RESUMO
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.
Assuntos
Subpopulações de Linfócitos B/imunologia , Sinalização do Cálcio/fisiologia , Interleucina-10/imunologia , Receptor Toll-Like 9/imunologia , Transcrição Gênica/fisiologia , Tirosina Quinase da Agamaglobulinemia , Subpopulações de Linfócitos B/citologia , Calcineurina/imunologia , Calcineurina/metabolismo , Inibidores de Calcineurina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Interleucina-10/metabolismo , Interleucina-6/biossíntese , Interleucina-6/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase SykRESUMO
Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.