play a critical role in this process. As part of the innate immune response, TLRs lead to cellular activation and cytokine production with subsequent initiation of an adaptive immune response. TLR7-9 recognize single-stranded RNA, nucleoside analogs and single-stranded CpG-DNA, respectively, and their activation initiates the immune response against viruses and bacteria. Furthermore, the stimulation of these TLRs may be exploited for adjuvant therapy, vaccination and anti-tumor responses. However, a role in the generation or perpetuation of autoimmune diseases such as systemic lupus erythematosus (SLE) has also been suggested.
Subject(s)
Nucleic Acids/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 9/metabolism , Amino Acid Sequence , Animals , CpG Islands , DNA, Single-Stranded/chemistry , Humans , Interleukin-6/biosynthesis , Mice , Models, Biological , Molecular Sequence Data , RNA/chemistry , Sequence Homology, Amino Acid , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 9/chemistryABSTRACT
The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9-/- mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9-/- and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9-/- mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.
Subject(s)
Gammaherpesvirinae , Herpesviridae Infections/immunology , Toll-Like Receptor 9/physiology , Animals , Dendritic Cells/immunology , Humans , Interferon-alpha/metabolism , Interleukin-12/metabolism , Interleukin-6/metabolism , Mice , Mice, Mutant Strains , Toll-Like Receptor 9/geneticsABSTRACT
Allergic diseases prevalence rates have increased dramatically over the last 50 years in developed countries and one explanation might be that modern practices in public health lead to a decreased exposure towards pathogens resulting in a misguided immune response. Recently, it has become evident that immune responses against pathogens are initiated by Toll-like receptors (TLRs) that recognize a variety of structures derived from viruses, bacteria, fungi or protozoa. In this review we will discuss TLR ligands, TLR signaling in regard to Th1 and Th2 immune responses, their involvement in the development and their therapeutic potential in treatment of allergic disease.