The critical DNA flanking sequences of a CpG oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity

Toll-like receptor 9 (TLR9) trafficking from the endoplasmic reticulum (ER) into endolysosomes is critical for eliciting cytidine-phosphate-guanosine (CpG) DNA-mediated immune responses. ADP-ribosylation factor 3 (ARF3) is a member of the Ras superfamily, which is crucial for a wide variety of cellular events including protein trafficking. In this study, we found that the inhibition of ARF3 by dominant mutants and siRNA impaired CpG oligodeoxynucleotide (ODN)-mediated responses whereas cells expressing the constitutively active ARF3 mutant enhanced CpG ODN-induced NF-κB activation and cytokine production. Further experiments with MyD88-overexpressing fibroblast cells transfected with a dominant-negative mutant and a constitutively active mutant of ARF3 demonstrated that ARF3 regulated CpG ODN-mediated signaling upstream of MyD88. Additional studies have shown that ARF3 inhibition impairs TLR9 trafficking from the ER into endolysosomes, thereby inhibiting the functional cleavage of TLR9, although it has no significant effect on CpG ODN uptake. Furthermore, activated ARF3 is associated with Unc93B1 and TLR9, suggesting that ARF3 conducts TLR9 trafficking by forming the TLR9-Unc93B1-ARF3 complex. Overall, our findings demonstrate that a novel ARF3 axis pathway mediates CpG ODN-induced responses by regulating TLR9 trafficking.

Download Full-text

Phosphodiester backbone of the CpG motif within immunostimulatory oligodeoxynucleotides augments activation of Toll-like receptor 9

Scientific Reports ◽

10.1038/s41598-017-15178-y ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 6

Author(s):

Jelka Pohar ◽

Duško Lainšček ◽

Ana Kunšek ◽

Miša-Mojca Cajnko ◽

Roman Jerala ◽

...

Keyword(s):

Dendritic Cells ◽

Binding Site ◽

Single Point ◽

Point Mutations ◽

Nonbridging Oxygen ◽

Mouse Bone Marrow ◽

Toll Like Receptor ◽

Phosphodiester Backbone ◽

Phosphodiester Linkage ◽

Cpg Motif

Abstract Toll-like receptor 9 (TLR9) stimulatory CpG-containing oligodeoxynucleotides (ODNs) with phosphorothioate backbones have successfully replaced the naturally occurring agonists of TLR9 in drug development due to their increased stability. Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. Here, we report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. Furthermore, we show that while single point mutations of W47, W96 and K690 within the TLR9 binding site retains full TLR9 activation by phosphodiester-based ODNs, activation by phosphorothioate-based ODNs is strongly impaired. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.

Download Full-text

Spatiotemporal regulation of intracellular trafficking of Toll-like receptor 9 by an inhibitory receptor, Ly49Q

Blood ◽

10.1182/blood-2008-12-192344 ◽

2009 ◽

Vol 114 (8) ◽

pp. 1518-1527 ◽

Cited By ~ 23

Author(s):

Mariko Yoshizaki ◽

Aya Tazawa ◽

Eiji Kasumi ◽

Shigemi Sasawatari ◽

Kenji Itoh ◽

...

Keyword(s):

Dendritic Cells ◽

Natural Killer ◽

Membrane Trafficking ◽

Plasmacytoid Dendritic Cells ◽

Type I ◽

Inhibitory Receptor ◽

Toll Like Receptor ◽

Spatiotemporal Regulation ◽

Cpg Oligodeoxynucleotide ◽

Tlr9 Signaling

Abstract Toll-like receptor (TLR) 9 recognizes unmethylated microorganismal cytosine guanine dinucleotide (CpG) DNA and elicits innate immune responses. However, the regulatory mechanisms of the TLR signaling remain elusive. We recently reported that Ly49Q, an immunoreceptor tyrosine-based inhibitory motif–bearing inhibitory receptor belonging to the natural killer receptor family, is crucial for TLR9-mediated type I interferon production by plasmacytoid dendritic cells. Ly49Q is expressed in plasmacytoid dendritic cells, macrophages, and neutrophils, but not natural killer cells. In this study, we showed that Ly49Q regulates TLR9 signaling by affecting endosome/lysosome behavior. Ly49Q colocalized with CpG in endosome/lysosome compartments. Cells lacking Ly49Q showed a disturbed redistribution of TLR9 and CpG. In particular, CpG-induced tubular endolysosomal extension was impaired in the absence of Ly49Q. Consistent with these findings, cells lacking Ly49Q showed impaired cytokine production in response to CpG-oligodeoxynucleotide. Our data highlight a novel mechanism by which TLR9 signaling is controlled through the spatiotemporal regulation of membrane trafficking by the immunoreceptor tyrosine-based inhibitory motif–bearing receptor Ly49Q.

Download Full-text

In VivoTLR9 Inhibition Attenuates CpG-Induced Myocardial Dysfunction

Mediators of Inflammation ◽

10.1155/2013/217297 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

O. Boehm ◽

P. Markowski ◽

M. van der Giet ◽

V. Gielen ◽

A. Kokalova ◽

...

Keyword(s):

Inflammatory Mediators ◽

Myocardial Dysfunction ◽

Toll Like Receptor ◽

Significant Drop ◽

Bacterial Dna ◽

Efficient Inhibitor ◽

Cardiac Inflammation ◽

Cardiac Depression ◽

Cpg Oligodeoxynucleotide

The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarifiedin vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances.

Download Full-text

The Toll-Like Receptor 9 Agonist, CpG-Oligodeoxynucleotide 1826, Ameliorates Cardiac Dysfunction After Trauma-Hemorrhage

Shock ◽

10.1097/shk.0b013e31825ce0de ◽

2012 ◽

Vol 38 (2) ◽

pp. 146-152 ◽

Cited By ~ 13

Author(s):

Xia Zhang ◽

Ming Gao ◽

Tuanzhu Ha ◽

John H. Kalbfleisch ◽

David L. Williams ◽

...

Keyword(s):

Cardiac Dysfunction ◽

Toll Like Receptor ◽

Cpg Oligodeoxynucleotide

Download Full-text

Toll-Like Receptor 9: A New Target of Ischemic Preconditioning in the Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600606 ◽

2008 ◽

Vol 28 (5) ◽

pp. 1040-1047 ◽

Cited By ~ 117

Author(s):

Susan L Stevens ◽

Thomas MP Ciesielski ◽

Brenda J Marsh ◽

Tao Yang ◽

Delfina S Homen ◽

...

Keyword(s):

Brain Injury ◽

Ischemic Injury ◽

Ischemic Brain Injury ◽

Dependent Manner ◽

Cpg Odn ◽

Toll Like Receptor ◽

Ischemic Brain ◽

Cpg Oligodeoxynucleotide ◽

Tlr9 Ligand

Preconditioning with lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, provides neuroprotection against subsequent cerebral ischemic brain injury, through a tumor necrosis factor (TNF)α-dependent process. Here, we report the first evidence that another TLR, TLR9, can induce neuroprotection. We show that the TLR9 ligand CpG oligodeoxynucleotide (ODN) can serve as a potent preconditioning stimulus and provide protection against ischemic brain injury. Our studies show that systemic administration of CpG ODN 1826 in advance of brain ischemia (middle cerebral artery occlusion (MCAO)) reduces ischemic damage up to 60% in a dose- and time-dependent manner. We also offer evidence that CpG ODN preconditioning can provide direct protection to cells of the central nervous system, as we have found marked neuroprotection in modeled ischemia in vitro. Finally, we show that CpG preconditioning significantly increases serum TNFα levels before MCAO and that TNFα is required for subsequent reduction in damage, as mice lacking TNFα are not protected against ischemic injury by CpG preconditioning. Our studies show that preconditioning with a TLR9 ligand induces neuroprotection against ischemic injury through a mechanism that shares common elements with LPS preconditioning via TLR4.

Download Full-text

Toll-Like Receptor Stimulation Enhances Phagocytosis and Intracellular Killing of Nonencapsulated and Encapsulated Streptococcus pneumoniae by Murine Microglia

Infection and Immunity ◽

10.1128/iai.01110-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 865-871 ◽

Cited By ~ 94

Author(s):

Sandra Ribes ◽

Sandra Ebert ◽

Tommy Regen ◽

Amit Agarwal ◽

Simone C. Tauber ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Microglial Activation ◽

Neonatal Period ◽

Toll Like Receptor ◽

Pneumococcal Infections ◽

Receptor Stimulation ◽

Bacterial Killing ◽

Cpg Oligodeoxynucleotide ◽

Murine Microglia ◽

The Brain

ABSTRACT Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam3CSK4), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.

Download Full-text