Toll-like receptor (TLR) 3 immune modulation by unformulated small interfering RNA or DNA and the role of CD14 (in TLR-mediated effects).

The Toll-like receptors (TLRs) 3, 7, 8 and 9 stimulate innate immune responses upon recognizing pathogen-derived nucleic acids. TLR3 is located on the cell surface and in cellular endosomes and recognizes double-stranded viral RNA or the synthetic mimic poly rI:rC. Recently, unformulated small interfering RNA (siRNA) has been reported as ligand for surface-expressed murine TLR3. Blockage of TLR3 is achieved by single-stranded DNA. We confirm and expand the observation that poly rI:rC-mediated TLR3 immune activation is blocked in a sequence-, length-, backbone- and CpG-dependent manner. However, human TLR3 is not activated by siRNA, which may be the result of differences in the amino acid composition of the TLR3 loop 1 of mice and humans. Although CD14 was previously described as a co-receptor for murine TLR3 and other nucleic acid-recognizing TLRs, human CD14 acts only as co-receptor to human TLR9, but not TLR3, TLR7 or TLR8. We show that CD14 up-regulates the TLR9 immune response of A, B and C-class oligodeoxynucleotides but down-regulates the phosphoro-diester version of B-class oligodeoxynucleotides.

Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor gammaRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.

Sequences derived from self-RNA containing certain natural modifications act as suppressors of RNA-mediated inflammatory immune responses.

The ability of the host to distinguish between self and foreign nucleic acids is one of the critical factors contributing to the recognition of pathogens by Toll-like receptors (TLRs). Under certain circumstances, eukaryotic self-RNA may reach TLR-containing compartments allowing for self-recognition. Specific modifications were previously demonstrated to suppress immune activation when placed at several positions in an immune stimulatory RNA or silencing RNA (siRNA). However, we show that even a simple natural modification such as a single 2'-O-methylation at different nucleotide positions throughout a sequence derived from a self-RNA strongly interferes with TLR-mediated effects. Such a single modification can even have an inhibitory effect in vitro and in vivo when placed in a different than the immune stimulatory RNA strand acting as suppressive RNA. Several safeguard mechanisms appear to have evolved to avoid cellular TLR-mediated activation by self-RNAs that may under other circumstances result in inflammatory or autoimmune responses. This knowledge can be used to include as few as a single 2'-O-methyl modification at a specific position in a siRNA sense or anti-sense strand to avoid TLR immune effects.

Characterization of conserved viral leader RNA sequences that stimulate innate immunity through TLRs.

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.

CpG oligodeoxynucleotides stimulate IFN-gamma-inducible protein-10 production in human B cells.

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-gamma-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-alpha2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-alpha in cells that express the IFN-alpha receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.

Impact of delivery systems on siRNA immune activation and RNA interference.

Small interfering RNAs (siRNAs) induce robust degradation of homologous mRNAs. Highly specific silencing of target genes makes siRNA an interesting tool in drug development. However, several non-specific effects complicate the use of RNA interference (RNAi). One of the most prevalent unspecific effects is triggering the innate immune system in mammals. In parallel, activating the immune system may open the possibility to develop dual siRNAs for treatment of a variety of diseases including cancer. Here, we show that the best use of unmodified siRNAs for RNAi and immune activation depends on the delivery system, formulation condition, sequence and siRNA design concerning ORN motifs. Testing several commercial delivery systems identified that the optimal siRNAs for dual functions should contain TLR7/8 ORN motifs at least in the antisense strand and be delivered by either Dharmafect or HiPerfect. Superfect delivery system only activates TLR7 and opens new capabilities in RNAi and immune activation.

Negative regulation of the type I interferon signaling pathway by synthetic Toll-like receptor 7 ligands.

Ten Toll-like receptor (TLR) family members have been reported in humans. Here, the endoplasmatic receptors TLR9, TLR8, TLR7, and TLR3 respond to nucleic acids and derivatives or to small molecules (TLR7 and 8). Another cytoplasmic RNA receptor, retinoic acid inducible gene I (RIG-I), is stimulated by 5' triphosphate double-stranded RNA. We discovered that TLR7 small-molecule agonists inhibit nucleic acid-mediated TLR3, TLR7, TLR9, or RIG-I-dependent interferon-α (IFN-α) immune response. Other cytokines and chemokines stimulated by nucleic acid agonists remained unaffected. The observed blockage of TLR3, TLR7, TLR9, and RIG-I-mediated IFN-α response appears to be driven by a competitive mechanism at the type I IFN pathway. Besides type I IFN, IFN response genes such as IFIT-1, Mx1, OAS1, or IRF7 were affected, which indicates that the key element driving the inhibition is located in the type I IFN pathway. Indeed, the heterotrimeric complex formation of phosphor-signal transducer and activator of transcription factor 1 (STAT1), phosphor-STAT2, and IRF9 (called ISGF3, IFN-stimulated gene factor 3) is inhibited through the TLR7 small-molecule agonists by phosphor-STAT2 blockage. These findings provide novel insights into the use of synthetic TLR7 or TLR7/8 small molecules as ligands for immune activation and suppression.

Dual or triple activation of TLR7, TLR8, and/or TLR9 by single-stranded oligoribonucleotides.

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.

Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses.

The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-alpha from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.

Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects.

Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level-mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense.

Neither inverted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing.

Transgene expression was analysed in Arabidopsis T-DNA transformants carrying defined numbers and arrangement of different reporter genes. All transgenes were placed under the control of the strong constitutive CaMV 35S promoter. High, stable transgene expression was observed in plants containing two copies of the beta-glucuronidase (GUS) gene, two or four copies of the green fluorescent protein (GFP) gene and two, four or six copies of the streptomycin phosphotransferase (SPT) gene. Thus, the mere presence of multiple promoter and/or transgene sequences did not result in gene silencing. In none of the cases analysed were tandem repeat arrangements of transgenes and/or inverted repeat (IR) T-DNA structures sufficient to trigger silencing of the different reporter genes. Instead, post-transcriptional gene silencing (PTGS) correlated with the copy number of the highly expressed transgenes. Twelve copies of the SPT and four copies of the GUS gene triggered silencing. Silencing is frequently associated with repetitive T-DNA structures. We favour the idea that in many cases this may be attributed to the high transgene doses rather than the repeat arrangements themselves.

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome.

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5'-upstream regions, coding sequences and 3'-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5'-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.