RESUMEN
The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.
Asunto(s)
Autoinmunidad/inmunología , Homeostasis/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Linfocitos T Reguladores/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/deficiencia , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genéticaRESUMEN
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.
Asunto(s)
Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , FN-kappa B/metabolismo , Linfocitos T/inmunología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Inmunomodulación , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Proteolisis , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
CARD-CC complexes involving BCL10 and MALT1 are major cellular signaling hubs. They govern NF-κB activation through their scaffolding properties as well as MALT1 paracaspase function, which cleaves substrates involved in NF-κB regulation. In human lymphocytes, gain-of-function defects in this pathway lead to lymphoproliferative disorders. CARD10, the prototypical CARD-CC protein in non-hematopoietic cells, is overexpressed in several cancers and has been associated with poor prognosis. However, regulation of CARD10 remains poorly understood. Here, we identified CARD10 as the first MALT1 substrate in non-hematopoietic cells and showed that CARD10 cleavage by MALT1 at R587 dampens its capacity to activate NF-κB. Preventing CARD10 cleavage in the lung tumor A549 cell line increased basal levels of IL-6 and extracellular matrix components in vitro, and led to increased tumor growth in a mouse xenograft model, suggesting that CARD10 cleavage by MALT1 might be a built-in mechanism controlling tumorigenicity.
RESUMEN
The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define proximal-promoters from a gene expression profiling study of murine experimental arthritis. Synovium expression profiles from progressing stages of collagen-induced arthritis (CIA) were classified into six distinct groups using k-means clustering. Using an algorithm based on local over-representation and comparative genomics, we identified putatively functional transcription factor-binding sites (TFBS) in TATA-dependent proximal-promoters. Applying a filter based on spacing between TATA box and transcription start site (TSS) combined with the presence of over-represented nuclear factor kappaB (NFkappaB), AP-1, or CCAAT/enhancer-binding protein beta (C/EBPbeta) sites, 382 candidate murine and human promoters were reduced to 66, corresponding to 45 genes. In vitro, 9 out of 10 computationally defined promoter regions conferred cytokine-inducible expression in murine cells and human synovial fibroblasts. Under these conditions, the serum amyloid A3 (Saa3) promoter showed the strongest transcriptional induction and strength. We applied this promoter for driving therapeutically efficacious levels of the interleukin-1 receptor antagonist (Il1rn) in a disease-regulated fashion. These results demonstrate the value of bioinformatics for guiding the selection of endogenous promoters for transcriptionally targeted gene therapy.
Asunto(s)
Artritis Reumatoide/terapia , Biología Computacional/métodos , Terapia Genética/métodos , Regiones Promotoras Genéticas/genética , Adenoviridae/genética , Algoritmos , Animales , Bovinos , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/fisiología , Lentivirus/genética , Masculino , Ratones , Modelos Genéticos , Células 3T3 NIH , Proteína Amiloide A Sérica/genéticaRESUMEN
A series of contiguous deletions were made in a cDNA encoding the ribonuclease restrictocin with the purpose of identifying the amino acids that are essential for the cleavage of the phosphodiester bond on the 3' side of G4325 in the alpha-sarcin/ricin domain of mammalian (rat) 28S rRNA. In all 93 of 149 amino acids, 62% of the residues in restrictocin, were not essential for the action of the toxin. Of the five residues that have been proposed to constitute the active site, three could be deleted without loss of activity if they were part of a deletion of three or five amino acids but not if they were removed singly. It is likely that the loss of these three residues is compensated for by a neighboring residue that occupies the structural space created by the larger amino acid deletions. This was demonstrated to be the case for the active site residue Glu95 which in the deletion mutant Delta91-95 is replaced by Asp90. Systematic deletion of amino acids is a rapid, cost effective method for identifying the residues in a protein likely to contribute directly to function and, hence, deserving of closer scrutiny. Moreover, a semiquantitative estimate of the contribution of the residue to function can be made. For this reason the method may be useful for functional proteomics.
Asunto(s)
Alérgenos , Citotoxinas/química , Proteínas Fúngicas/química , Ribonucleasas/química , Ribosomas/química , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Antígenos de Plantas , Citotoxinas/genética , ADN Complementario/química , Proteínas Fúngicas/genética , Eliminación de Gen , Lisina/química , Datos de Secuencia Molecular , Mutación , Plásmidos , Estructura Secundaria de Proteína , ARN Ribosómico 28S/química , Conejos , Ratas , Reticulocitos/química , Ribonucleasas/genéticaRESUMEN
IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Lipopolisacáridos/farmacología , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Inhibidor NF-kappaB alfa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Repeated injection of streptococcal cell wall (SCW) fragments results in chronic arthritis in mice. The objective of this study was to identify genes and pathways that determine disease progression based on gene expression profiling in this model. METHODS: Chronic arthritis was induced in mice by 4 injections of SCW fragments. RNA samples were isolated from synovial tissue obtained at various time points and were analyzed using mouse genome array and quantitative reverse transcription-polymerase chain reaction techniques. The functional role of potential key genes was evaluated in mice with specific gene deletions. RESULTS: Gene expression analyses revealed a shift in molecular signature. In contrast to an up-regulation of the inflammatory response pathway, the pathways involved in oxidative metabolism were significantly down-regulated during the chronic phase of arthritis. Since oxidative metabolism determines the mode of macrophage activation, we investigated phenotype switching in macrophages. Markers of alternatively activated macrophages, such as arginase 1, were at maximal levels during acute inflammation. In contrast, induction of markers of classically activated macrophages (M1), such as interleukin-1beta (IL-1beta) and inducible nitric oxide synthase (iNOS), was relatively low during the acute phase of disease, but highly increased toward the chronic phase. M1 polarization during the chronic phase was accompanied by a Th1 signature, characterized by IL-12p40, IL-12p35, and interferon-gamma. However, the absence of IL-12p40, but not IL-12p35, significantly inhibited the chronic phase of arthritis and was marked by a reduction in IL-17 and iNOS levels, as well as restored expression of oxidative metabolism genes. CONCLUSION: M1 polarization accompanied by a decline in oxidative metabolism determine the chronic phase of arthritis. IL-12p40, most likely acting through the IL-23/IL-17 axis, plays a critical role in this process.
Asunto(s)
Artritis Experimental/fisiopatología , Subunidad p40 de la Interleucina-12/fisiología , Activación de Macrófagos/fisiología , Enfermedad Aguda , Animales , Artritis Experimental/metabolismo , Biomarcadores/análisis , Enfermedad Crónica , Regulación hacia Abajo , Expresión Génica , Interleucina-17/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/análisis , Oxidación-Reducción , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.