RESUMEN
BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.
Asunto(s)
Agammaglobulinemia/genética , Síndromes Periódicos Asociados a Criopirina/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Inmunidad Adaptativa , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Bacterianas/inmunología , Células Cultivadas , Humanos , Inmunidad Innata , Leucocidinas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Proteínas NLR , Nigericina/inmunología , Proteínas Tirosina Quinasas/genética , Proteómica , Dominio Pirina/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptor de Lamina BRESUMEN
MicroRNAs are important posttranscriptional regulators of gene expression, which have been shown to fine-tune innate immune responses downstream of pattern recognition receptor (PRR) signaling. This study identifies miR-650 as a novel PRR-responsive microRNA that is downregulated upon stimulation of primary human monocyte-derived dendritic cells (MDDCs) with a variety of different microbe-associated molecular patterns. A comprehensive target search combining in silico analysis, transcriptional profiling, and reporter assays reveals that miR-650 regulates several well-known interferon-stimulated genes, including IFIT2 and MXA. In particular, downregulation of miR-650 in influenza A infected MDDCs enhances the expression of MxA and may therefore contribute to the establishment of an antiviral state. Together these findings reveal a novel link between miR-650 and the innate immune response in human MDDCs.
Asunto(s)
Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Virus de la Influenza A/inmunología , MicroARNs/inmunología , Proteínas de Resistencia a Mixovirus/biosíntesis , Células Cultivadas , Células Dendríticas/metabolismo , Regulación hacia Abajo , Citometría de Flujo , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Innata/inmunología , Immunoblotting , MicroARNs/genética , Microscopía Confocal , Proteínas de Resistencia a Mixovirus/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Reconocimiento de Patrones/inmunología , Transducción de Señal , TransfecciónRESUMEN
Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor κB signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization.
