RESUMO
Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.
Assuntos
Citocinas/metabolismo , Inativação Gênica , Imunização/métodos , Oligorribonucleotídeos/síntese química , RNA Interferente Pequeno/genética , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Motivos de Aminoácidos , Animais , Buffy Coat , Citocinas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Oligorribonucleotídeos/genética , Oligonucleotídeos Fosforotioatos/química , RNA Interferente Pequeno/metabolismo , Uridina/químicaRESUMO
Toll-like receptors (TLRs) are mediators of innate immune responses detecting conserved pathogen-associated molecules. Whereas most TLRs are expressed on the cell surface, TLR3, 7, 8 and 9 are predominantly localized in endosomal compartments. Recent studies reported that TLRs are also expressed by T lymphocytes, resulting in direct co-stimulation of isolated CD4(+) T cells for example by Pam3CSK4 (TLR2 ligand) or flagellin (TLR5 ligand). We here describe enhanced IFN-γ production and T cell proliferation by anti-CD3 T cell receptor (TCR) or antigenic stimulation of purified human CD4(+) T cells upon co-culture with TLR7/8 specific single-stranded oligoribonucleotides or small molecule ligands. Surprisingly, TLR7/8 stimulation of CD4(+) T cells within a whole peripheral mononuclear cell (PBMC) environment did not result in enhanced T cell proliferation, but in a lack of proliferation that was cell-cell contact dependent. Immune cell depletion assays pointed towards a monocyte-mediated effect. Different TLR ligands influenced T cell proliferation differently. The effect of inhibition of T cell proliferation was most prominently seen for TLR7 ligands whereas the effects were minimal for TLR8 and TLR9 ligands indicating that the suppressive phenotype is unique only for certain TLRs. Our results strongly suggest that co-stimulation of T cell proliferation by TLR7/8 agonists is dependent on the specific cellular context.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Interferon gama/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Imidazóis/farmacologia , Interferon gama/genética , Lipopeptídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Receptor Cross-Talk/imunologia , Receptores de Antígenos de Linfócitos T/agonistasRESUMO
Most of the therapeutic applications for synthetic oligodeoxynucleotides (ODN) and oligoribonucleotides (ORN) relate to mechanisms of manipulating gene expression based on Watson-Crick base pairing to endogenous nucleic acids. However, in recent years it has become apparent that the immune system has evolved defense mechanisms against infections that are based on the detection of infecting viral and bacterial nucleic acids. In some cases, synthetic ODN and ORN can trigger these defenses and, therefore, can interfere with or distort the mechanism of action of antisense ODN or small interfering RNAs.