RESUMEN
Suppressory B-cell function controls immune responses and is mainly dependent on IL-10 secretion. Pharmacological manipulation of B-cell-specific IL-10 synthesis could, thus, be therapeutically useful in B-cell chronic lymphocytic leukemia, transplantation, autoimmunity and sepsis. TLR are thought to play a protagonistic role in the formation of IL-10-secreting B cells. The aim of the study was to identify the molecular events selectively driving IL-10 production in TLR9-stimulated human B cells. Our data highlight the selectivity of calcineurin inhibitors in blocking TLR9-induced B-cell-derived IL-10 transcription and secretion, while IL-6 transcription and release, B-cell proliferation, and differentiation remain unaffected. Nevertheless, TLR9-induced IL-10 production was found to be independent of calcineurin phosphatase activity and was even negatively regulated by NFAT. In contrast to TLR9-induced IL-6, IL-10 secretion was highly sensitive to targeting of spleen tyrosine kinase (syk) and Bruton's tyrosine kinase. Further analyses demonstrated increased phosphorylation of Ca(2+) /calmodulin kinase II (CaMKII) in TLR9-stimulated B cells and selective reduction of TLR9-induced secretion of IL-10 upon treatment with CaMKII inhibitors, with negligible impact on IL-6 levels. Altogether, our results identify calcineurin antagonists as selective inhibitors of IL-10 transcription and syk/Bruton´s tyrosine kinase-induced Ca(2+) /calmodulin- and CaMKII-dependent signaling as a pathway regulating the release of TLR9-induced B-cell-derived IL-10.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Señalización del Calcio/fisiología , Interleucina-10/inmunología , Receptor Toll-Like 9/inmunología , Transcripción Genética/fisiología , Agammaglobulinemia Tirosina Quinasa , Subgrupos de Linfocitos B/citología , Calcineurina/inmunología , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/inmunología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proliferación Celular , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Interleucina-10/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Quinasa SykRESUMEN
Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/microbiología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Interleucina-10/biosíntesis , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Proteína Estafilocócica A/farmacología , Animales , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Infecciones Estafilocócicas/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
IRAK4, a serine/threonine kinase is a central adaptor protein in TLR signaling. To better understand the clinical significance of IRAK4 deficiency we examined the impact of IRAK4 on bacterial recognition in human monocytes. We show that IRAK4 knockdown modulates monocyte-derived cytokine secretion in response to Staphylococcus aureus and Streptococcus pneumoniae, resulting in decreased IL-12 and elevated IL-10 production, a finding also reproducible with ligands for TLR2 and TLR4. In contrast, silencing of MyD88 leads to a complete loss of cytokine secretion, indicating that IRAK4 acts as a differential regulator of bacteria/TLR-induced cytokine secretion downstream of MyD88. Further analysis revealed that this modulatory function results from IRAK4-mediated suppression of protein kinase B (PKB/Akt). Release of suppression upon IRAK4 silencing (but not MyD88 knockdown) increases phosphorylation of PKB/Akt, counteracts NF-κB activation and finally results in a monocyte phenotype with tolerogenic features, thus unleashing Akt- and mTOR-dependent release of IL-10, along with concomitant phosphorylation of FOXO transcription factors. In line with these observations IRAK4-deficient monocytes failed to induce allogeneic CD8(+) and CD4(+) T-cell responses, an effect reverted by neutralization of IL-10. Taken together, our data highlight an unexpected role of IRAK4, Akt, and mTOR in the regulation of tolerance in human monocytes.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones Neumocócicas/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Streptococcus pneumoniae/inmunología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Células Cultivadas , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Activación de Linfocitos/genética , Monocitos/microbiología , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Serina-Treonina Quinasas TOR/inmunologíaRESUMEN
Re-expression of recombinase activating genes (RAG) in mature B cells may support autoreactivity by enabling revision of the B-cell receptor (BCR). Recent reports suggest that administration of Toll-like receptor 9 (TLR9) -stimulating CpG oligodeoxynucleotides (ODN) could trigger the manifestation of autoimmune disease and that TLR are involved in the selection processes eliminating autoreactive BCR. The mechanisms involved remain to be elucidated. This prompted us to ask, whether TLR9 could be involved in receptor revision. We found that phosphorothioate-modified CpG ODN (CpG(PTO)) induced expression of Ku70 and re-expression of RAG-1 in human peripheral blood B lymphocytes and Igλ expression in sorted Igκ(+) B cells. Further results revealed unselective binding specificity of CpG(PTO) -induced immunoglobulin and suggested that CpG(PTO) engage and/or mimic IgM receptor signalling, an important prerequisite for the initialization of receptor editing or revision. Altogether, our data describe a potential role for TLR9 in receptor revision and suggest that CpG(PTO) could mimic chromatin-bearing autoantigens by simultaneously engaging the BCR and TLR9 on IgM(+) B cells.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Oligodesoxirribonucleótidos/inmunología , Receptor Toll-Like 9/inmunología , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Antígenos Nucleares/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Western Blotting , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Humanos , Cadenas lambda de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Autoantígeno Ku , Microscopía Fluorescente , Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos Fosforotioatos/inmunología , Oligonucleótidos Fosforotioatos/farmacología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 9/agonistasRESUMEN
In contrast to murine B cells, Toll-like receptor (TLR) expression in human B cells is mainly restricted to endosomally localized TLR7 and -9, receptors for RNA and DNA, respectively. Most importantly, B lymphocytes lack classical phagocytic receptors and instead internalize antigen only via the B cell receptor (BCR), a surface immunoglobulin specific for a defined antigen. BCR ligation triggers internalization of particulate antigens and physically associated molecules among them bacterial DNA or RNA. Thereby, this process provides access to endosomal nucleic acid-sensing TLRs. Co-stimulation of BCR and TLR ultimately leads to T cell-independent B cell activation. Here, we explain how this process can be experimentally mimicked in human peripheral blood B cells, e.g., using a microsphere-based system that promotes uptake of nucleic acid-based TLR ligands via BCR engagement.
Asunto(s)
Linfocitos B/metabolismo , Ácidos Nucleicos/metabolismo , Diferenciación Celular , Proliferación Celular , Humanos , Receptores de Antígenos de Linfocitos B/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismoRESUMEN
MicroRNAs (miRNAs) are fine-tuners in cellular processes, including those of the immune response. To study their functions and effects in immune cells, it is necessary to achieve specific silencing of individual miRNAs. To date, introduction of antisense microRNAs (antagomiRs) into primary cells is based on electroporation, lipofection, and viral vectors. However, these techniques often compromise viability, proliferative capacity, and differentiation. Furthermore, efficiency varies depending on the cell type and some are not suitable for in vivo approaches. To overcome these limitations we exploited the property of phosphorothioate (PTO)-modified DNA oligodeoxynucleotides (ODN) to enter cells with high efficacy: we developed and evaluated ODN/antagomiR constructs that consist of a PTO-ODN carrier covalently linked to a fully methylated antagomiR RNA sequence. Using these constructs, we achieved transfection efficiency of approximately 99% in leukocytes-in particular, in B lymphocytes that are hard to transfect with other methods. Our data demonstrate that miRNA silencing by the antagomiR portion of the constructs was specific and efficient, which could be further confirmed by an increase in target protein under silencing conditions. The constructs were successfully tested in human B cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells, thus demonstrating their versatility. Moreover, introduction of stimulatory CpG sequences into the ODN portion conveys immune stimulatory quality when intended. Thus, bifunctional ODN/antagomiR constructs represent a highly efficient, versatile, and easy-to-handle tool to manipulate cellular miRNA expression levels and to allow the subsequent investigation of specific miRNA functions.
Asunto(s)
Técnicas de Silenciamiento del Gen , MicroARNs/genética , Oligonucleótidos Antisentido/genética , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Caenorhabditis elegans , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Lipopolisacáridos/farmacología , MicroARNs/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Cultivo Primario de CélulasRESUMEN
High mobility group box protein B1 (HMGB1), a nuclear protein reportedly involved in the structural organisation of DNA, is released from necrotic cells or upon cellular activation. After its release into the extracellular space, HMGB1 serves as a mediator of inflammation. In contrast to necrotic cells, apoptotic ones usually do not release HMGB1. Formation and release of membranous vesicles is a well-known feature of apoptotic cell death. Only recently, subcellular membrane vesicles, such as those released during apoptotic cell death have been identified as immune regulators and as mediators of cell to cell communication. We and others have previously detected nuclear antigens within apoptosis-released membranous vesicles and HMGB1 together with nuclear antigens has been discussed to be a key player in etiology and pathogenesis of autoimmune diseases. On this background, we analysed whether HMGB1 is included in the membranous vesicles generated by apoptosing cells. Employing immune blots we observed abundand amounts of HMGB1 in the fraction of the small membraneous particles isolated from cell culture supernatants and conclude that HMGB1 is translocated into vesicles generated during apoptosis.