RESUMEN
A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.
Asunto(s)
Artritis Reumatoide , Trampas Extracelulares , Lupus Eritematoso Sistémico , Humanos , Animales , Ratones , Citrulinación , Artritis Reumatoide/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Inflamación/metabolismo , Autoinmunidad/genética , Trampas Extracelulares/metabolismoRESUMEN
The intestinal epithelium is a single cell layer that is constantly renewed and acts as a physical barrier that separates intestinal microbiota from underlying tissues. In inflammatory bowel disease (IBD) in humans, as well as in experimental mouse models of IBD, this barrier is impaired, causing microbial infiltration and inflammation. Deficiency in OTU deubiquitinase with linear linkage specificity (OTULIN) causes OTULIN-related autoinflammatory syndrome (ORAS), a severe inflammatory pathology affecting multiple organs including the intestine. We show that mice with intestinal epithelial cell (IEC)-specific OTULIN deficiency exhibit increased susceptibility to experimental colitis and are highly sensitive to TNF toxicity, due to excessive apoptosis of OTULIN deficient IECs. OTULIN deficiency also increases intestinal pathology in mice genetically engineered to secrete excess TNF, confirming that chronic exposure to TNF promotes epithelial cell death and inflammation in OTULIN deficient mice. Mechanistically we demonstrate that upon TNF stimulation, OTULIN deficiency impairs TNF receptor complex I formation and LUBAC recruitment, and promotes the formation of the cytosolic complex II inducing epithelial cell death. Finally, we show that OTULIN deficiency in IECs increases susceptibility to Salmonella infection, further confirming the importance of OTULIN for intestinal barrier integrity. Together, these results identify OTULIN as a major anti-apoptotic protein in the intestinal epithelium and provide mechanistic insights into how OTULIN deficiency drives gastrointestinal inflammation in ORAS patients.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Mucosa Intestinal , Animales , Humanos , Ratones , Apoptosis , Muerte Celular , InflamaciónRESUMEN
The anti-inflammatory protein A20 serves as a critical brake on NF-κB signaling and NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-κB activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-κB signaling to control osteoclast differentiation, assuring proper bone development and turnover.
Asunto(s)
FN-kappa B , Humanos , Animales , RatonesRESUMEN
OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.
Asunto(s)
Endopeptidasas/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Animales , Muerte Celular/genética , Citocinas/metabolismo , Endopeptidasas/genética , Proteína de Dominio de Muerte Asociada a Fas , Técnicas de Sustitución del Gen , Homeostasis , Inflamación/patología , Interferón Tipo I , Interleucina-1beta , Ratones , Necroptosis , Fragmentos de Péptidos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Piel/patología , Células Madre/metabolismo , Análisis de Sistemas , Ubiquitina/metabolismoRESUMEN
Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.
Asunto(s)
Ácidos Grasos/metabolismo , Obesidad/patología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Tejido Adiposo Blanco/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hidroliasas/genética , Hidroliasas/metabolismo , Resistencia a la Insulina , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Obesidad/metabolismo , Consumo de Oxígeno , Palmitatos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficiencia , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.
Asunto(s)
Apoptosis , Enterobacteriaceae/crecimiento & desarrollo , Enterobacteriaceae/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Intestinos/citología , Intestinos/microbiología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/patología , Femenino , Enfermedades Transmitidas por los Alimentos/microbiología , Vida Libre de Gérmenes , Interacciones Huésped-Patógeno , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Masculino , Ratones , Mucositis/inducido químicamente , Salmonella/enzimología , Salmonella/genética , Salmonella/crecimiento & desarrollo , Salmonella/metabolismo , Transcriptoma , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
Asunto(s)
Inflamación/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Epiteliales/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Poliubiquitina/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Dedos de Zinc/fisiologíaRESUMEN
Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) significantly protects mice from developing liver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Endopeptidasas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/antagonistas & inhibidores , Hepatitis/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Células CHO , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Cricetulus , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Hepatitis/genética , Hepatitis/patología , Humanos , Interferón Tipo I/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de SeñalRESUMEN
The original version of this article contained an error in the name of one of the co-authors (Wim Declercq). This has been corrected in the PDF and HTML versions.
RESUMEN
A20 is a potent anti-inflammatory protein, acting by inhibiting nuclear factor κB (NF-κB) signaling and inflammatory gene expression and/or by preventing cell death. Mutations in the A20/TNFAIP3 gene have been associated with a plethora of inflammatory and autoimmune pathologies in humans and in mice. Although the anti-inflammatory role of A20 is well accepted, fundamental mechanistic questions regarding its mode of action remain unclear. Here, we review new findings that further clarify the molecular and cellular mechanisms by which A20 controls inflammatory signaling and cell death, and discuss new evidence for its involvement in inflammatory and autoimmune disease development.
Asunto(s)
Autoinmunidad , Muerte Celular , Predisposición Genética a la Enfermedad , Inflamación/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antiinflamatorios/farmacología , Enfermedades Autoinmunes/inmunología , Humanos , Ratones , Ratones Noqueados , Mutación , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Polimorfismo de Nucleótido Simple , Conformación Proteica , Dominios Proteicos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Ubiquitina/químicaRESUMEN
The cytokine TNF promotes inflammation either directly by activating the MAPK and NF-κB signaling pathways, or indirectly by triggering cell death. A20 is a potent anti-inflammatory molecule, and mutations in the gene encoding A20 are associated with a wide panel of inflammatory pathologies, both in human and in the mouse. Binding of TNF to TNFR1 triggers the NF-κB-dependent expression of A20 as part of a negative feedback mechanism preventing sustained NF-κB activation. Apart from acting as an NF-κB inhibitor, A20 is also well-known for its ability to counteract the cytotoxic potential of TNF. However, the mechanism by which A20 mediates this function and the exact cell death modality that it represses have remained incompletely understood. In the present study, we provide in vitro and in vivo evidences that deletion of A20 induces RIPK1 kinase-dependent and -independent apoptosis upon single TNF stimulation. We show that constitutively expressed A20 is recruited to TNFR1 signaling complex (Complex I) via its seventh zinc finger (ZF7) domain, in a cIAP1/2-dependent manner, within minutes after TNF sensing. We demonstrate that Complex I-recruited A20 protects cells from apoptosis by stabilizing the linear (M1) ubiquitin network associated to Complex I, a process independent of its E3 ubiquitin ligase and deubiquitylase (DUB) activities and which is counteracted by the DUB CYLD, both in vitro and in vivo. In absence of linear ubiquitylation, A20 is still recruited to Complex I via its ZF4 and ZF7 domains, but this time protects the cells from death by deploying its DUB activity. Together, our results therefore demonstrate two distinct molecular mechanisms by which constitutively expressed A20 protect cells from TNF-induced apoptosis.
Asunto(s)
Receptores Tipo I de Factores de Necrosis Tumoral/efectos adversos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/uso terapéutico , Ubiquitina/efectos de los fármacos , Animales , Apoptosis , Humanos , Ratones , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Alelos , Animales , Humanos , Ratones , FosforilaciónRESUMEN
The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20-/- cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20-/- cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis.
Asunto(s)
Homeostasis , Células Asesinas Naturales/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Supervivencia Celular , Linfopenia/metabolismo , Linfopenia/patología , Ratones , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficienciaRESUMEN
Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1ß release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.
Asunto(s)
Artritis/genética , Inflamación/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Animales , Artritis/inducido químicamente , Artritis/patología , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación/inducido químicamente , Inflamación/patología , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Mutación , FN-kappa B/genética , Necrosis/genética , Necrosis/patología , Unión Proteica , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Ubiquitina/genéticaRESUMEN
Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Repeticiones WD40/genética , Animales , Autofagia/inmunología , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Endoscopía , Femenino , Homeostasis/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/citología , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/inmunología , Proteómica , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunología , Repeticiones WD40/inmunologíaRESUMEN
Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-κB) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microglia-confined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS)-like disease, due to hyperactivation of the Nlrp3 inflammasome leading to enhanced interleukin-1ß secretion and CNS inflammation. Finally, we confirm a Nlrp3 inflammasome signature and IL-1ß expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.
Asunto(s)
Inflamasomas/inmunología , Microglía/inmunología , Esclerosis Múltiple/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-1beta/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Microglía/patología , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/patología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Transducción de Señal/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
A20 is a negative regulator of NF-κB signaling; it controls inflammatory responses and ensures tissue homeostasis. A20 is thought to restrict NF-κB activation both by its ubiquitin-editing activity as well as by its nonenzymatic activities. Besides its role in NF-κB signaling, A20 also acts as a protective factor inhibiting apoptosis and necroptosis. Because of the ability of A20 to both ubiquitinate and deubiquitinate substrates, and its involvement in many cellular processes, we hypothesized that deletion of A20 might generally impact on protein levels, thereby disrupting cellular signaling. We performed a differential proteomics study on bone marrow-derived macrophages (BMDMs) from control and myeloid-specific A20 knockout mice, both in untreated conditions and after LPS or TNF treatment, and demonstrated A20-dependent changes in protein expression. Several inflammatory proteins were found up-regulated in the absence of A20, even without an inflammatory stimulus, but, depending on the treatment and the treatment time, more proteins were found regulated. Together these protein changes may affect normal signaling events, which may disturb tissue homeostasis and induce (autoimmune) inflammation, in agreement with A20s proposed identity as a susceptibility gene for inflammatory disease. We further verify that immune-responsive gene 1 (IRG1) is up-regulated in the absence of A20 and that its levels are transcriptionally regulated.
Asunto(s)
Hidroliasas/metabolismo , Proteómica/métodos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficiencia , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Hidroliasas/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Transcripción Genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
OBJECTIVES: A20 is an important endogenous regulator of inflammation. Single nucleotide polymorphisms in A20 have been associated with various immune-mediated inflammatory diseases, and cell-specific deletion of A20 results in diverse inflammatory phenotypes. Our goal was to delineate the underlying mechanisms of joint inflammation in myeloid-specific A20-deficient mice (A20myelKO mice). METHODS: Inflammation in A20myelKO mice was assessed in a time-dependent manner. Western blot analysis and quantitative PCR analysis were performed on bone marrow-derived macrophages from A20myelKO and littermate control mice to study the effect of A20 on STAT1/STAT3 expression and STAT1/STAT3-dependent gene transcription in myeloid cells. The in vivo role of Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling in the development of enthesitis in A20myelKO mice was assessed following administration of a JAK inhibitor versus placebo control. RESULTS: Enthesitis was found to be an early inflammatory lesion in A20myelKO mice. A20 negatively modulated STAT1-dependent, but generally not STAT3-dependent gene transcription in myeloid cells by suppressing STAT1 but not STAT3 expression, both in unstimulated conditions and after interferon-γ or interleukin-6 stimulation. The increase in STAT1 gene transcription in the absence of A20 was shown to be JAK-STAT-dependent. Moreover, JAK inhibition in vivo resulted in significant reduction of enthesitis, both clinically and histopathologically. CONCLUSIONS: Our data reveal an important and novel interplay between myeloid cells and tissue resident cells at entheseal sites that is regulated by A20. In the absence of A20, STAT1 but not STAT3 expression is enhanced leading to STAT1-dependent inflammation. Therefore, A20 acts as a novel endogenous regulator of STAT1 that prevents onset of enthesitis.