RESUMEN
Sepsis often causes cell death via pyroptosis and hence results in septic cardiomyopathy. Triggering receptors expressed in myeloid cells-1 (TREM-1) may initiate cellular cascade pathways and, in turn, induce cell death and vital organ dysfunction in sepsis, but the evidence is limited. We set to investigate the role of TREM-1 on nucleotide-binding oligomerization domain-like receptors with pyrin domain-3 (NLRP3) inflammasome activation and cardiomyocyte pyroptosis in sepsis models using cardiac cell line (HL-1) and mice. In this study, TREM-1 was found to be significantly increased in HL-1 cells challenged with lipopolysaccharide (LPS). Pyroptosis was also significantly increased in the HL-1 cells challenged with lipopolysaccharide and an NLRP3 inflammasome activator, nigericin. The close interaction between TREM-1 and structural maintenance of chromosome 4 (SMC4) was also identified. Furthermore, inhibition of TREM-1 or SMC4 prevented the upregulation of NLRP3 and decreased Gasdermin-D, IL-1ß and caspase-1 cleavage. In mice subjected to caecal ligation and puncture, the TREM-1 inhibitor LR12 decreased the expression of NLRP3 and attenuated cardiomyocyte pyroptosis, leading to improved cardiac function and prolonged survival of septic mice. Our work demonstrates that, under septic conditions, TREM-1 plays a critical role in cardiomyocyte pyroptosis. Targeting TREM-1 and its associated molecules may therefore lead to novel therapeutic treatments for septic cardiomyopathy.
Asunto(s)
Inflamasomas , Miocitos Cardíacos , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , Sepsis , Receptor Activador Expresado en Células Mieloides 1 , Animales , Humanos , Ratones , Adenosina Trifosfatasas/inmunología , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/inmunología , Caspasa 1/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Cromosomas Humanos Par 4/inmunología , Inflamasomas/agonistas , Inflamasomas/genética , Inflamasomas/inmunología , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/farmacología , Células Mieloides/inmunología , Miocitos Cardíacos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/agonistas , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Piroptosis/genética , Piroptosis/inmunología , Sepsis/complicaciones , Sepsis/genética , Sepsis/inmunología , Receptor Activador Expresado en Células Mieloides 1/antagonistas & inhibidores , Receptor Activador Expresado en Células Mieloides 1/genética , Receptor Activador Expresado en Células Mieloides 1/inmunologíaRESUMEN
High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.
Asunto(s)
Anticuerpos , Aurora Quinasa B , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Fragmentos Fab de Inmunoglobulinas , Complejos Multiproteicos , Survivin , Anticuerpos/química , Anticuerpos/genética , Aurora Quinasa B/análisis , Aurora Quinasa B/inmunología , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/inmunología , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/inmunología , Citoesqueleto/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Mitosis , Complejos Multiproteicos/análisis , Complejos Multiproteicos/inmunología , Biblioteca de Péptidos , Fosforilación , Huso Acromático/metabolismo , Survivin/química , Survivin/metabolismoRESUMEN
Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.
Asunto(s)
Linfocitos B/patología , ADN Intergénico/genética , Predisposición Genética a la Enfermedad , Mieloma Múltiple/genética , Proteínas de Neoplasias/genética , Células Plasmáticas/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica , Linfocitos B/inmunología , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Cromatina/química , Cromatina/inmunología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , ADN Intergénico/inmunología , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Patrón de Herencia , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Proteínas de Neoplasias/inmunología , Células Plasmáticas/inmunología , Polimorfismo Genético , Cultivo Primario de Células , Sitios de Carácter Cuantitativo , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Medición de Riesgo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/inmunologíaRESUMEN
The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.
Asunto(s)
Linfocitos B/inmunología , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas Cromosómicas no Histona/genética , Dosificación de Gen , Centro Germinal/inmunología , Haploinsuficiencia , Linfoma de Células B Grandes Difuso/genética , Células Plasmáticas/inmunología , Animales , Linfocitos B/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/inmunología , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas Cromosómicas no Histona/inmunología , Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Bases de Datos Genéticas , Dioxigenasas/genética , Dioxigenasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Centro Germinal/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/metabolismo , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Fenotipo , Células Plasmáticas/metabolismo , Transcripción GenéticaRESUMEN
Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren's syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1-96.6%) sensitivity with 100.0% (95% CI: 96.4-100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman's rho = 0.89, 95% CI: 0.77.9-0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria.
Asunto(s)
Anticuerpos Antinucleares/aislamiento & purificación , Proteínas Cromosómicas no Histona/inmunología , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Esclerodermia Sistémica/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/inmunología , Estudios de Casos y Controles , Niño , Diagnóstico Diferencial , Estudios de Factibilidad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/instrumentación , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Juego de Reactivos para Diagnóstico , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/inmunología , Adulto JovenRESUMEN
In both plant and animal innate immune responses, surveillance of pathogen infection is mediated by membrane-associated receptors and intracellular nucleotide-binding domain and leucine-rich-repeat receptors (NLRs). Homeostasis of NLRs is under tight multilayered regulation to avoid over-accumulation or over-activation, which often leads to autoimmune responses that have detrimental effects on growth and development. How NLRs are regulated epigenetically at the chromatin level remains unclear. Here, we report that SWP73A, an ortholog of the mammalian switch/sucrose nonfermentable (SWI/SNF) chromatin-remodeling protein BAF60, suppresses the expression of NLRs either directly by binding to the NLR promoters or indirectly by affecting the alternative splicing of some NLRs through the suppression of cell division cycle 5 (CDC5), a key regulator of RNA splicing. Upon infection, bacteria-induced small RNAs silence SWP73A to activate a group of NLRs and trigger robust immune responses. SWP73A may function as a H3K9me2 reader to enhance transcription suppression.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas NLR/metabolismo , Inmunidad de la Planta , Animales , Proteínas de Arabidopsis/inmunología , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Plantas , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de TranscripciónRESUMEN
Anticentromere antibodies (ACA) were associated with lower oocyte maturation rates and cleavage rates, while the mechanism was not clear. Aims of this study were to examine whether active immunization with centromere protein C could elicit the CENP-C autoantibody in mice and the impacts of the CENP-C autoantibody on oocyte meiosis. Mice were divided into two groups, one was the experimental group immunized with human centromere protein C and Freund's adjuvant (CFA), and the other was the control group injected with CFA only. Serum and oocytes of BALB/c mice immunized with human centromere protein C (CENP-C) in complete Freund's adjuvant (CFA) or injected with only CFA were studied for the development of the CENP-C antibody. Rates of germinal vesicle breakdown (GVBD), first polar body (Pb1) extrusion, abnormal spindle morphology, and chromosome misalignment were compared between the experimental group and the control group. The CENP-C antibody was only observed in serum and oocytes of mice immunized with the centromere protein C antigen. The first polar body (Pb1) extrusion rate was lower in the experimental group (P < 0.01). A higher percentage of spindle defects and chromosome congression failure were also detected in the experimental group (spindle defects: 64.67 ± 1.16% vs. 9.27 ± 2.28% control; chromosome misalignment: 50.80 ± 2.40% vs. 8.30 ± 1.16% control; P < 0.01 for both). Oocyte meiosis was severely impaired by the CENP-C antibody, which may be the main mechanism of adverse reproductive outcomes for ACA-positive women who have no clinical symptoms of any autoimmune diseases.
Asunto(s)
Proteínas Cromosómicas no Histona/inmunología , Aberraciones Cromosómicas , Segregación Cromosómica , Meiosis/genética , Meiosis/inmunología , Oocitos/metabolismo , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Diferenciación Celular , Femenino , Inmunización , Ratones , Oocitos/citología , Oogénesis/genética , Oogénesis/inmunologíaAsunto(s)
Antígeno B7-H1/inmunología , Proteínas de Ciclo Celular/inmunología , Proteínas Cromosómicas no Histona/inmunología , Neoplasias/inmunología , Proteínas Nucleares/inmunología , Antígeno B7-H1/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/inmunología , Proteínas Cromosómicas no Histona/genética , Inestabilidad Genómica/genética , Inestabilidad Genómica/inmunología , Humanos , Neoplasias/terapia , Proteínas Nucleares/genética , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , CohesinasRESUMEN
Exposure to heavy metals may have toxic effects on several human organs causing morbidity and mortality. Metals may trigger or exacerbate autoimmunity in humans. Inbred mouse strains with certain H-2 haplotypes are susceptible to xenobiotic-induced autoimmunity; and their immune response to metals such as mercury, gold, and silver have been explored. Serum antinuclear antibodies (ANA), polyclonal B-cell activation, hypergammaglobulinemia and tissue immune complex deposition are the main features of metal-induced autoimmunity in inbred mice. However, inbred mouse strains do not represent the genetic heterogeneity in humans. In this study, outbred Swiss Webster (SW) mice exposed to gold or mercury salts showed immune and autoimmune responses. Intramuscular injection of 22.5 mg/kg.bw aurothiomalate (AuTM) induced IgG ANA in SW mice starting after 5 weeks that persisted until week 15 although with a lower intensity. This was accompanied by elevated serum levels of total IgG antibodies against chromatin and total histones. Exposure to gold led to development of serum IgG autoantibodies corresponding to H1 and H2A histones, and dsDNA. Both gold and mercury induced polyclonal B-cell activation. Eight mg/L mercuric chloride (HgCl2) in drinking water, caused IgG antinucleolar antibodies (ANoA) after 5 weeks in SW mice accompanied by immune complex deposition in kidneys and spleen. Serum IgG antibodies corresponding to anti-fibrillarin, and anti-PM/Scl-100 antibodies, were observed in mercury-exposed SW mice. Gold and mercury trigger systemic autoimmune response in genetically heterogeneous outbred SW mice and suggest them as an appropriate model to study xenobiotic-induced autoimmunity.
Asunto(s)
Anticuerpos Antinucleares/sangre , Autoinmunidad/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Tiomalato Sódico de Oro/toxicidad , Inmunoglobulina G/sangre , Activación de Linfocitos/efectos de los fármacos , Cloruro de Mercurio/toxicidad , Administración Oral , Animales , Complejo Antígeno-Anticuerpo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Cromatina/inmunología , Proteínas Cromosómicas no Histona/inmunología , Exorribonucleasas/inmunología , Complejo Multienzimático de Ribonucleasas del Exosoma/inmunología , Femenino , Tiomalato Sódico de Oro/administración & dosificación , Histonas/inmunología , Inyecciones Intramusculares , Riñón/efectos de los fármacos , Riñón/inmunología , Cloruro de Mercurio/administración & dosificación , Ratones , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
OBJECTIVES: Recent evidences have revealed that anti-SSA/SSB antibodies, the major autoantibodies in Sjögren's syndrome (SS), are produced in salivary glands. This study aims to clarify overall of autoantibody production at lesion site, including anti-centromere antibody (ACA)-positive SS. METHODS: Antibodies of antibody-secreting cells in human salivary glands were produced as recombinant antibodies. The reactivity of these antibodies and their revertants were investigated by ELISA and newly developed antigen-binding beads assay, which can detect conformational epitopes. The target of uncharacterised antibodies was identified by immunoprecipitation and mass spectrometry. Autoantibody-secreting cells in salivary gland tissue were identified by immunohistochemistry using green fluorescent protein-autoantigen fusion proteins. RESULTS: A total of 256 lesion antibodies were generated, and 69 autoantibodies including 24 ACAs were identified among them. Beads assay could detect more autoantibodies than ELISA, suggesting autoantibodies target to antigens with native conformation. After somatic hypermutations were reverted, autoantibodies drastically decreased antigen reactivity. We showed that MIS12 complex, a novel target of ACA, and CENP-C are major targets of ACA produced in salivary glands by examining cloned antibodies and immunohistochemistry, whereas few anti-CENP-B antibodies were detected. The target profiling of serum ACA from 269 patients with SS, systemic sclerosis (SSc), primary biliary cirrhosis (PBC) and healthy controls revealed that ACA-positive patients have antibodies against various sites of centromere complex regardless of disease. CONCLUSION: We showed direct evidences of antigen-driven maturation of anti-SSA/SSB antibody and ACA in SS lesion. ACA recognises centromere 'complex' rather than individual protein, and this feature is common among patients with SS, SSc and PBC.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Formación de Anticuerpos/inmunología , Centrómero/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Células Productoras de Anticuerpos , Autoanticuerpos/inmunología , Proteína A Centromérica/inmunología , Proteína B del Centrómero/inmunología , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/inmunología , Femenino , Humanos , Cirrosis Hepática Biliar/inmunología , Masculino , Persona de Mediana Edad , Glándulas Salivales/citología , Esclerodermia Sistémica/inmunologíaRESUMEN
BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease with undefined pathogenesis. Non-SMC condensin I complex subunit D2 (NCAPD2) and non-SMC condensin II complex subunit D3 (NCAPD3) play pivotal roles in chromosome assembly and segregation during both mitosis and meiosis. To date, there has been no relevant report about the functional role of NCAPD2 and NCAPD3 in UC. AIM: To determine the level of NCAPD2/3 in intestinal mucosa and explore the mechanisms of NCAPD2/3 in UC. METHODS: Levels of NCAPD2/3 in intestinal tissue were detected in 30 UC patients and 30 healthy individuals with in situ hybridization (ISH). In vitro, NCM60 cells were divided into the NC group, model group, si-NCAPD2 group, si-NCAPD3 group and si-NCAPD2+si-NCAPD3 group. Inflammatory cytokines were measured by ELISA, IKK and NF-κB were evaluated by western blot, and IKK nucleation and NF-κB volume were analyzed by immunofluorescence assay. RESULTS: Compared with expression in healthy individuals, NCAPD2 and NCAPD3 expression in intestinal tissue was significantly upregulated (P < 0.001) in UC patients. Compared with levels in the model group, IL-1ß, IL-6 and TNF-α in the si-NCAPD2, si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups were significantly downregulated (P < 0.01). IKK and NF-κB protein expression in the si-NCAPD2, si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups was significantly decreased (P < 0.01). Moreover, IKK nucleation and NF-κB volume were suppressed upon si-NCAPD2, si-NCAPD3 and si-NCAPD2+ si-NCAPD3 transfection. CONCLUSION: NCAPD2/3 is highly expressed in the intestinal mucosa of patients with active UC. Overexpression of NCAPD2/3 promotes the release of pro-inflammatory cytokines by modulating the IKK/NF-κB signaling pathway.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Colitis Ulcerosa/inmunología , Mucosa Intestinal/patología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Adolescente , Adulto , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Colitis Ulcerosa/patología , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Quinasa I-kappa B/metabolismo , Mucosa Intestinal/inmunología , Masculino , FN-kappa B/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Estudios Retrospectivos , Transducción de Señal/inmunología , Regulación hacia Arriba , Adulto JovenRESUMEN
Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.
Asunto(s)
Ensamble y Desensamble de Cromatina/inmunología , Tumor Rabdoide/genética , Tumor Rabdoide/inmunología , Linfocitos T/inmunología , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Humanos , Inmunohistoquímica/métodos , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Systemic sclerosis (SSc) is a rare autoimmune disease with high mortality, characterized by chronic inflammation and fibrosis, which are processes associated with higher serum tumor necrosis factor-α (sTNF-α) levels. TNFA -308G>A and -238G>A polymorphisms have been associated with higher sTNF-α levels. In this study, we genotyped the TNFA -308G>A and -238G>A polymorphisms in 53 SSc patients and 115 unrelated control subjects (CS) from southern Mexico. The TNFA mRNA expression and sTNF-α levels were also quantified by qPCR and enzyme-linked immunosorbent assays, respectively. TNFA -308GA genotype was associated with disease susceptibility according to a codominant genetic model (OR = 3.2, 95% CI 1.05-9.75, p = 0.03), and with higher anti-fibrillarin antibodies (p = 0.01), and higher skin thickening (p = 0.006). TNFA -238GA was not associated with SSc risk. TNFA mRNA expression and sTNF-α levels were similar between SSc patients and CS and were not statistically associated with the TNFA polymorphisms; however, a correlation (rho = 0.362, p = 0.009) between sTNF-α levels with anti-RNA polymerase III antibodies was observed in the SSc patients. In conclusion, the -308G>A polymorphism is a genetic marker of SSc susceptibility in population from southern Mexico, and it is associated with skin thickening and anti-fibrillarin antibodies. In addition, sTNF-α levels correlate positively with the anti-RNA pol III antibodies levels.
Asunto(s)
Autoanticuerpos/metabolismo , Esclerodermia Sistémica/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Adulto , Estudios de Casos y Controles , Proteínas Cromosómicas no Histona/inmunología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , México , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , ARN Polimerasa III/inmunología , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/inmunologíaRESUMEN
Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.
Asunto(s)
Antígenos de Neoplasias/genética , Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Citotóxicos/inmunología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Fusión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/inmunología , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Secuenciación Completa del GenomaRESUMEN
Organism aging is characterized by increased inflammation and decreased stem cell function, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells (HSPCs) exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signaling, which increases chromatin accessibility in the vicinity of NF-κB target genes in response to inflammation. Rad21 is required for normal differentiation, but limits self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB-dependent manner. HSCs from aged mice fail to down-regulate Rad21/cohesin and inflammation/differentiation signals in the resolution phase of inflammation. Inhibition of cohesin/NF-κB reverts hypersensitivity of aged HSPCs to inflammation-induced differentiation and myeloid-biased HSCs with disrupted/reduced expression of Rad21/cohesin are increasingly selected during aging. Together, Rad21/cohesin-mediated NF-κB signaling limits HSPC function during aging and selects for cohesin-deficient HSCs with myeloid-skewed differentiation.
Asunto(s)
Envejecimiento/inmunología , Proteínas de Ciclo Celular/inmunología , Proliferación Celular , Proteínas Cromosómicas no Histona/inmunología , Células Madre Hematopoyéticas/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Envejecimiento/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Inflamación/genética , Inflamación/inmunología , Ratones , Ratones Noqueados , FN-kappa B/genética , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Transducción de Señal/genética , CohesinasRESUMEN
The BAF57 subunit, an indispensable member of the BAF complex, is functionally implicated in apoptosis, cell cycle, and T cell development through chromosomal remodeling. However, the precise roles of BAF57 in the T cell receptor (TcR)-mediated signaling pathway have not been elucidated. In this study, a nucleus-transducible form of BAF57, absent the proline-rich and HMG domains (ntBAF57-ΔPH), was generated to interfere with the interaction between BAF57 and its binding protein, BAF155. ntBAF57-ΔPH was effectively delivered into mouse CD4+ T cells in a dose- and time-dependent manner, without cellular toxicity. Inhibition of T cell activation by ntBAF57-ΔPH was mediated by its disruption of the interaction between BAF155 and BAF57, leading to the degradation of endogenous BAF57 and BAF155. This phenomenon led to alterations in gene expression similar to those associated with Ciclosporin A treatment. In vivo administration of ntBAF57-ΔPH enhanced survival rate of sepsis-induced mice and reduced the LPS-induced secretion of pro-inflammatory cytokines and the expression of endogenous BAF57. These results reveal a novel function of BAF57 as an essential regulator of T cell activation. ntBAF57-ΔPH represents a novel immune-suppressive drug candidate with potential uses in the treatment of autoimmunity and graft rejection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteínas Cromosómicas no Histona/inmunología , Terapia de Inmunosupresión , Activación de Linfocitos , Animales , Linfocitos T CD4-Positivos/patología , Proteínas Cromosómicas no Histona/genética , Ciclosporina/farmacología , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Sepsis/tratamiento farmacológico , Sepsis/genética , Sepsis/inmunología , Sepsis/patología , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.
Asunto(s)
Antígenos Nucleares/inmunología , Proteínas de Ciclo Celular/inmunología , Proteínas Cromosómicas no Histona/inmunología , Interacciones Huésped-Patógeno , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/inmunología , Rotavirus/inmunología , Esferoides Celulares/inmunología , Antígenos Nucleares/genética , Sistemas CRISPR-Cas , Células CACO-2 , Proteínas de Ciclo Celular/genética , Núcleo Celular/inmunología , Núcleo Celular/virología , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Eliminación de Gen , Edición Génica , Regulación de la Expresión Génica , Genoma Humano , Células HEK293 , Células HT29 , Células HeLa , Humanos , Interferones/genética , Interferones/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Quinasas Janus/genética , Quinasas Janus/inmunología , Proteínas de la Membrana/genética , Nucleotidiltransferasas/genética , Rotavirus/crecimiento & desarrollo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/inmunología , Transducción de Señal , Esferoides Celulares/virología , CohesinasRESUMEN
Atherosclerotic plaque formation is characterized by the persistence of lipid-laden macrophages on the inner walls of arteries. Chronic inflammation and imbalanced macrophage function are likely to play a critical role. Herein, we investigated whether bromodomain-containing protein 7 (Brd7), a member of the bromodomain-containing protein family, regulates atherosclerosis, and if so, which mechanisms are responsible for the process. We found that Brd7 is expressed in mouse atherosclerotic plaques, and mostly in macrophages. Inhibition of Brd7 accelerates atherosclerotic lesion formation in ApoE-/- mice by promoting NF-κB-mediated inflammation. Furthermore, Brd7 inhibition alters the phenotype of macrophages and promotes plaque instability, at least partly via STAT6 signaling. Our data define a previously undescribed role of Brd7 in the development of atherosclerosis.
Asunto(s)
Aortitis/inmunología , Aterosclerosis/inmunología , Proteínas Cromosómicas no Histona/inmunología , Macrófagos/inmunología , Macrófagos/patología , FN-kappa B/inmunología , Animales , Aortitis/patología , Apolipoproteínas E/genética , Aterosclerosis/patología , Proteínas Cromosómicas no Histona/deficiencia , Masculino , Ratones , Ratones NoqueadosRESUMEN
OBJECTIVE: The nuclear oncoprotein DEK is an autoantigen associated with juvenile idiopathic arthritis (JIA), especially the oligoarticular subtype. DEK is a secreted chemotactic factor. Abundant levels of DEK and DEK autoantibodies are found in inflamed synovium in JIA. We undertook this study to further characterize the nature of DEK autoantibodies in screening serum samples from 2 different cohorts that consisted mostly of patients with JIA. METHODS: DEK autoantibody levels were analyzed in sera from 33 JIA patients, 13 patients with other inflammatory conditions, and 11 healthy controls, as well as in 89 serum samples from JIA patients receiving anti-tumor necrosis factor (anti-TNF) therapy. Recombinant His-tagged full-length DEK protein (1-375 amino acids [aa]) and the 187-375-aa and 1-350-aa His-tagged DEK fragments made in a baculovirus system were used for enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The C-terminal 25-aa fragment of DEK was expressed in a glutathione S-transferase-tagged vector. ELISA results were calculated as area under the curve by the trapezoidal rule. RESULTS: DEK autoantibody levels were significantly higher in patients with polyarticular JIA than in those with oligoarticular JIA, and were higher in patients with polyarticular JIA who had more active disease after cessation of anti-TNF therapy. Immunoblotting against the C-terminal 25-aa fragment of DEK confirmed that this section of the DEK molecule is the most immunogenic domain. CONCLUSION: DEK autoantibody levels are higher in patients with polyarticular JIA than in those with oligoarticular JIA, and higher in patients who have disease flares after cessation of anti-TNF therapy. The C-terminal 25-aa fragment is the most immunogenic portion of DEK. These findings are significant with respect to the nature of DEK autoantibodies, their contribution to JIA pathogenesis, and their implications for JIA management.