Analysis of NFAT5 expression and activity in response to toll-like receptors

Stimulation of Toll-like receptors (TLRs) in cells of the innate immune system activates the expression of a proinflammatory and antimicrobial gene program controlled by a network of transcriptional regulators. We show that NFAT5, which belongs to the Rel family of transcription factors and was previously characterized as an osmostress responsive factor, is required for the expression of a group of TLR-responsive genes in macrophages, such as Nos2, Il6 and Tnf. NFAT5 recruitment to its target genes is dependent on IKKβ activity, de novo protein synthesis and is sensitive to histone deacetylases. Interestingly, NFAT5 is essential in the response to low doses of TLR ligands, regulating specific gene subsets depending on the stimulus strength. We also show that macrophages use NFAT5 to facilitate chromatin accessibility, allowing the recruitment of transcriptional regulators such as p65/NF-κB, c-Fos and p300 to its target regions. We use Nos2 as a gene whose induction is NFAT5-dependent especially at low doses of LPS to demonstrate that NFAT5 controls the recruitment of p65 by facilitating the activity of H3K27 demethylases, without influencing the binding of Polycomb repressive complex 2 or JMJD3. Altogether, this thesis characterizes NFAT5 as a novel regulator of the immune response to low pathogen load involved in the control of local chromatin accessibility.En las células del sistema inmunitario innato, la estimulación de los receptores de tipo Toll (TLR) activa la expresión de un programa génico pro-inflamatorio y antimicrobiano que está controlado por una red de reguladores transcripcionales. Hemos demostrado que el NFAT5, perteneciente a la familia de factores de transcripción Rel y previamente caracterizado como un factor de respuesta a estrés osmótico, es importante para la expresión de un grupo de genes de respuesta a TLRs, entre ellos Nos2, Il6 y Tnf. El reclutamiento del NFAT5 a sus genes diana requiere la actividad de IKKβ, la síntesis de novo de proteínas y es sensible a la acción de las deacetilasas de histonas. Resulta interesante el hecho de que el NFAT5 es esencial para responder a bajas dosis de ligando de los TLRs, y que regula grupos de genes específicos dependiendo de la intensidad del estímulo. También mostramos que NFAT5 facilita la accesibilidad de la cromatina en macrófagos, permitiendo el reclutamiento de reguladores transcripcionales como p65/NF-kB, c-Fos y p300 a sus regiones diana. Utilizando Nos2 como un gen cuya inducción es más dependiente de NFAT5 a bajas dosis de LPS, demostramos que el NFAT5 controla el reclutamiento de p65 gracias a que facilita la actividad de las demetilasas de H3K27, pero sin influir en la unión del complejo Polycomb 2 ni JMJD3. En conclusión, esta tesis caracteriza al NFAT5 como un nuevo regulador del sistema inmunitario implicado en el control de la accesibilidad local de la cromatina en respuesta a baja carga de patógenos

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.C. López-Rodríguez was supported by the Ramón y Cajal and I3 Researchers Programmes and grants from the Spanish Government (SAF2006-04913 and SAF2009-08066) and European Union (MCIRG516308). J. Aramburu was supported by grants from the Spanish Government (BFU2008-01070 and SAF2011-24268), and Distinció de la Generalitat de Catalunya per a la Promoció de la Recerca Universitària. Research in the laboratories of C. López-Rodríguez and J. Aramburu is also supported by Fundació la Marató TV3 (030230/31, 080730), Spanish Ministry of Health (ISCIII-RETIC RD06/0009-FEDER), and Generalitat de Catalunya (2009 SGR 601). M. Buxadé was supported by a Beatriu de Pinós postdoctoral contract from Generalitat de Catalunya. S. Iborra was supported by a Sara Borrell postdoctoral contract from the Ministry of Health of Spain. J. Minguillón and R. Berga-Bolaños were supported by predoctoral fellowships from the Spanish Government

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.C. López-Rodríguez was supported by the Ramón y Cajal and I3 Researchers Programmes and grants from the Spanish Government (SAF2006-04913 and SAF2009-08066) and European Union (MCIRG516308). J. Aramburu was supported by grants from the Spanish Government (BFU2008-01070 and SAF2011-24268), and Distinció de la Generalitat de Catalunya per a la Promoció de la Recerca Universitària. Research in the laboratories of C. López-Rodríguez and J. Aramburu is also supported by Fundació la Marató TV3 (030230/31, 080730), Spanish Ministry of Health (ISCIII-RETIC RD06/0009-FEDER), and Generalitat de Catalunya (2009 SGR 601). M. Buxadé was supported by a Beatriu de Pinós postdoctoral contract from Generalitat de Catalunya. S. Iborra was supported by a Sara Borrell postdoctoral contract from the Ministry of Health of Spain. J. Minguillón and R. Berga-Bolaños were supported by predoctoral fellowships from the Spanish Government