Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2′-O-methyl gapmer oligonucleotides

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2′OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2′OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2′OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.     

ICOS-induced B7h shedding on B cells is inhibited by TLR7/8 and TLR9

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-gamma and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-gamma production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.     

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.     

Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines

Among the 11 human TLRs, a subfamily TLR7, TLR8, and TLR9 display similarities in structure and endosomal localization. Natural agonists consisting of nucleic acids, such as ssRNA or DNA with CpG motifs, activate the innate immune cells through these TLRs. Immune response modifiers (IRMs) of imidazoquinoline class compounds 3M-001, 3M-002, and 3M-003 have been shown to activate the innate immune system via TLR7, TLR8, and TLR7/8, respectively. In looking at the effect of the agonists of the TLR7, TLR8, and TLR9 on the activation of NF-kappaB of transfected HEK cells, we discovered that some oligodeoxynucleotides (ODNs) could modulate imidazoquinoline effects in a negative or positive manner. In this study we demonstrate that poly(T) ODNs can inhibit TLR7 and enhance TLR8 signaling events involving NF-kappaB activation in HEK cells and cytokine production (IFN-alpha, TNF, and IL-12) by human primary PBMC. In contrast, TLR3 agonist poly(I:C) does not affect imidazoquinoline-induced responses. The modulation of TLR7 and TLR8 responses is independent of CpG motifs or the nature of the ODN backbone structure. Furthermore, we show that to be an effective modulator, the ODNs need to be in the cell at the same time with either of the TLR7 or TLR8 agonist. We have also demonstrated that there is a physical interaction between IRMs and ODNs. The cross-talk between ODNs, IRMs, and TLR7 and TLR8 uncovered by this study may have practical implications in the field of microbial infections, vaccination, and tumor therapy.     

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.     

Intra-acrosomal inhibition of boar acrosin by synthetic proteinase inhibitors

Thirteen serine proteinase inhibitors of the guanidine, monoamidine and diamidine type were tested for their ability to inhibit the proteinase acrosin present in the acrosome of ejaculated and capacitated boar spermatozoa. All compounds studied proved to be potent in-vitro inhibitors of acrosin. Inhibition constants (Ki) in the range of 1.2 x 10(-7) to 6 x 10(-8) M were found for the reversible inhibitors. The intra-acrosomal inhibition of acrosin was assessed by the gelatin substrate film method: 2 guanidinobenzoates, one monoamidine and one diamidine derivative proved to inhibit acrosin completely in intact spermatozoa. Intravenous injection of 6-amidino-2-(4-amidinophenyl)-indole had no effect on fertilization, but application of 4-nitrophenyl-4-guanidinobenzoate in a vaginal suppository gave a 50% reduction of fertilization.     

MiRNA在TLR信号通路中的负性调控作用

TLR信号是生物体重要的病原体模式识别信号,在免疫识别和炎症反应中具有重要作用,其信号异常会导致许多免疫和炎症相关疾病的发生,因此探讨和明确TLR信号通路的调控机制具有非常重要的意义。近年来研究发现,作为重要的基因表达调控的小分子RNA,微RNA(microRNA,miRNA)能与TLR信号通路中众多靶基因mRNA的3’UTR区结合,从而抑制翻译过程或降解mRNA来发挥负性调控作用。本文就miRNA对TLR信号通路中的一些受体、信号分子、调节因子和细胞因子的负性调控作用方面进行阐述。     

TLR3 but not TLR7/8 ligand induces allergic sensitization to inhaled allergen

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.     

Preliminary evaluation of a 3H imidazoquinoline library as dual TLR7/TLR8 antagonists

Toll-like receptors (TLR) -7 and -8 are thought to play an important role in immune activation processes underlying the pathophysiology of HIV and several clinically important autoimmune diseases. Based on our earlier findings of TLR7-antagonistic activity in a 3H imidazoquinoline, we sought to examine a pilot library of 3H imidazoquinolines for dual TLR7/8 antagonists, since they remain a poorly explored chemotype. 2D-NOE experiments were employed to unequivocally characterize the compounds. A quinolinium compound 12, bearing p-methoxybenzyl substituents on N3 and N5 positions was identified as a lead. Compound 12 was found to inhibit both TLR7 and TLR8 at low micromolar concentrations. Our preliminary results suggest that alkylation with electron-rich substituents on the quinoline N5, or conversely, elimination of the fixed charge of the resultant quaternary amine on the quinolinium may yield more active compounds.     

Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8

Although TLR7 and TLR8 are phylogenetically and structurally related, their relative functions are largely unknown. The role of TLR7 has been established using TLR7-deficient mice and small molecule TLR7 agonists. The absence of TLR8-selective agonists has hampered our understanding of the role of TLR8. In this study TLR agonists selective for TLR7 or TLR8 were used to determine the repertoire of human innate immune cells that are activated through these TLRs. We found that TLR7 agonists directly activated purified plasmacytoid dendritic cells and, to a lesser extent, monocytes. Conversely, TLR8 agonists directly activated purified myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells (GM-CSF/IL-4/TGF-beta). Accordingly, TLR7-selective agonists were more effective than TLR8-selective agonists at inducing IFN-alpha- and IFN-regulated chemokines such as IFN-inducible protein and IFN-inducible T cell alpha chemoattractant from human PBMC. In contrast, TLR8 agonists were more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as TNF-alpha, IL-12, and MIP-1alpha. Thus, this study demonstrated that TLR7 and TLR8 agonists differ in their target cell selectivity and cytokine induction profile.     

Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-beta, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.     

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.     

The TLR7/8 ligand resiquimod targets monocyte-derived dendritic cell differentiation via TLR8 and augments functional dendritic cell generation

Imidazoquinolone compounds, such as resiquimod are Toll-like receptor (TLR) 7/8 ligands representing novel immune response modifiers undergoing clinical testing. Resiquimod has been reported to modulate conventional human monocyte-derived DC (moDC) differentiation, but the role of TLR7 and TLR8 is unclear. We directly dissected the TLR7- and TLR8-dependency by employing selective TLR7 ligands and resiquimod-coculture experiments with inhibitory oligonucleotides (iODN) suppressing TLR7, TLR7+8 or TLR7+8+9. Selective TLR7 ligands did not affect conventional moDC differentiation as analyzed by CD14/CD1a expression. iODN experiments confirmed that resiquimod’s effects during DC differentiation were antagonized only with TLR8 iODNs. Direct comparison of resiquimod DC with TLR7- and control-DC revealed significantly higher T-cell costimulatory molecule and MHC class II expression. Resiquimod DC promoted significantly stronger allogeneic T-cell proliferation and stronger naïve CD4⁺ T-cell proliferation. These results indicate the relevance of TLR8 for human monocyte-derived DC differentiation and maturation and may be relevant for clinical trials employing resiquimod.     

Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16–q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined TLR7/8 and TLR9 Ligands Potentiate the Activity of a Schistosoma japonicum DNA Vaccine

Background

Toll-like receptor (TLR) ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST).

Methodology/Principal Findings

In this study, we evaluated a TLR7/8 ligand (R848) and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG) as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens.

Conclusions

Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.     

Encountering unpredicted off-target effects of pharmacological inhibitors

Arkadia, also known as ring finger 111 (Rnf111), is an E3 ubiquitin ligase that amplifies transforming growth factor (TGF)-β family signalling through degradation of negative TGF-β signal regulators, i.e. Smad7, c-Ski and SnoN. Arkadia plays critical roles in early embryonic development through modulation of nodal signalling, as well as progression of tissue fibrosis and cancer through regulation of TGF-β signalling. Recent findings suggest that, similar to other ubiquitin ligases, including Smurf1 and 2, Arkadia regulates signalling pathways other than those of the TGF-β family. Arkadia interacts with the clathrin-adaptor 2 (AP2) complex and regulates endocytosis of certain cell surface receptors, leading to modulation of epidermal growth factor (EGF) and possibly other signalling pathways.     

Targeting viral infection by microRNA inhibition

An inhibitor of microRNA-122 reduces viral load in chimpanzees that are chronically infected with hepatitis C virus, suggesting that such an approach might have therapeutic potential in humans.     

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.