RESUMEN
Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.
Asunto(s)
ADN Bacteriano/inmunología , ADN/inmunología , Inmunidad Innata/inmunología , Interleucinas/inmunología , Células Th17/inmunología , Receptor Toll-Like 9/inmunología , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Ratones , Psoriasis/inmunología , Receptores de Interleucina/inmunología , Receptores de Interleucina/metabolismoRESUMEN
Plasmacytoid dendritic cells (pDCs) are known mainly for their secretion of type I IFN upon viral encounter. We describe a CD2hiCD5+CD81+ pDC subset, distinguished by prominent dendrites and a mature phenotype, in human blood, bone marrow, and tonsil, which can be generated from CD34+ progenitors. These CD2hiCD5+CD81+ cells express classical pDC markers, as well as the toll-like receptors that enable conventional pDCs to respond to viral infection. However, their gene expression profile is distinct, and they produce little or no type I IFN upon stimulation with CpG oligonucleotides, likely due to their diminished expression of IFN regulatory factor 7. A similar population of CD5+CD81+ pDCs is present in mice and also does not produce type I IFN after CpG stimulation. In contrast to conventional CD5-CD81- pDCs, human CD5+CD81+ pDCs are potent stimulators of B-cell activation and antibody production and strong inducers of T-cell proliferation and Treg formation. These findings reveal the presence of a discrete pDC population that does not produce type I IFN and yet mediates important immune functions previously attributed to all pDCs.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular , Proliferación Celular/fisiología , Células Dendríticas/fisiología , Activación de Linfocitos , Linfocitos T/fisiología , Animales , Antígenos CD2/metabolismo , Antígenos CD5/metabolismo , Separación Celular , Citometría de Flujo , Humanos , Interferón Tipo I/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/inmunología , Tetraspanina 28/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
The intracellular location of nucleic acid sensors prevents recognition of extracellular self-DNA released by dying cells. However, on forming a complex with the endogenous antimicrobial peptide LL37, extracellular DNA is transported into endosomal compartments of plasmacytoid dendritic cells, leading to activation of Toll-like receptor-9 and induction of type I IFNs. Whether LL37 also transports self-DNA into nonplasmacytoid dendritic cells, leading to type I IFN production via other intracellular DNA receptors is unknown. Here we found that LL37 very efficiently transports self-DNA into monocytes, leading the production of type I IFNs in a Toll-like receptor-independent manner. This type I IFN induction was mediated by double-stranded B form DNA, regardless of its sequence, CpG content, or methylation status, and required signaling through the adaptor protein STING and TBK1 kinase, indicating the involvement of cytosolic DNA sensors. Thus, our study identifies a novel link between the antimicrobial peptides and type I IFN responses involving DNA-dependent activation of cytosolic sensors in monocytes.
Asunto(s)
Catelicidinas/metabolismo , Citosol/metabolismo , ADN/inmunología , ADN/metabolismo , Espacio Extracelular/metabolismo , Monocitos/metabolismo , Péptidos Catiónicos Antimicrobianos , Catelicidinas/inmunología , Citosol/inmunología , Humanos , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Microscopía Confocal , Monocitos/inmunología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell-mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10-producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10-producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.
Asunto(s)
Células Dendríticas/inmunología , Interleucina-10/biosíntesis , Proteínas/metabolismo , Linfocitos T Reguladores/inmunología , Adulto , Antígenos CD , Diferenciación Celular , Células Dendríticas/clasificación , Células Dendríticas/metabolismo , Humanos , Técnicas In Vitro , Ligando Coestimulador de Linfocitos T Inducibles , Células Mieloides/clasificación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Plasmáticas/clasificación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Células Th2/inmunología , Regulación hacia ArribaRESUMEN
Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Autoantígenos/metabolismo , Enfermedades Autoinmunes/metabolismo , Autoinmunidad , ADN/metabolismo , Células Dendríticas/metabolismo , Psoriasis/metabolismo , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Catelicidinas , ADN/inmunología , Células Dendríticas/inmunología , Endocitosis , Endosomas/inmunología , Endosomas/metabolismo , Humanos , Psoriasis/inmunología , Psoriasis/patología , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
Raised serum levels of interferon (IFN)-alpha have been observed in systemic lupus erythematosus (SLE) patients, and these levels are correlated with both disease activity and severity. The origin of this IFN-alpha is still unclear, but increasing evidence suggests the critical involvement of activated plasmacytoid predendritic cells (PDCs). In SLE patients, DNA and RNA viruses, as well as immune complexes (ICs), that consist of autoantibodies specific to self-DNA and RNA protein particles can stimulate production of IFN-alpha. We have developed three series of oligonucleotide (ODN)-based inhibitors of Toll-like receptor (TLR) signaling. These ODNs include inhibitors of TLR9, inhibitors of TLR7 but not TLR9, and sequences that inhibit both TLR7 and TLR9. Specificity of these inhibitors is confirmed by inhibition of IFN-alpha production by PDCs in response to DNA or RNA viruses. We show that mammalian DNA and RNA, in the form of ICs, are potent self-antigens for TLR9 and TLR7, respectively, and induce IFN-alpha production by PDCs. This work suggests that TLRs may have a critical role in the promotion of lupus through the induction of IFN-alpha by PDCs. These inhibitors of TLR signaling thus represent novel therapeutic agents with potential for the treatment of lupus.
Asunto(s)
Interferón-alfa/sangre , Lupus Eritematoso Sistémico/etiología , Oligonucleótidos/metabolismo , Transducción de Señal , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus/inmunologíaRESUMEN
Immunostimulatory DNA sequences (ISS) containing CpG motifs induce interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) from human peripheral blood mononuclear cells and stimulate human B cells to proliferate and produce IL-6. We studied the motif and structural requirements for both types of activity using novel chimeric immunomodulatory compounds (CICs), which contain multiple heptameric ISS connected by non-nucleoside spacers in both linear and branched configurations. We found that the optimal motifs and structure for IFN-alpha production versus B cell activation differed. IFN-alpha production was optimal for CICs containing the sequences 5'-TCGXCGX and 5'-TCGXTCG, where X is any nucleotide. The presentation of multiple copies of these heptameric ISS with free 5'-ends via long, hydrophilic spacers, such as hexaethylene glycol, significantly enhanced the induction of IFN-alpha. Conversely, human B cell activity was predominantly dependent on ISS motif, with 5'-TCGTXXX and 5'-AACGTTC being the most active sequences. Thus, we found CICs could be 'programmed' for IFN-alpha production or B cell activation as independent variables. Additionally, CICs with separate human- and mouse-specific motifs were synthesized and these were used to confirm in vivo activity in mice. CICs may offer unique advantages over conventional ISS because identification of the optimal motifs, spacers and structures for different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Islas de CpG/genética , Oligonucleótidos/farmacología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/genética , Animales , Secuencia de Bases , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interferones/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tionucleótidos/química , Tionucleótidos/genética , Tionucleótidos/farmacologíaRESUMEN
CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/efectos de los fármacos , Citocinas/biosíntesis , Expresión Génica/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Adyuvantes Inmunológicos/genética , Animales , Proteínas Reguladoras de la Apoptosis , Linfocitos B/inmunología , Proliferación Celular , Islas de CpG/genética , Femenino , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Expresión Génica/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Interferón-alfa/metabolismo , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Ganglios Linfáticos/química , Ganglios Linfáticos/metabolismo , Ratones , Proteínas de Resistencia a Mixovirus , Oligodesoxirribonucleótidos/genética , Papio , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Recent reports have identified two major classes of CpG motif-containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)-alpha, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)-derived IFN-alpha but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN-gamma/IFN-alpha from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN-alpha. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG-containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN-alpha/IFN-gamma induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Oligodesoxirribonucleótidos/clasificación , Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos/farmacología , Adyuvantes Inmunológicos/química , Antígenos CD/biosíntesis , Linfocitos B/inmunología , Secuencia de Bases , Células Cultivadas , Quimiocinas/biosíntesis , Quimiocinas/genética , Citocinas/biosíntesis , Células Dendríticas/inmunología , Expresión Génica/efectos de los fármacos , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Interferón gamma/biosíntesis , Interferón gamma/genética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Oligodesoxirribonucleótidos/química , Oligonucleótidos/química , Oligonucleótidos/clasificación , ARN Mensajero/biosíntesisRESUMEN
Monocytes rapidly infiltrate inflamed tissues and differentiate into CD209(+) inflammatory dendritic cells (DCs) that promote robust immunity or, if unregulated, inflammatory disease. Previous studies in experimental animal models indicate that inflammatory DC depletion through systemic elimination of their monocyte precursors with clodronate-loaded liposomes ameliorates the development of psoriasis and other diseases. However, translation of systemic monocyte depletion strategies is difficult due to the importance of monocytes during homeostasis and infection clearance. Here, we describe a strategy that avoids the monocyte intermediates to deplete inflammatory DCs through antibody-loaded toxin. Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with anti-CD209 antibody conjugated to saporin, a potent ribosome inactivator. The results demonstrate depletion of CD209(+) DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Células Dendríticas/inmunología , Inmunotoxinas/inmunología , Lectinas Tipo C/antagonistas & inhibidores , Procedimientos de Reducción del Leucocitos , Receptores de Superficie Celular/antagonistas & inhibidores , Proteínas Inactivadoras de Ribosomas Tipo 1/inmunología , Animales , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Inflamación/inmunología , Inflamación/metabolismo , Lectinas Tipo C/metabolismo , Ratones , Receptores de Superficie Celular/metabolismo , SaporinasRESUMEN
Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer death among women. Despite its immunogenicity, effective antitumor responses are limited, due, in part, to the presence of forkhead box protein 3-positive (Foxp3(+)) T regulatory (Treg) cells in the tumor microenvironment. However, the mechanisms that regulate the accumulation and the suppressive function of these Foxp3(+) Treg cells are poorly understood. Here, we found that the majority of Foxp3(+) Treg cells accumulating in the tumor microenvironment of EOCs belong to the subset of Foxp3(+) Treg cells expressing inducible costimulator (ICOS). The expansion and the suppressive function of these cells were strictly dependent on ICOS-L costimulation provided by tumor plasmacytoid dendritic cells (pDC). Accordingly, ICOS(+) Foxp3(+) Treg cells were found to localize in close vicinity of tumor pDCs, and their number directly correlated with the numbers of pDCs in the tumors. Furthermore, pDCs and ICOS(+) Foxp3(+) Treg cells were found to be strong predictors for disease progression in patients with ovarian cancer, with ICOS(+) Treg cell subset being a stronger predictor than total Foxp3(+) Treg cells. These findings suggest an essential role for pDCs and ICOS-L in immunosuppression mediated by ICOS(+) Foxp3(+) Treg cells, leading to tumor progression in ovarian cancer.
Asunto(s)
Células Dendríticas/inmunología , Factores de Transcripción Forkhead/fisiología , Proteína Coestimuladora de Linfocitos T Inducibles/fisiología , Neoplasias Ováricas/inmunología , Linfocitos T Reguladores/patología , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Neoplasias Ováricas/patologíaRESUMEN
Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.
Asunto(s)
Células Dendríticas/inmunología , Lupus Eritematoso Sistémico/inmunología , Neutrófilos/inmunología , Anticuerpos Antinucleares/sangre , Complejo Antígeno-Anticuerpo/sangre , Péptidos Catiónicos Antimicrobianos , Autoantígenos/sangre , Linfocitos B/inmunología , Estudios de Casos y Controles , Catelicidinas/inmunología , ADN/sangre , ADN/inmunología , Humanos , Activación de Linfocitos , Péptidos/sangre , Péptidos/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
Interleukin-17 (IL-17) producing T helper cells (T(H)-17) comprise a newly recognized T cell subset with an emerging role in adaptive immunity to a variety of fungi. Whether different airborne fungi trigger a common signaling pathway for T(H)-17 induction, and whether this ability is related to the inherent pathogenic behavior of each fungus is currently unknown. Here we show that, as opposed to primary pathogenic fungi (Histoplasma capsulatum), opportunistic fungal pathogens (Aspergillus and Rhizopus) trigger a common innate sensing pathway in human dendritic cells (DCs) that results in robust production of IL-23 and drives T(H)-17 responses. This response requires activation of dectin-1 by the fungal cell wall polysaccharide b-glucan that is selectively exposed during the invasive growth of opportunistic fungi. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma not only abrogates the pathogenicity of this fungus, but also triggers the induction of IL-23 producing DCs. Thus, b-glucan exposure in the fungal cell wall is essential for the induction of IL-23/T(H)-17 axis and may represent a key factor that regulates protective immunity to opportunistic but not pathogenic fungi.
Asunto(s)
Células Dendríticas/inmunología , Hongos/patogenicidad , Interleucina-23/inmunología , Micosis/inmunología , Células Th17/inmunología , Microbiología del Aire , Células Cultivadas , Células Dendríticas/microbiología , Hongos/inmunología , Humanos , Lectinas Tipo C , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Proteínas de la Membrana/inmunología , Micosis/microbiología , Proteínas del Tejido Nervioso/inmunología , beta-Glucanos/inmunologíaRESUMEN
Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/ß)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.
Asunto(s)
Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Piel/inmunología , Cicatrización de Heridas/inmunología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas/deficiencia , Catelicidinas/genética , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Femenino , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Ácidos Nucleicos/inmunología , Ácidos Nucleicos/metabolismo , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/lesiones , Receptor Toll-Like 7/deficiencia , Receptor Toll-Like 7/genéticaRESUMEN
Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid-recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA-LL37 complexes activate TLR7 and, like self-DNA-LL37 complexes, trigger the secretion of IFN-alpha without inducing maturation or the production of IL-6 and TNF-alpha. In contrast to self-DNA-LL37 complexes, self-RNA-LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-alpha and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA-LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Células Dendríticas/inmunología , Sustancias Macromoleculares/metabolismo , Psoriasis/inmunología , ARN/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Catelicidinas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Luciferasas , Sustancias Macromoleculares/inmunología , Datos de Secuencia Molecular , ARN/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunologíaRESUMEN
In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-alpha and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Leucocitos Mononucleares/inmunología , Subgrupos Linfocitarios/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Sepsis/mortalidad , Sepsis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Separación Celular , Islas de CpG/inmunología , Proteínas de Unión al ADN/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Guanosina/administración & dosificación , Guanosina/farmacología , Humanos , Inyecciones Subcutáneas , Interferón-alfa/antagonistas & inhibidores , Interleucina-10/fisiología , Ratones , Ratones Endogámicos BALB C , Conformación de Ácido Nucleico , Receptores de Superficie Celular/fisiología , Sepsis/inmunología , Sepsis/patología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Tionucleótidos/administración & dosificación , Tionucleótidos/farmacología , Receptor Toll-Like 9RESUMEN
Recent reports have shown that immunostimulatory sequences (ISS) containing CpG motifs have minimal length requirements (>/=12 bases) for the exertion of immune-enhancing function upon mammalian cells. Herein we demonstrate that short ISS (5-7 bases), which exhibit no activity on their own, induce IFN-gamma and IFN-alpha secretion from human peripheral blood mononuclear cells when adsorbed to the surface of cationic poly(D,L-lactide-co-glycolide) microparticles (cPLGA). Utilizing this technique, we discovered a minimal ISS sequence for induction of IFN-gamma and IFN-alpha from human cells: 5'-TCGXX-3'. These short ISS/cPLGA formulations targeted PDC in similar fashion to longer ISS ODN, the activity of which does not require (but is enhanced by) cPLGA. PDC stimulated with short ISS/cPLGA responded with enhanced uptake of ISS and elevated production of cytokines, including IFN-alpha. However, ISS-responsive B cells did not respond to short ISS/cPLGA, underlining the plasmacytoid dendritic cell selectivity of this formulation. These results describe a novel technique for formulating active, but very short, ISS oligodeoxynucleotide that allows for the dissection and characterization of minimal immunostimulatory CpG motifs.