RESUMEN
Endosomally translocated host (self) DNA activates Toll-like receptor 9 (TLR9), while extracellular self-DNA does not. This inconsistency reflects poor endosomal DNA translocation but also implies that host DNA contains DNA sequences that function as ligands for TLR9. Herein we report that contrary to phosphorothioate (PS)-stabilized oligonucleotides (ODN), "natural" phosphodiester (PD) ODN lacking CpG motifs activate TLR9. CpG motif-independent TLR9 activation of Flt3-L-induced dendritic cells (DC) was dependent on enforced endosomal translocation and triggered upregulation of CD40 and CD69 as well as production of IL-6 and IFN-alpha. Binding studies utilizing surface plasmon resonance technology (Biacore) revealed low TLR9 binding to single-stranded (ss) PD-ODN lacking CpG motifs. At higher concentrations their TLR9 binding activity compared well with TLR9 binding of canonical ss PD CpG-ODN. These results imply that both the chemical modification of the DNA backbone as well as the amount of endosomally translocated DNA represent determining factors that allow CpG motif-independent activation of TLR9 by ss PD-DNA.
Asunto(s)
Islas de CpG/inmunología , ADN de Cadena Simple/inmunología , Células Dendríticas/inmunología , Endosomas/inmunología , Oligodesoxirribonucleótidos/inmunología , Receptor Toll-Like 9/inmunología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/inmunología , Células Cultivadas , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/farmacología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Endosomas/metabolismo , Ratones , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Resonancia por Plasmón de Superficie/métodos , Receptor Toll-Like 9/metabolismoRESUMEN
Cellular recognition of immuno-stimulatory microbial products alarming the host immune system upon infection, as well as endogenous molecular patterns representing perturbation of regular homeostasis such as through necrosis of host cells is mediated by innate pattern recognition receptors to which toll-like receptors (TLRs) belong. A variety of agonists has been attributed to TLR2. We raised monoclonal antibodies (mAbs) toward the murine TLR2 extracellular domain (mT2ECD) in order to analyze murine TLR2 expression. Murine macrophages were stained TLR2-specifically with distinct mAbs as shown by flow cytometry, immuno precipitation, and immuno-cytochemical analysis. TLR2-specific murine macrophage activation was inhibited through pre-incubation with a mAb mT2.4 while another mTLR2-specific mAb mT2.7 did not affect cell activation through TLR2. Plasmon resonance based analysis showed inhibition of lipopeptide binding to mT2ECD if complex formation with mT2.4 preceded binding analysis. Systemic induction of IL-6, IL-12p40, and GROalpha/KC release to the serum upon lipopeptide challenge of mice was inhibited by systemic administration of mT2.4. Furthermore, 120 mg/kg of mT2.4 protected mice from lethal shock-like syndrome in an experimental low-dose model of septic shock. This result validates blockage of cell surface TLR2 for inhibition of immune cell over-activation upon microbial challenge.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Animales , Línea Celular , Humanos , Ratones , Ratones Noqueados , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Tasa de Supervivencia , Receptor Toll-Like 2RESUMEN
Toll-like receptors (TLR) are activated by microbial components and transmit signals that induce cell activation and differentiation. A number of recent reports further indicate that TLR also have the potential to induce apoptosis upon ligand binding. Here we investigate the apoptosis-inducing capacity of TLR9, the receptor for microbial CpG-DNA. Unlike ligands for TLR2 and TLR4, CpG-DNA failed to induce apoptosis in RAW264.7 mouse macrophages. In human embryonic kidney fibroblasts transfected stably to express TLR9, CpG-DNA weakly induced apoptosis in one clone but not others without an obvious allocation to differences in TLR-signaling events. Analysis of the apoptotic signaling showed that the mitochondrial pathway of apoptosis was triggered by TLR9, as mitochondrial Bax was activated upstream of caspase-cleavage. CpG-DNA-induced apoptosis was reduced by cycloheximide suggesting that de novo protein synthesis was required. Strikingly, stimulation with CpG-DNA resulted in a strongly increased sensitivity of TLR9-expressing fibroblasts to apoptosis induced by staurosporine and UV-irradiation. These results identify a mitochondrial pathway to apoptosis that can be triggered by TLR9 and that may serve to sensitize cells from the innate immune system to apoptosis in the course of an immune response.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/fisiología , Fibroblastos/fisiología , Receptores de Superficie Celular/fisiología , Animales , Apoptosis/genética , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Receptor Toll-Like 9RESUMEN
Toll-like receptors (TLR) recognize bacterial and viral components, but direct interaction of receptor and ligand is unclear. Here, we demonstrate that TLR9 binds directly and sequence-specifically to single-stranded unmethylated CpG-DNA containing a phosphodiester backbone. TLR9-CpG-DNA interaction occurs at the acidic pH (6.5-5.0) found in endosomes and lysosomes. By sequence comparison we identified a potential CpG-DNA binding domain homologous to that described for methyl-CpG-DNA binding proteins. Amino acid substitutions in this region abrogated CpG-DNA binding and led to loss of NF-kappaB activation. Furthermore, chloroquine and quinacrine, therapeutic agents for autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, directly blocked TLR9-CpG-DNA interaction but not TLR2-Pam3Cys binding. Our results demonstrate direct binding of TLR9 to CpG-DNA and suggest that the therapeutic activity of chloroquine and quinacrine in autoimmune diseases may be due to its activity as a TLR9 antagonist and inhibitor of endosomal acidification.
Asunto(s)
Islas de CpG , ADN de Cadena Simple/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cloroquina/farmacología , Humanos , Concentración de Iones de Hidrógeno , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Quinacrina/farmacología , Receptores de Superficie Celular/química , Receptor Toll-Like 2 , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMEN
Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.
Asunto(s)
Glicoproteínas de Membrana/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Choque Séptico/inmunología , Choque Séptico/prevención & control , Animales , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Epítopos/genética , Epítopos/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Ligandos , Activación de Macrófagos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Choque Séptico/etiología , Transducción de Señal , Receptor Toll-Like 2 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.
Asunto(s)
Anticuerpos Antinucleares/biosíntesis , ADN Bacteriano/farmacología , Proteínas de Unión al ADN/fisiología , ADN/inmunología , Inmunoglobulina G/biosíntesis , Nefritis Lúpica/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptores de Superficie Celular/fisiología , Animales , Anticuerpos Antinucleares/sangre , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Quimiocinas/biosíntesis , Quimiocinas/genética , Proteínas de Unión al ADN/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Escherichia coli/genética , Inmunoglobulina G/sangre , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Nefritis Lúpica/sangre , Nefritis Lúpica/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Receptores de Superficie Celular/efectos de los fármacos , Receptor Toll-Like 9RESUMEN
Recognition by innate immune cells of the pathogen associated molecular patterns (PAMP) lipopolysaccharide (LPS) from Gram-negative bacteria and bacterial CpG-DNA depends on Toll-like receptor4 (TLR4) and TLR9, respectively. To define differences in the response to these distinct PAMP we compared a key intracellular event, namely recruitment of myeloid differentiation marker 88 (MyD88) to the respective PAMP-initiated TLR signaling. Using MyD88-GFP fusion protein expressing macrophages we demonstrate that LPS and CpG-DNA trigger signaling from two different cellular locations: theformer at the cell membrane and the latter at the lysosomal compartment. While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG-DNA to activate TLR9. In support of these data TLR9 is not localized at the cell surface, but intracellularily. These data stress the need to characterize individual TLR at the very beginning of signal initiation in order to understand their diverse biological functions.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Islas de CpG , ADN Bacteriano/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Transporte Biológico , Compartimento Celular , Línea Celular , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Receptor Toll-Like 4 , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMEN
The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.