RESUMEN
The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.
Asunto(s)
Estudios de Asociación Genética , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Proteínas S100/genética , Investigación Biomédica Traslacional , Adulto , Animales , Anticuerpos Bloqueadores/farmacología , Polaridad Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dermatitis/diagnóstico , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/prevención & control , Modelos Animales de Enfermedad , Epítopos/efectos de los fármacos , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/prevención & control , Memoria Inmunológica/efectos de los fármacos , Interleucina-13/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/patología , Proteína de Unión al Calcio S100A4 , Proteínas S100/deficiencia , Células Th2/citología , Células Th2/inmunologíaRESUMEN
The Th2 cytokine IL-13 plays a key role in allergy, by regulating IgE, airway hyper secretion, eosinophils and mast cells. In this study, we aimed to identify novel transcription factors (TFs) that potentially regulated IL-13. We analyzed Th2 polarized naïve T cells from four different blood donors with gene expression microarrays to find clusters of genes that were correlated or anti-correlated with IL13. These clusters were further filtered, by selecting genes that were functionally related. In these clusters, we identified three transcription factors (TFs) that were predicted to regulate the expression of IL13, namely CEBPB, E2F6 and AHR. siRNA mediated knockdowns of these TFs in naïve polarized T cells showed significant increases of IL13, following knockdown of CEBPB and E2F6, but not AHR. This suggested an inhibitory role of CEBPB and E2F6 in the regulation of IL13 and allergy. This was supported by analysis of E2F6, but not CEBPB, in allergen-challenged CD4+ T cells from six allergic patients and six healthy controls, which showed decreased expression of E2F6 in patients. In summary, our findings indicate an inhibitory role of E2F6 in the regulation of IL-13 and allergy. The analytical approach may be generally applicable to elucidate the complex regulatory patterns in Th2 cell polarization and allergy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor de Transcripción E2F6/metabolismo , Interleucina-13/metabolismo , Rinitis Alérgica Estacional/metabolismo , Adulto , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Linfocitos T CD4-Positivos/metabolismo , Análisis por Conglomerados , Factor de Transcripción E2F6/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Interleucina-5/análisis , Activación de Linfocitos , Masculino , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Hidrocarburo de Aril/genética , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.