RESUMO
In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Poliubiquitina/metabolismo , Príons/metabolismo , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/metabolismo , Vírus Sendai , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Cells express a plethora of interferon-stimulated genes (ISGs) in response to viral infection. Among these is ISG15, a ubiquitin-like protein (UBL) that can be covalently attached to both host and viral proteins. Here we review recent advances toward understanding the role and mechanism of ISG15 modification in antiviral defense.
Assuntos
Citocinas/imunologia , Ubiquitinas/imunologia , Viroses/imunologia , Vírus/imunologia , Animais , Humanos , CamundongosRESUMO
Nuclear factor kappa enhancer binding protein (NF-kappaB) regulates diverse biological processes including immunity, inflammation, and apoptosis. A vast array of cellular stimuli converges on NF-kappaB, and ubiquitination plays an essential role in the coordination of these signals to regulate NF-kappaB activity. At least three steps in NF-kappaB activation directly involve ubiquitination: proteasomal degradation of inhibitor of NF-kappaB (IkappaB), processing of NF-kappaB precursors, and activation of the transforming growth factor (TGF)-beta-activated kinase (TAK1) and IkappaB kinase (IKK) complexes. In this review, we discuss recent advances in the identification and characterization of ubiquitination and deubiquitination machinery that regulate NF-kappaB. Particular emphasis is given to proteasome-independent functions of ubiquitin, specifically its role in the activation of protein kinase complexes and in coordination of cell survival and apoptosis signals downstream of tumor necrosis factor alpha (TNFalpha).
Assuntos
NF-kappa B/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Animais , Apoptose , Sobrevivência Celular , Humanos , UbiquitinaçãoRESUMO
OBJECTIVES: Polymorphism in a coding region of deoxyribonuclease I-like III (DNASE1L3), causing amino acid substitution of Arg-206 to Cys (R206C), is a robustly replicated heritable risk factor for SSc and other autoimmune diseases. DNASE1L3 is secreted into the circulation, where it can digest genomic DNA (gDNA) in apoptosis-derived membrane vesicles (AdMVs). We sought to determine the impact of DNASE1L3 R206C on digestion of circulating gDNA in SSc patients and healthy controls (HCs). METHODS: The ability of DNASE1L3 to digest AdMV-associated gDNA was tested in vitro. The effect of R206C substitution on extracellular secretion of DNASE1L3 was determined using a transfected cell line and primary monocyte-derived dendritic cells from SSc patients. Plasma samples from SSc patients and HCs with DNASE1L3 R206C or R206 wild type were compared for their ability to digest AdMV-associated gDNA. The digestion status of endogenous gDNA in plasma samples from 123 SSc patients and 74 HCs was determined by measuring the proportion of relatively long to short gDNA fragments. RESULTS: The unique ability of DNASE1L3 to digest AdMV-associated gDNA was confirmed. Extracellular secretion of DNASE1L3 R206C was impaired. Plasma from individuals with DNASE1L3 R206C had reduced ability to digest AdMV-associated gDNA. The ratio of long: short gDNA fragments was increased in plasma from SSc patients with DNASE1L3 R206C, and this ratio correlated inversely with DNase activity. CONCLUSION: Our results confirm that circulating gDNA is a physiological DNASE1L3 substrate and show that its digestion is reduced in SSc patients with the DNASE1L3 R206C variant.
Assuntos
Ácidos Nucleicos Livres , Escleroderma Sistêmico , Humanos , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , DNA/genética , Genômica , Escleroderma Sistêmico/genética , DigestãoRESUMO
OBJECTIVES: SSc is associated with increased health-care resource utilization and economic burden. The Collaborative National Quality and Efficacy Registry (CONQUER) is a US-based collaborative that collects longitudinal follow-up data on SSc patients with <5 years of disease duration enrolled at scleroderma centres in the USA. The objective of this study was to investigate the relationship between gastrointestinal tract symptoms and self-reported resource utilization in CONQUER participants. METHODS: CONQUER participants who had completed a baseline and 12-month Gastrointestinal Tract Questionnaire (GIT 2.0) and a Resource Utilization Questionnaire (RUQ) were included in this analysis. Patients were categorized by total GIT 2.0 severity: none-to-mild (0-0.49); moderate (0.50-1.00), and severe-to-very severe (1.01-3.00). Clinical features and medication exposures were examined in each of these categories. The 12-month RUQ responses were summarized by GIT 2.0 score categories at 12 months. RESULTS: Among the 211 CONQUER participants who met the inclusion criteria, most (64%) had mild GIT symptoms, 26% had moderate symptoms, and 10% severe GIT symptoms at 12 months. The categorization of GIT total severity score by RUQ showed that more upper endoscopy procedures and inpatient hospitalization occurred in the CONQUER participants with severe GIT symptoms. These patients with severe GIT symptoms also reported the use of more adaptive equipment. CONCLUSION: This report from the CONQUER cohort suggests that severe GIT symptoms result in more resource utilization. It is especially important to understand resource utilization in early disease cohorts when disease activity, rather than damage, primarily contributes to health-related costs of SSc.
Assuntos
Gastroenteropatias , Escleroderma Sistêmico , Humanos , Gastroenteropatias/etiologia , Inquéritos e Questionários , Autorrelato , Sistema de Registros , Escleroderma Sistêmico/complicaçõesRESUMO
OBJECTIVES: Determine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time. METHODS: A total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions. RESULTS: Gene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up. CONCLUSIONS: Skin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design.
Assuntos
Esclerodermia Difusa , Esclerodermia Localizada , Escleroderma Sistêmico , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Esclerodermia Difusa/patologia , Esclerodermia Localizada/metabolismo , Escleroderma Sistêmico/patologia , Pele/patologiaRESUMO
OBJECTIVES: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease. METHODS: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated. RESULTS: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression. CONCLUSIONS: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.
Assuntos
Imunidade Adaptativa/genética , Imunidade Inata/genética , Esclerodermia Difusa/genética , Esclerodermia Difusa/imunologia , Adulto , Biomarcadores/análise , Biópsia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos , Sistema de Registros , Análise de Regressão , Esclerodermia Difusa/patologia , Análise de Sequência de RNA , Índice de Gravidade de Doença , Pele/imunologia , Pele/patologia , TranscriptomaRESUMO
Systemic Sclerosis (Scleroderma, SSc) is a multifaceted disease characterized by autoimmunity, vasculopathy, and fibrosis affecting the skin and internal organs. Despite advances in the understanding and treatment of SSc in recent years, SSc continues to cause reduced quality of life and premature mortality. Type I interferons (IFNs), a family of cytokines with essential roles in the immune response to microbial infection, play a pathogenic role in certain autoimmune diseases (reviewed elsewhere in this edition). Polymorphisms in interferon-regulatory factors confer an increased risk of SSc, and IFN excess is evident in the blood and skin of a large percentage of SSc patients. Here we describe the evidence of Type I IFN dysregulation in SSc, revealed predominately by genetics and gene expression profiling. We also discuss evidence regarding mechanisms by which Type I IFN might contribute to SSc pathogenesis, mechanisms driving excess Type I IFN production in SSc, and the potential roles of Type I IFNs as biomarkers and therapeutic targets in SSc.
Assuntos
Interferon Tipo I/fisiologia , Escleroderma Sistêmico/etiologia , Animais , Biomarcadores , Humanos , Fatores Reguladores de Interferon/genética , Interferon Tipo I/metabolismo , Camundongos , Polimorfismo Genético , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/terapia , Transdução de SinaisRESUMO
Central tolerance checkpoints are critical for the elimination of autoreactive B cells and the prevention of autoimmunity. When autoreactive B cells encounter their Ag at the immature B cell stage, BCR cross-linking induces receptor editing, followed by apoptosis if edited cells remain autoreactive. Although the transcription factor Foxo1 is known to promote receptor editing, the role of the related factor Foxo3 in central B cell tolerance is poorly understood. We find that BCR-stimulated immature B cells from Foxo3-deficient mice demonstrate reduced apoptosis compared with wild type cells. Despite this, Foxo3-/- mice do not develop increased autoantibodies. This suggests that the increased survival of Foxo3-/- immature B cells allows additional rounds of receptor editing, resulting in more cells "redeeming" themselves by becoming nonautoreactive. Indeed, increased Igλ usage and increased recombining sequence recombination among Igλ-expressing cells were observed in Foxo3-/- mice, indicative of increased receptor editing. We also observed that deletion of high-affinity autoreactive cells was intact in the absence of Foxo3 in the anti-hen egg lysozyme (HEL)/membrane-bound HEL model. However, Foxo3 levels in B cells from systemic lupus erythematosus (SLE) patients were inversely correlated with disease activity and reduced in patients with elevated anti-dsDNA Abs. Although this is likely due in part to increased B cell activation in these SLE patients, it is also possible that low-affinity B cells that remain autoreactive after editing may survive inappropriately in the absence of Foxo3 and become activated to secrete autoantibodies in the context of other SLE-associated defects.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Proteína Forkhead Box O3/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Diferenciação Celular/imunologia , Feminino , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologiaRESUMO
PURPOSE OF REVIEW: To discuss recent advances in identification of biomarkers in systemic sclerosis for disease severity, prognosis, and treatment response. RECENT FINDINGS: Recent reports describe novel circulating markers of disease severity, autoantibody associations with specific manifestations including cancer, and skin gene expression-based predictors of modified Rodnan skin score progression and treatment response. Moreover, there is converging evidence that C-reactive protein and pneumoproteins such as Krebs von den Lungen-6 and chemokine ligand 18 could serve as prognostic biomarkers in systemic sclerosis-associated interstitial lung disease. SUMMARY: Several novel biomarkers show promise in improving the assessment of systemic sclerosis (SSc) disease severity, prognosis, and treatment response. Their potential utility in prospective selection of patients for clinical trials and in individual patient management require additional research.
Assuntos
Mucina-1/metabolismo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Autoanticorpos , Biomarcadores/metabolismo , Humanos , PrognósticoRESUMO
OBJECTIVES: There is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis. METHODS: SSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed. RESULTS: IRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-ß signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression. CONCLUSIONS: IRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-ß-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc.
Assuntos
Inflamação/genética , Fator Regulador 7 de Interferon/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Pele/patologia , Animais , Bleomicina , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Camundongos Knockout , Escleroderma Sistêmico/induzido quimicamente , Transdução de Sinais/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20's seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.
Assuntos
Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , NF-kappa B/metabolismo , Proteínas Nucleares , Transdução de Sinais/genética , Dedos de Zinco/genética , Autoimunidade/genética , Proteínas de Ligação a DNA , Ativação Enzimática/genética , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Imunoprecipitação , Inflamação/genética , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Poliubiquitina , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , UbiquitinaçãoRESUMO
OBJECTIVES: Patients with clinically evident autoimmune disease are at increased risk for premature cardiovascular disease (CVD). Markers of serological autoimmunity such as anti-nuclear antibodies (ANA) are found in approximately 25% of the general population. Yet, the vast majority will not develop clinical autoimmune disease. Serological autoimmunity is a risk factor for CVD death in individuals without autoimmune disease; however, the mechanisms mediating this excess CVD risk have not been elucidated. METHODS: We examined associations of ANA with traditional cardiovascular risk factors, inflammatory mediators, and vascular biomarkers in the Dallas Heart Study - a large, representative multiethnic population-based cohort. Plasma ANA were measured by enzyme linked immunosorbent assay in 3,488 Dallas Heart Study participants aged 30 to 65 years who do not have known rheumatologic disease. Associations of ANA with demographic characteristics, cardiovascular risk factors, and biomarkers were assessed using univariable and multivariable linear regression. RESULTS: Factors independently associated with higher ANA include female sex, African-American race/ethnicity, soluble intracellular adhesion molecule-1, soluble CD40 ligand, chemokine CXCL-2, and Cystatin C (p<0.05 for each). ANA was not associated with traditional cardiovascular risk factors, high sensitivity C-reactive protein, coronary artery calcium scores, or aortic wall thickness. CONCLUSION: ANA are associated with inflammatory mediators and biomarkers of vascular activation, but not with traditional cardiovascular risk factors in a multiethnic population-based cohort. These findings suggest that the cardiovascular risk associated with ANA may involve pathways distinct from traditional risk factors and include dysregulation of endothelial cells and the immune system.
Assuntos
Anticorpos Antinucleares/imunologia , Autoimunidade , Doenças Cardiovasculares/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Adulto , Idoso , Anticorpos Antinucleares/sangue , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Feminino , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Inflamação/epidemiologia , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Medição de Risco , Fatores de Risco , Texas/epidemiologiaRESUMO
Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Ets1 transcription factor acts in B cells to prevent PC differentiation. Ets1(-/-) mice accumulate PCs and produce autoantibodies. Ets1 expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Ets1 levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoantibodies, and an autoimmune phenotype similar to that of Ets1(-/-) mice. Defects in inhibitory signaling molecules, including Lyn and Ets1, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. In this study, we explore the effect of partial disruption of the Lyn/Ets1 pathway on B cell tolerance and find that Lyn(+/-)Ets1(+/-) mice demonstrate greater and earlier production of IgM, but not IgG, autoantibodies compared with Lyn(+/-) or Ets1(+/-) mice. We also show that Btk-dependent downregulation of Ets1 is important for normal PC homeostasis when inhibitory signaling is intact. Ets1 deficiency restores the decrease in steady state PCs and Ab levels observed in Btk(-/-) mice. Thus, depending on the balance of activating and inhibitory signals to Ets1, there is a continuum of effects on autoantibody production and PC maintenance. This ranges from full-blown autoimmunity with complete loss of Ets1-maintaining signals to reduced PC and Ab levels with impaired Ets1 downregulation.
Assuntos
Anticorpos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteína Proto-Oncogênica c-ets-1/imunologia , Quinases da Família src/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Anticorpos/metabolismo , Autoanticorpos/sangue , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Epistasia Genética , Citometria de Fluxo , Expressão Gênica/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Esplenomegalia/genética , Esplenomegalia/imunologia , Esplenomegalia/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Lysine-63 (K63)-linked polyubiquitination has emerged as a mechanism regulating diverse cellular functions, including activation of the protein kinase IKK in the NF-kappaB pathways. However, genetic evidence for a key role of K63 polyubiquitination in IKK activation is lacking. Here, we devise a tetracycline-inducible RNAi strategy to replace endogenous ubiquitin with a K63R mutant in a human cell line. We demonstrate that K63 of ubiquitin and the catalytic activity of Ubc13, an E2 that catalyzes K63 polyubiquitination, are required for IKK activation by IL-1beta, but surprisingly, not by TNFalpha. We further show that IKK activation by TNFalpha requires Ubc5, which functions with the E3 cIAP1 to catalyze polyubiquitination of RIP1 not restricted to K63 of ubiquitin. These results indicate that distinct ubiquitin-dependent mechanisms are employed for IKK activation by different pathways. The ubiquitin replacement methodology described here provides a means to investigate the function of polyubiquitin topology in various cellular processes.
Assuntos
Técnicas Genéticas , Quinase I-kappa B/metabolismo , Interleucina-1beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina/genética , Ubiquitina/metabolismo , Biocatálise/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Introdução de Genes , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Lisina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poliubiquitina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Tetraciclina/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Fibroblasts constitute a heterogeneous population of cells. In this study, we integrated single-cell RNA-sequencing and bulk RNA-sequencing data as well as clinical information to study the role of individual fibroblast populations in systemic sclerosis (SSc). SSc skin demonstrated an increased abundance of COMP+, COL11A1+, MYOC+, CCL19+, SFRP4/SFRP2+, and PRSS23/SFRP2+ fibroblasts signatures and decreased proportions of CXCL12+ and PI16+ fibroblast signatures in the Prospective Registry of Early Systemic Sclerosis and Genetics versus Environment in Scleroderma Outcome Study cohorts. Numerical differences were confirmed by multicolor immunofluorescence for selected fibroblast populations. COMP+, COL11A1+, SFRP4/SFRP2+, PRSS23/SFRP2+, and PI16+ fibroblasts were similarly altered between normal wound healing and patients with SSc. The proportions of profibrotic COMP+, COL11A1+, SFRP4/SFRP2+, and PRSS23/SFRP2+ and proinflammatory CCL19+ fibroblast signatures were positively correlated with clinical and histopathological parameters of skin fibrosis, whereas signatures of CXCL12+ and PI16+ fibroblasts were inversely correlated. Incorporating the proportions of COMP+, COL11A1+, SFRP4/SFRP2+, and PRSS23/SFRP2+ fibroblast signatures into machine learning models improved the classification of patients with SSc into those with progressive versus stable skin fibrosis. In summary, the profound imbalance of fibroblast subpopulations in SSc may drive the progression of skin fibrosis. Specific targeting of disease-relevant fibroblast populations may offer opportunities for the treatment of SSc and other fibrotic diseases.
Assuntos
Fibroblastos , Escleroderma Sistêmico , Pele , Humanos , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Feminino , Pele/patologia , Pele/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto , Fibrose , Estudos Prospectivos , Análise de Célula Única , CicatrizaçãoRESUMO
Dermal fibrosis is a cardinal feature of systemic sclerosis (SSc) for which there are limited treatment strategies. This is in part due to our fragmented understanding of how dermal white adipose tissue (DWAT) contributes to skin fibrosis. We identified elevated sine oculis homeobox homolog 1 (SIX1) expression in SSc skin samples from the GENISOS and PRESS cohorts, the expression of which correlated with adipose-associated genes and molecular pathways. SIX1 localization studies identified increased signals in the DWAT area in SSc and in experimental models of skin fibrosis. Global and adipocyte specific Six1 deletion abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage induced by SQ bleomycin treatment. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. However, SIX1 deletion did not appear to affect cellular trans differentiation. Taken together these results point at SIX1 as a potential target for dermal fibrosis in SSc.
RESUMO
Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive multiorgan fibrosis. While the cause of SSc remains unknown, a perturbed vasculature is considered a critical early step in the pathogenesis. Using fibrinogen as a marker of vascular leakage, we found extensive extravascular fibrinogen deposition in the dermis of both limited and diffuse systemic sclerosis disease, and it was present in both early and late-stage patients. Based on a timed series of excision wounds, retention on the fibrin deposit of the splice variant domain, fibrinogen αEC, indicated a recent event, while fibrin networks lacking the αEC domain were older. Application of this timing tool to SSc revealed considerable heterogeneity in αEC domain distribution providing unique insight into disease activity. Intriguingly, the fibrinogen-αEC domain also accumulated in macrophages. These observations indicate that systemic sclerosis is characterized by ongoing vascular leakage resulting in extensive interstitial fibrin deposition that is either continually replenished and/or there is impaired fibrin clearance. Unresolved fibrin deposition might then incite chronic tissue remodeling.
RESUMO
OBJECTIVE: To assess the predictive significance of blood neutrophil count and the ratio between neutrophil and lymphocyte count (neutrophil-to-lymphocyte ratio [NLR]) for disease severity and mortality in systemic sclerosis (SSc). METHODS: Neutrophil and lymphocyte counts were prospectively measured in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) and the Scleroderma Lung Study II (SLS II). Forced vital capacity percent predicted (FVC%) and modified Rodnan skin thickness score (MRSS) were used as surrogate measures for disease severity. Longitudinal analyses were performed using generalized linear mixed models. Cox proportional hazards models evaluated the predictive significance of these cell counts for mortality. RESULTS: Of the 447 SSc patients in the GENISOS cohort at the time of analysis, 377 (84.3%) had available baseline blood neutrophil and lymphocyte counts. Higher baseline neutrophil count and NLR predicted lower serially obtained FVC% (b = -4.74, P = 0.009 and b = -2.68, P = 0.028, respectively) and higher serially obtained MRSS (b = 4.07, P < 0.001 and b = 2.32, P < 0.001, respectively). Longitudinal neutrophil and NLR measurements also significantly correlated with lower concurrently obtained FVC% measurements and higher concurrently obtained MRSS. Baseline neutrophil count and NLR predicted increased risk of long-term mortality, even after adjustment for baseline demographic and clinical factors (hazard ratio [HR] 1.42, P = 0.02 and HR 1.48, P < 0.001, respectively). The predictive significance of higher baseline neutrophil count and NLR for declining FVC% and increased long-term mortality was confirmed in the SLS II. CONCLUSION: Higher blood neutrophil count and NLR are predictive of more severe disease course and increased mortality, indicating that these easily obtainable laboratory studies might be a reflection of pathologic immune processes in SSc.
Assuntos
Neutrófilos , Escleroderma Sistêmico , Humanos , Linfócitos , Progressão da Doença , Pele , Contagem de LinfócitosRESUMO
Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.