RESUMEN
Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.
Asunto(s)
Proteínas ADAM , Infecciones por Cardiovirus , Virus de la Encefalomiocarditis , Inmunidad Innata , Interferón Tipo I , Helicasa Inducida por Interferón IFIH1 , Proteínas de la Membrana , Miocarditis , Animales , Ratones , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Infecciones por Cardiovirus/inmunología , Infecciones por Cardiovirus/virología , Virus de la Encefalomiocarditis/inmunología , Células HEK293 , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/inmunología , Miocarditis/virología , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial. OBJECTIVES: To determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura. METHODS: Fluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti-ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis-based Western blotting were employed to assess plasma ADAMTS-13 antigen. RESULTS: We showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen. CONCLUSION: We conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.
Asunto(s)
Proteínas ADAM , Proteína ADAMTS13 , Autoanticuerpos , Ensayo de Inmunoadsorción Enzimática , Púrpura Trombocitopénica Trombótica , Proteína ADAMTS13/sangre , Proteína ADAMTS13/inmunología , Humanos , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/inmunología , Púrpura Trombocitopénica Trombótica/diagnóstico , Púrpura Trombocitopénica Trombótica/enzimología , Autoanticuerpos/sangre , Masculino , Proteínas ADAM/sangre , Proteínas ADAM/inmunología , Proteínas ADAM/deficiencia , Adulto , Femenino , Persona de Mediana Edad , Inmunoglobulina G/sangre , Transferencia Resonante de Energía de Fluorescencia , Western Blotting , Factor de von Willebrand/metabolismo , Factor de von Willebrand/análisis , AncianoRESUMEN
Introduction: Vascular smooth muscle cells (VSMCs) are highly involved in airway vascular remodeling in asthma. Objectives: This study aimed to investigate the mechanisms underlying the effects of a disintegrin and metalloproteinase-33 (ADAM33) gene on the migration capacity and inflammatory cytokine secretion of VSMCs. Methods: Human aortic smooth muscle cells (HASMCs) were transfected with lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 or negative control vectors. The migration capacity of HASMCs was evaluated by a transwell assay. The levels of secreted inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative polymerase chain reaction and Western blot assays were performed to detect mRNA and protein expression levels. Results: Silencing of ADAM33 significantly inhibited the migration of HASMCs. The expression of tumor necrosis factor alpha (TNF-α) in the supernatant of HASMCs was decreased, while that of interferon gamma (IFN-γ) was increased after the transfection of shRNA targeting ADAM33. Insufficient ADAM33 expression also suppressed the expression levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTOR), Rho-associated protein kinases, phospho-forkhead box protein O1 (FOXO1), and cyclin D1, but it did not affect the levels of AKT, mTOR, or Rho. Conclusion: Silencing of the ADAM33 gene inhibited HASMC migration and regulated inflammatory cytokine secretion via targeting the PI3K/AKT/mTOR pathway and its downstream signaling. These data contribute to a better understanding of the regulatory mechanisms of airway vascular remodeling in asthma.
Asunto(s)
Proteínas ADAM , Remodelación de las Vías Aéreas (Respiratorias) , Asma , Silenciador del Gen , Músculo Liso Vascular , Remodelación Vascular , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/genética , Asma/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Silenciador del Gen/fisiología , Humanos , Músculo Liso Vascular/inmunología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/farmacología , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunología , Remodelación Vascular/genética , Remodelación Vascular/inmunologíaRESUMEN
Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.
Asunto(s)
Proteínas ADAM/inmunología , Colitis/inmunología , Matriz Extracelular/metabolismo , Fibroblastos/inmunología , Mucosa Intestinal/inmunología , Células Madre Mesenquimatosas/inmunología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Animales , Diferenciación Celular , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/inmunología , Colon/patología , Matriz Extracelular/inmunología , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Inflamación , Interleucina-11/genética , Interleucina-11/inmunología , Mucosa Intestinal/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Dodecil Sulfato de Sodio/administración & dosificación , Transcripción Genética , Transcriptoma , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunologíaRESUMEN
For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.
Asunto(s)
Adhesión Celular/fisiología , Leucocitos/enzimología , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/fisiología , Proteínas ADAM/inmunología , Proteínas ADAM/fisiología , ADP-Ribosil Ciclasa/inmunología , ADP-Ribosil Ciclasa/fisiología , ADP-Ribosil Ciclasa 1/inmunología , ADP-Ribosil Ciclasa 1/fisiología , Antígenos CD/inmunología , Antígenos CD/fisiología , Antígenos CD13/inmunología , Antígenos CD13/fisiología , Adhesión Celular/inmunología , Membrana Celular/enzimología , Membrana Celular/inmunología , Dipeptidil Peptidasa 4/inmunología , Dipeptidil Peptidasa 4/fisiología , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/fisiología , Humanos , Leucocitos/inmunología , Leucocitos/fisiología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Modelos BiológicosRESUMEN
ADAM15 is highly expressed in malignant tumors and is correlated with tumor progression. However, the role of ADAM15 in hepatocellular carcinoma (HCC) remains unclear. In the study, our results indicated that ADAM15 was highly expressed in HCC tissues and cells compared with corresponding tissues and liver cells. Overexpression of ADAM15 was linked to poor prognosis, and was an independent risk factor for HCC prognosis. Besides, analysis of immune infiltration indicated that ADAM15 expression was related to tumor infiltrating lymphocytes based on the TIMER, TISIDB and GEPIA databases. Many immune checkpoint gene expression was associated with ADAM15 expression. Functional enrichment analyses indicated that apoptosis, cell adhesion was enriched. ADAM15 knockdown promoted apoptosis and suppressed proliferation, migration and invasion of liver cancer cells. The findings of western blot showed that ADAM15 knockdown reduced the expression of Bcl-2, Vimentin, N-Cadherin and Snail, and elevated the expression of Bax, E-cadherin and ZO-1. However, overexpression of ADAM15 had the opposite results. Collectively, our findings demonstrated that ADAM15 was connected with poor prognosis of HCC patients, and could be considered as a potential biomarker for the diagnosis and treatment of HCC.
Asunto(s)
Proteínas ADAM/inmunología , Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/inmunología , Proteínas de la Membrana/inmunología , Proteínas ADAM/genética , Anciano , Anciano de 80 o más Años , Apoptosis , Cadherinas/genética , Cadherinas/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/fisiopatología , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunologíaRESUMEN
The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.
Asunto(s)
Proteínas ADAM/inmunología , Caspasas/inmunología , Colitis , Furina/inmunología , Enfermedad Injerto contra Huésped , Inmunoterapia , Linfocitos T Reguladores/inmunología , Animales , Ingeniería Celular , Colitis/inmunología , Colitis/patología , Colitis/terapia , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/terapia , Humanos , Ratones , Linfocitos T Reguladores/patologíaRESUMEN
Objective: The inflammatory mechanisms underpinning asthma-chronic obstructive pulmonary disease (COPD) overlap syndrome (ACOS) have not been fully elucidated. Here, we examined the levels of cysteinyl leukotrienes (cys-LTs), prostaglandin D2 (PG-D2), prostaglandin E2 (PG-E2), interleukin 5 (IL-5), and a disintegrin and metalloprotease domain (ADAM 33) in ACOS patients to determine the relationship between levels of these inflammatory markers and pulmonary functions.Methods: Blood samples were obtained from asthma, COPD, and ACOS patients who received combined therapy and were stable for the last month to measure cys-LTs, PG-D2, PG-E2, IL-5, and ADAM33 levels. Differences between groups and their correlations with pulmonary function tests were evaluated.Results: In total, 24 ACOS, 27 asthma, and 35 COPD patients were included. . PG-D2 levels were higher in ACOS (120.9 ± 117.2 ng/L) and asthma (119.6 ± 111.7 ng/L) patients than in COPD (82.6 ± 46.7 ng/L) patients (p = 0.036 and p = 0.038, respectively). In ACOS patients, PG-D2, cys-LTs, and ADAM33 levels were negatively correlated with FEV1/FVC% values (p = 0.021, p = 0.008, and p = 0.028, respectively). In COPD patients, a negative correlation was detected between PG-E2 and FEV1/FVC% (p = 0.007), whereas positive correlations were detected between IL-5 and pulmonary function tests, including FVC, FVC%, FEV1, FEV1%, FEF25-75, and FEF25-75% (p = 0.047, p = 0.005, p = 0.002, p = 0.002, p = 0.010, and p = 0.005, respectively). In asthma patients, cys-LTs levels were negatively correlated with FEV1 and FEF25-75 values (p = 0.045 and p = 0.037, respectively).Conclusions: PG-D2 levels may be a valuable biomarker to differentiate COPD in asthma and ACOS patients.
Asunto(s)
Síndrome de Superposición de la Enfermedad Pulmonar Obstructiva Crónica-Asmática/diagnóstico , Asma/diagnóstico , Mediadores de Inflamación/sangre , Prostaglandina D2/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Proteínas ADAM/sangre , Proteínas ADAM/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Asma/sangre , Asma/inmunología , Síndrome de Superposición de la Enfermedad Pulmonar Obstructiva Crónica-Asmática/sangre , Síndrome de Superposición de la Enfermedad Pulmonar Obstructiva Crónica-Asmática/inmunología , Biomarcadores/sangre , Estudios Transversales , Diagnóstico Diferencial , Dinoprostona/sangre , Dinoprostona/inmunología , Femenino , Humanos , Inflamación/sangre , Inflamación/diagnóstico , Inflamación/inmunología , Mediadores de Inflamación/inmunología , Interleucina-5/sangre , Interleucina-5/inmunología , Masculino , Persona de Mediana Edad , Prostaglandina D2/inmunología , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Espirometría , Adulto JovenRESUMEN
From the time of their discovery in 1999, the aggrecanases, and ADAMTS-5 in particular, have been heavily investigated as targets for disease-modifying osteoarthritis drug (DMOAD) development. Here, we provide a brief narrative review of the discovery efforts to target these enzymes, and how this led to the current ongoing programmes that hold promise for the future. We discuss a comparison of inhibition of collagen breakdown versus inhibition of aggrecan breakdown. We then summarise existing programmes that target ADAMTS-5, including small molecule inhibitors, monoclonal neutralising antibodies and nanobodies, and gene editing technologies. We also briefly discuss the potential analgesic effects this strategy may offer in addition to its joint-protective effects.
Asunto(s)
Proteínas ADAM , Endopeptidasas/metabolismo , Osteoartritis/enzimología , Procolágeno N-Endopeptidasa , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/inmunología , Proteína ADAMTS4 , Agrecanos/metabolismo , Humanos , Osteoartritis/tratamiento farmacológico , Osteoartritis/inmunologíaAsunto(s)
Proteínas ADAM/inmunología , Asma/inmunología , Células Epiteliales/inmunología , Factor de Crecimiento Transformador beta/inmunología , Proteínas ADAM/genética , Alérgenos/inmunología , Animales , Células COS , Chlorocebus aethiops , Pulmón/inmunología , Ratones Transgénicos , Dominios Proteicos , Pyroglyphidae/inmunología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
OBJECTIVE: To investigate the effects of interferon-γ (IFN-γ) on the proliferation and viability of human embryonic lung Mrc-5 fibroblasts in vitro and the expression of the A Disintegrin and Metalloprotease 33 (ADAM33) gene and to explore the mechanism of airway remodeling. METHODS: Mrc-5 fibroblasts were sensitized with Dermatophagoides farinae 1 (Derf1) in vitro to mimic in vivo conditions observed in bronchial asthma. An inverted fluorescence microscope was used to observe changes in cell morphology before and after treatment. The viability of Mrc-5 cells was tested using the Cell Counting kit-8 (CCK8). Expression of the ADAM33 gene and protein in Mrc-5 cells was assessed using qPCR and Western blotting, respectively. RESULTS: Different concentrations of Derf1 increased cell growth and the expression of the ADAM33 gene in Mrc-5 cells, and these changes were most obvious in the 10 µg/ml group. In contrast, IFN-γ decreased cell growth and the expression of the ADAM33 gene in both Mrc-5 cells and Derf1-induced Mrc-5 cells, and these changes were most obvious in the 10 ng/ml group. The negative effects of 10 ng/ml IFN-γ were the most significant at 32 hours. CONCLUSIONS: Derf1-induced Mrc-5 cells successfully imitated the in vivo conditions observed in patients with asthma. IFN-γ inhibited the proliferation and viability of Mrc-5 cells, and Derf1-induced Mrc-5 cells were more sensitive to IFN-γ treatment. IFN-γ treatment significantly downregulated the expression of the ADAM33 gene in a concentration- and time-dependent manner. IFN-γ may participate in airway remodeling in asthma by regulating the expression of the ADAM33 gene.
Asunto(s)
Proteínas ADAM/metabolismo , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/tratamiento farmacológico , Interferón gamma/farmacología , Proteínas ADAM/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Alérgenos/inmunología , Antígenos Dermatofagoides/inmunología , Asma/inmunología , Asma/patología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Fibroblastos , Humanos , Interferón gamma/metabolismo , Interferón gamma/uso terapéutico , Pulmón/inmunología , Pulmón/patología , Miocitos del Músculo LisoRESUMEN
Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Proteínas ADAM/sangre , Proteínas ADAM/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Ácidos Carboxílicos/metabolismo , Células Cultivadas , Humanos , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Metilación , Ratones , Inhibidores de Proteasas/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Especificidad por Sustrato , Transferrina/análogos & derivados , Transferrina/metabolismoRESUMEN
As the first line of defence, marginal zone (MZ) B cells play principal roles in clearing blood-borne pathogens during infection and are over-primed in autoimmune diseases. However, the basic mechanisms underlying MZ B-cell development are still unclear. We found here that CD19 deficiency blocked the differentiation of marginal zone precursors (MZP) to MZ B cells, whereas CD19 expression in CD19-deficient MZP rescues MZ B-cell generation. Furthermore, CD19 regulates Notch2 cleavage by up-regulating ADAM28 expression in MZP. Finally, we found that CD19 suppressed Foxo1 expression to promote ADAM28 expression in MZP. These results suggest that CD19 controls the differentiation of MZP to MZ B cells by regulating ADAM28-mediated Notch2 cleavage. Thus, we demonstrated the basic mechanisms underlying the differentiation of MZP to MZ B cells.
Asunto(s)
Proteínas ADAM/genética , Antígenos CD19/genética , Linfocitos B/inmunología , Tejido Linfoide/inmunología , Receptor Notch2/genética , Proteínas ADAM/inmunología , Animales , Antígenos CD19/inmunología , Linfocitos B/citología , Diferenciación Celular/inmunología , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/inmunología , Regulación de la Expresión Génica , Tejido Linfoide/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteolisis , Receptor Notch2/inmunología , Transducción de SeñalRESUMEN
OBJECTIVES: Multiomics study was conducted to elucidate the crucial molecular mechanisms of primary Sjögren's syndrome (SS) pathology. METHODS: We generated multiple data set from well-defined patients with SS, which includes whole-blood transcriptomes, serum proteomes and peripheral immunophenotyping. Based on our newly generated data, we performed an extensive bioinformatic investigation. RESULTS: Our integrative analysis identified SS gene signatures (SGS) dysregulated in widespread omics layers, including epigenomes, mRNAs and proteins. SGS predominantly involved the interferon signature and ADAMs substrates. Besides, SGS was significantly overlapped with SS-causing genes indicated by a genome-wide association study and expression trait loci analyses. Combining the molecular signatures with immunophenotypic profiles revealed that cytotoxic CD8 -T cells- were associated with SGS. Further, we observed the activation of SGS in cytotoxic CD8 T cells isolated from patients with SS. CONCLUSIONS: Our multiomics investigation identified gene signatures deeply associated with SS pathology and showed the involvement of cytotoxic CD8 T cells. These integrative relations across multiple layers will facilitate our understanding of SS at the system level.
Asunto(s)
Epigenómica , Perfilación de la Expresión Génica , Inmunofenotipificación , Proteómica , ARN Mensajero/metabolismo , Síndrome de Sjögren/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Proteínas ADAM/metabolismo , Adulto , Anciano , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Biología Computacional , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Linfocitos T Citotóxicos/metabolismo , TranscriptomaRESUMEN
The metalloproteinase ADAM8 serves as a pivotal catalyst in the development of inflammatory diseases and cancer metastasis. The cyclic peptide cyclo(RLsKDK) has been shown to inhibit the enzymatic activity of ADAM8 with high specificity and potency. Herein we report a structure-activity relationship (SAR) study of cyclo(RLsKDK) that involves the synthesis and biological evaluation of the lead compound and structural analogues thereof. This study provides insight into the ligand-receptor interactions that govern the binding of cyclo(RLsKDK) to the ADAM8 disintegrin domain and represents a stepping stone for the development of new treatments for inflammatory diseases and cancer metastasis.
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Proteínas ADAM/antagonistas & inhibidores , Antiinflamatorios/química , Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Metástasis de la Neoplasia/tratamiento farmacológico , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Proteínas ADAM/inmunología , Animales , Células COS , Chlorocebus aethiops , Humanos , Inflamación/inmunología , Proteínas de la Membrana/inmunología , Metástasis de la Neoplasia/inmunología , Peptidomiméticos/química , Peptidomiméticos/farmacología , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To obtain recombinant mouse collagenase ADAMTS2 (ADAM metallopeptidase with thrombospondin type 1 motif, 2) C terminal (1109-1213), and prepare the corresponding rabbit anti-ADAMTS2 polyclonal antibodies. METHODS: The recombinant expression plasmid pGEX-6p-1-ADAMTS2 (1109-1213) was transformed into E.coli. The target protein was induced by IPTG and identified by mass spectrometry following affinity purification. The expressed and purified ADAMTS2 (1109-1213) protein was used to immunize New Zealand rabbits to prepare anti-ADAMTS2 polyclonal antibodies. The antibody titers were detected by ELISA and the antibody specificity by Western blotting. RESULTS: The protein ADAMTS2 (1109-1213) was expressed in E.coli after IPTG induction, and with the purified protein, we prepared antiserum in the immunized rabbits. The titer of the antiserum reached over 1:160 000. The antiserum showed a good specificity. CONCLUSION: The high titer and specific rabbit anti-ADAMTS2 antibody has been prepared successfully.
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Proteínas ADAM/inmunología , Anticuerpos Monoclonales/inmunología , Sueros Inmunes/inmunología , Procolágeno N-Endopeptidasa/inmunología , Proteínas Recombinantes/inmunología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Proteína ADAMTS4 , Animales , Especificidad de Anticuerpos/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Masculino , Ratones Endogámicos C57BL , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Conejos , Proteínas Recombinantes/metabolismoRESUMEN
Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11.
Asunto(s)
Proteínas ADAM/inmunología , Secretasas de la Proteína Precursora del Amiloide/inmunología , Interleucina-11/inmunología , Elastasa de Leucocito/inmunología , Proteínas de la Membrana/inmunología , Monocitos/inmunología , Mieloblastina/inmunología , Receptores de Interleucina-11/inmunología , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Antiinflamatorios/farmacología , Línea Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inflamación , Interleucina-11/genética , Elastasa de Leucocito/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/patología , Mieloblastina/genética , Unión Proteica , Proteolisis , Receptores de Interleucina-11/genética , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Transducción de SeñalRESUMEN
Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11- and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR- or ASYILIRD-reactive CD4(+)T cells are present in peripheral blood mononuclear cells from HLA-DRB1*11 and HLA-DRB1*03-positive subjects with acquired TTP. The presence of ADAMTS13-reactive CD4(+)T cells was addressed by flow cytometry and the expression of activation marker CD40 ligand by CD4(+)T cells. FINVAPHAR-reactive CD4(+)T cells were identified in an HLA-DRB1*11-positive patient during the acute phase of the disease whereas ASYILIRD-positive CD4(+)T cells were identified in a DRB1*03-positive patient with acquired TTP. Frequencies of CUB2 domain-reactive CD4(+)T cells ranged from 3.3% to 4.5%. Control peptides in which the anchor residues were modified did not induce activation of CD4(+)T cells. Taken together, our data provide evidence for the involvement of CUB2 domain-reactive CD4(+)T cells in the etiology of acquired TTP.
Asunto(s)
Proteínas ADAM/inmunología , Linfocitos T CD4-Positivos/inmunología , Péptidos/inmunología , Púrpura Trombocitopénica Trombótica/inmunología , Proteínas ADAM/química , Proteína ADAMTS13 , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/patología , Cadenas HLA-DRB1/inmunología , Humanos , Datos de Secuencia Molecular , Péptidos/química , Estructura Terciaria de Proteína , Púrpura Trombocitopénica Trombótica/patologíaRESUMEN
Antiphospholipid syndrome (APS) is an autoimmune disease in which antiphospholipid antibodies (aPLs) are generated. Previous studies show concurrence of APS and thrombotic thrombocytopenic purpura; therefore it is plausible to assume that anti-ADAMTS13 autoantibody is also involved in the pathophysiology of APS. We investigated the clinical significance of ADAMTS13 activity and anti-ADAMTS13 antibody in patients with aPLs. Two hundred and sixteen patients with positive lupus anticoagulant and/or anticardiolipin antibody were included. ADAMTS13 activity and anti-ADAMTS13 antibody were measured using fluorescence resonance energy-transfer technology and ELISA, respectively. Reduced ADAMTS13 activity was observed in 40.3% (87/216) of patients with aPLs. Although 33.8% (73/216) of patients were positive for anti-ADAMTS13 antibody, 41 of these 73 patients had normal levels of ADAMTS13 activity. Reduced ADAMTS13 activity was a significant risk factor for thrombotic events. Thrombotic events and age contributed to the reduced level of ADAMTS13 activity. Presence of anti-ADAMTS13 antibody did not show any association with the level of ADAMTS13 activity. Patients with autoimmune diseases tended to show higher levels of anti-ADAMTS13 antibody. Our findings suggest that reduced ADAMTS13 activity is a significant thrombotic risk factor in patients with aPLs irrespective of the presence of anti-ADAMTS13 antibody. Presence of anti-ADAMTS13 antibody is not seen with reduced activity and it tends to be increased in patients with autoimmune diseases.
Asunto(s)
Proteínas ADAM/inmunología , Anticuerpos Antifosfolípidos/inmunología , Inhibidor de Coagulación del Lupus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Factores de Riesgo , Trombosis , Adulto JovenRESUMEN
OBJECTIVE: The primary goal of this study was to test the disease-modifying effect of blocking a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 with a neutralizing monoclonal antibody (mAb) starting 4 weeks after destabilization of the medial meniscus (DMM) in the mouse. We also investigated whether ADAMTS-5 blockade reversed mechanical allodynia and decreased monocyte chemoattractant protein (MCP)-1 production by dorsal root ganglia (DRG) cells. METHODS: Ten-week old male C57BL/6 mice underwent DMM surgery and were either left untreated or treated with anti-ADAMTS-5 mAb or IgG2c isotype control mAb starting 4 weeks after surgery. Knees were collected for histopathology 4 or 12 weeks later. Mechanical allodynia was monitored biweekly in the ipsilateral hind paw through 16 weeks. DRG were collected and cultured 8 weeks after DMM for analysis of MCP-1 production. RESULTS: By 4 weeks after DMM, mild cartilage degeneration was evident in the medial compartment, small osteophytes were present, and subchondral bone sclerosis was established. By 16 weeks after surgery, significant cartilage deterioration was apparent on the medial tibial plateaux and medial femoral condyles, osteophyte size had increased, and subchondral bone sclerosis was maintained. Treatment with ADAMTS-5 mAb from week 4 to 16 after surgery slowed cartilage degeneration and osteophyte growth but did not affect subchondral bone sclerosis. Moreover, ADAMTS-5 blockade resulted in temporary reversal of mechanical allodynia, which correlated with decreased MCP-1 production by cultured DRG cells. CONCLUSIONS: This study suggests therapeutic efficacy of an ADAMTS-5 mAb in the DMM model, when therapy starts early in disease.