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1.
Eur J Immunol ; 44(5): 1285-98, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24470136

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 Syk
2.
J Immunol ; 190(4): 1591-602, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23325892

RESUMEN

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/inmunología
3.
Eur J Immunol ; 43(6): 1630-42, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23519847

RESUMEN

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/inmunología
4.
J Immunol ; 189(4): 1747-56, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22786771

RESUMEN

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.


Asunto(s)
Apoptosis/inmunología , Micropartículas Derivadas de Células/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/metabolismo , Separación Celular , ADN/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Interferón Tipo I/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Immunology ; 139(2): 166-78, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23289794

RESUMEN

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/agonistas
6.
PLoS One ; 7(1): e29806, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22238657

RESUMEN

Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations.


Asunto(s)
Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ligandos , Mitógenos de Phytolacca americana/farmacología , Receptores Toll-Like/agonistas , Animales , Linfocitos B/metabolismo , Linfocitos B/fisiología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
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