RESUMO
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.
Assuntos
Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/química , Receptor 7 Toll-Like/efeitos dos fármacos , Receptor 8 Toll-Like/efeitos dos fármacos , Receptor Toll-Like 9/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocinas/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Feminino , Células HEK293/efeitos dos fármacos , Células HEK293/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Oligonucleotídeos Fosforotioatos/administração & dosagem , Oligonucleotídeos Fosforotioatos/química , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODN are recognized by Toll-like receptor 9 (TLR9). Three classes of TLR9 agonists have been described: B-Class CpG ODN that induce strong B- and NK-cell activation and A-Class ODN that induce very high levels of IFN-alpha by plasmacytoid dendritic cells. The recently described C-Class ODN combine most efficiently properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN-alpha secretion comparable to A-Class ODN. Here, we investigate sequence requirements of C-Class ODN regarding optimal IFN-alpha secretion. Sequence as well as backbone modifications like 2'-O-methyl modifications especially in the 5' part of the ODN influence IFN-alpha-producing capacity. Kinetic studies on mRNA level for CD69, IFN-gamma, IP-10 and IL-18 by semi-quantitative PCR demonstrated differences in mRNA transcription for some cytokines suggesting different regulatory mechanisms for different ODN classes. High amounts of IP-10 mRNA and protein as well as up-regulation of IL-18 mRNA were observed especially for the A- and C-Classes. According to these data, C-Class ODN can be described as strong Th1 inducers with the stimulation of type I and II interferon as well as IP-10 production and strong NK activation. These characteristics can be availed to induce potent anti-tumor or anti-viral effects. Consequently, C-Class CpG ODN represent ideal drug candidates for anti-viral and/or anti-tumor therapy.
Assuntos
Oligodesoxirribonucleotídeos/química , RNA Mensageiro/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Quimiocina CXCL10 , Quimiocinas CXC/metabolismo , Meios de Cultura , Regulação para Baixo , Humanos , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Cinética , Lectinas Tipo C , Glicoproteínas de Membrana/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptor Toll-Like 9 , Receptores Toll-Like , Regulação para CimaRESUMO
Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune-stimulatory effects of bacterial DNA and are recognized by Toll-like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9-dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9-mediated immune response, but this response is insufficient to induce optimal interferon-alpha-mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure-activity relationship for TLR9 agonist compounds with diverse immune effects.
Assuntos
Linfócitos B/efeitos dos fármacos , Ilhas de CpG/imunologia , Inosina/análogos & derivados , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Motivos de Aminoácidos/imunologia , Animais , Linfócitos B/imunologia , Linhagem Celular , Feminino , Compostos Heterocíclicos/química , Compostos Heterocíclicos/imunologia , Compostos Heterocíclicos/farmacologia , Humanos , Inosina/química , Inosina/imunologia , Inosina/farmacologia , Interferon-alfa/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Estrutura Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared. The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells. CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antivirais/farmacologia , Proteínas de Ligação a DNA/agonistas , Imunidade Celular/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Receptores de Superfície Celular/agonistas , Ribavirina/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Cultivadas , Quimiocinas/biossíntese , Citocinas/biossíntese , Humanos , NF-kappa B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Receptor Toll-Like 9 , TransfecçãoRESUMO
Locked nucleic acid (LNA) is an RNA derivative that when introduced into oligodeoxynucleotides (ODN), mediates high efficacy and stability. CpG ODNs are potent immune stimulators and are recognized by toll-like receptor-9 (TLR9). Some phosphorothioate antisense ODNs bearing CpG dinucleotides have been shown to possess immune modulatory capacities. We investigated the effects of LNA substitutions on immune stimulation mediated by antisense ODN G3139 or CpG ODN 2006. LNA ODNs were tested for their ability to stimulate cytokine secretion from human immune cells or TLR9-dependent signaling. Phosphorothioate chimeric LNA/DNA antisense ODNs with phosphodiester-linked LNA nucleobases at both ends showed a marked decrease of immune modulation with an increasing number of 3' and 5' LNA bases. In addition, guanosine-LNA and cytosine-LNA or simply cytosine-LNA substitutions in the CpG dinucleotides of ODN 2006 led to strong decrease or near complete loss of immune modulation. TLR9-mediated signaling was similarly affected. These data indicate that increasing amounts of LNA residues in the flanks or substitutions of CpG nucleobases with LNA reduce or eliminate the immune stimulatory effects of CpG-containing phosphorothioate ODN.
Assuntos
Ilhas de CpG/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Células Cultivadas , Ilhas de CpG/genética , Ilhas de CpG/imunologia , Citocinas/biossíntese , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll-like receptor 9 (TLR9). CpG ODN of the B-Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN-alpha secretion by plasmacytoid DC were found to occur only with A-Class ODN. A third class of CpG ODN combines the immune effects of A- and B-Class CpG ODN. C-Class ODN strongly stimulate B cell or NK cell activation and IFN-alpha production. In contrast to the A-Class, the C-Class is wholly phosphorothioate, has no poly-G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9-dependent signaling, but with strikingly different dose-response relationships that are quite in contrast to those observed for IFN-alpha. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9-deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C-Class ODN are very potent Th1 adjuvants. C-Class ODN may represent new therapeutic drugs that combine the effects of A- and B-Class ODN for broad applications in infectious disease or cancer therapy.