Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The K_D value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis,and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.     

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.     

Mechanism‐Based Inhibitor of DNA Cytosine‐5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2‐Amino‐4‐Halopyridine‐C‐Nucleoside

In chromatin, 5‐methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine‐5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP) and oligodeoxyribonucleotides (ODNs) containing d^XP as a novel mechanism‐based inhibitor of DNMTs. The designed ODN containing ^XPpG forms a complex with DNMTs by covalent bonding through a nucleophilic aromatic substitution (S_NAr) reaction, and its cell proliferation activity is investigated. This study suggests that d^XP in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a S_NAr reaction would be useful for DNMTs and other protein–DNA interactions.     

Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: results from an NMR study

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.     

Intra-Peritoneal Administration of Mitochondrial DNA Provokes Acute Lung Injury and Systemic Inflammation via Toll-Like Receptor 9

The pathogenesis of sepsis is complex. Mitochondrial dysfunction, which is responsible for energy metabolism, intrinsic apoptotic pathway, oxidative stress, and systemic inflammatory responses, is closely related with severe sepsis induced death. Mitochondria DNA (mtDNA) contain un-methylated cytosine phosphate guanine (CpG) motifs, which exhibit immune stimulatory capacities. The aim of this study was to investigate the role and mechanism of mtDNA release on lipopolysaccharide (LPS) induced acute lung injury (ALI) and systemic inflammation. Following LPS injection, plasma mtDNA copies peak at 8 h. Compared with wild-type (WT) mice, mtDNA in toll like receptor 4 knockout (TLR4 KO) mice were significantly decreased. MtDNA intra-peritoneal administration causes apparent ALI as demonstrated by increased lung injury score, bronchoalveolar lavage fluid (BALF) total protein and wet/dry (W/D) ratio; mtDNA injection also directly provokes systemic inflammation, as demonstrated by increased IL-1β, IL-6, high-mobility group protein B1 (HMGB1) level; while nuclear DNA (nDNA) could not induce apparent ALI and systemic inflammation. However, compared with WT mice, TLR4 KO could not protect from mtDNA induced ALI and systemic inflammation. Specific TLR9 inhibitor, ODN 2088 pretreatment can significantly attenuate mtDNA induced ALI and systemic inflammation, as demonstrated by improved lung injury score, decreased lung wet/dry ratio, BALF total protein concentration, and decreased systemic level of IL-1β, IL-6 and HMGB1. MtDNA administration activates the expression of p-P38 mitogen-activated protein kinases (MAPK) in lung tissue and specific TLR9 inhibitor pretreatment can attenuate this activation. Thus, LPS-induced mtDNA release occurs in a TLR4-dependent manner, and mtDNA causes acute lung injury and systemic inflammation in a TLR9-dependent and TLR4-independent manner.     

Magic Peptide: Unique Properties of the LRR11 Peptide in the Activation of Leukotriene Synthesis in Human Neutrophils

Neutrophil-mediated innate host defense mechanisms include pathogen elimination through bacterial phagocytosis, which activates the 5-lipoxygenase (5-LOX) product synthesis. Here, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs), which mimic the receptor-recognized sites of bacterial (CpG-ODNs) and genomic (G-rich ODNs) DNAs released from the inflammatory area, on the neutrophil functions after cell stimulation with Salmonella typhimurium. A possible mechanism for ODN recognition by Toll-like receptor 9 (TLR9) and RAGE receptor has been proposed. We found for the first time that the combination of the magic peptide LRR11 from the leucine-rich repeat (LRR) of TLR9 with the CpG-ODNs modulates the uptake and signaling from ODNs, in particular, dramatically stimulates 5-LOX pathway. Using thickness shear mode acoustic method, we confirmed the specific binding of CpG-ODNs, but not G-rich ODN, to LRR11. The RAGE receptor has been shown to play an important role in promoting ODN uptake. Thus, FPS-ZM1, a high-affinity RAGE inhibitor, suppresses the synthesis of 5-LOX products and reduces the uptake of ODNs by neutrophils; the inhibitor effect being abolished by the addition of LRR11. The results obtained revealed that the studied peptide-ODN complexes possess high biological activity and can be promising for the development of effective vaccine adjuvants and antimicrobial therapeutics.     

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.     

Hybrid Cells Derived from Human Breast Cancer Cells and Human Breast Epithelial Cells Exhibit Differential TLR4 and TLR9 Signaling

TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-β, TNF-α, IL-1β and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.     

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

Endosome-localized Toll-like receptors (TLRs) 3 and 9 are expressed and functionally active in adipocytes. The functionality and role of TLR7 in adipocyte biology and innate immunity of adipose tissue (AT) is poorly characterized. We analyzed TLR7 mRNA and protein expression in murine 3T3-L1 and primary adipocytes, in co-cultures of 3T3-L1 adipocytes with murine J774A.1 monocytes and in human AT. The effects of TLR7 agonists imiquimod (IMQ) and cell-free nucleic acids (cfDNA) on adipokine concentration in cell-culture supernatants and gene expression profile were investigated. We found that TLR7 expression is strongly induced during adipocyte differentiation. TLR7 gene expression in adipocytes and AT stroma-vascular cells (SVC) seems to be independent of TLR9. IMQ downregulates resistin concentration in adipocyte cell-culture supernatants and modulates gene expression of glucose transporter Glut4. Adipocyte-derived cfDNA reduces adiponectin and resistin in cell-culture supernatants and potentially inhibits Glut4 gene expression. The responsiveness of 3T3-L1 adipocytes to imiquimod is preserved in co-culture with J774A.1 monocytes. Obesity-related, adipocyte-derived cfDNA engages adipocytic pattern recognition receptors (PRRs), modulating AT immune and metabolic homeostasis during adipose inflammation.     

Structure‐Based Design of Novel Human Toll‐like Receptor 8 Agonists

Toll‐like receptor (TLR)‐8 agonists activate adaptive immune responses by inducing robust production of T helper 1‐polarizing cytokines, suggesting that TLR8‐active compounds might be promising candidate vaccine adjuvants. Recently, a C2‐butyl furo[2,3‐c]quinoline was reported with purely TLR8 agonistic activity. This compound was successfully co‐crystallized with the human TLR8 ectodomain, and the co‐crystal structure revealed ligand‐induced reorganization of the binding pocket of TLR8. The loss of a key hydrogen bond between the oxygen atom of the furanyl ring of the agonist and Thr 574 in TLR8 suggested that the furan ring is dispensable. Employing a disconnection strategy, 3‐ and 4‐substituted aminoquinolines were investigated. Focused structure‐based ligand design studies led to the identification of 3‐pentyl‐quinoline‐2‐amine as a novel, structurally simple, and highly potent human TLR8‐specific agonist (EC₅₀=0.2 μM ). Preliminary evaluation of this compound in ex vivo human blood assay systems revealed that it retains prominent cytokine‐inducing activity. Together, these results indicate the suitability of this compound as a novel vaccine adjuvant, warranting further investigation.     

Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase

Dnmt3a‐C, the catalytic domain of the Dnmt3a DNA‐(cytosine‐C5)‐methyltransferase, is active in an isolated form but, like the full‐length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a‐C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation‐sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a‐C by optimization of the sequence of the DNA substrate, we analyzed its flanking‐sequence preference in detail by bisulfite DNA‐methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence‐preference profile for Dnmt3a‐C from the ?6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position ?2, A over G at ?1, a pyrimidine at +1, and A and T over G at +3. We designed one “good” substrate optimized for methylation and one “bad” substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a‐C substrate can be applied in enzymatic high‐throughput assays with Dnmt3a‐C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.     

Roles of B Cell-Intrinsic TLR Signals in Systemic Lupus Erythematosus

Toll-like receptors (TLRs) are a large family of pattern recognition receptors. TLR signals are involved in the pathogenesis of systemic lupus erythematosus. Mouse and human B cells constitutively express most TLRs. Many B cell subpopulations are highly responsive to certain TLR ligation, including B-1 B cells, transitional B cells, marginal zone B cells, germinal center B cell and memory B cells. The B cell-intrinsic TLR signals play critical roles during lupus process. In this review, roles of B cell-intrinsic TLR2, 4, 7, 8 and 9 signals are discussed during lupus pathogenesis in both mouse model and patients. Moreover, mechanisms underlying TLR ligation-triggered B cell activation and signaling pathways are highlighted.     

Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.