RESUMEN
Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.
Asunto(s)
Artritis Reumatoide , Fibroblastos , Proteína Proto-Oncogénica c-ets-1 , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Ligando RANK/genética , Factores de Transcripción/metabolismoRESUMEN
Autoreactive T cells are eliminated in the thymus to prevent autoimmunity by promiscuous expression of tissue-restricted self-antigens in medullary thymic epithelial cells. This expression is dependent on the transcription factor Fezf2, as well as the transcriptional regulator Aire, but the entire picture of the transcriptional program has been obscure. Here, we found that the chromatin remodeler Chd4, also called Mi-2ß, plays a key role in the self-antigen expression in medullary thymic epithelial cells. To maximize the diversity of self-antigen expression, Fezf2 and Aire utilized completely distinct transcriptional mechanisms, both of which were under the control of Chd4. Chd4 organized the promoter regions of Fezf2-dependent genes, while contributing to the Aire-mediated induction of self-antigens via super-enhancers. Mice deficient in Chd4 specifically in thymic epithelial cells exhibited autoimmune phenotypes, including T cell infiltration. Thus, Chd4 plays a critical role in integrating Fezf2- and Aire-mediated gene induction to establish central immune tolerance.
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Autoantígenos/inmunología , Tolerancia Central/fisiología , Regulación de la Expresión Génica/inmunología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/inmunología , Animales , Autoantígenos/biosíntesis , ADN Helicasas/inmunología , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin ß-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.
Asunto(s)
Fibroblastos/inmunología , Linfocitos T/inmunología , Timo/metabolismo , Animales , Autoantígenos/inmunología , Autoinmunidad , Células Cultivadas , Microambiente Celular , Selección Clonal Mediada por Antígenos , Dipeptidil Peptidasa 4/metabolismo , Tolerancia Inmunológica , Receptor beta de Linfotoxina/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Timo/citologíaRESUMEN
The periosteum is the layer of cells that covers nearly the entire surface of every bone. Upon infection, injury or malignancy the bone surface undergoes new growth-the periosteal reaction-but the mechanism and physiological role of this process remain unknown1,2. Here we show that the periosteal reaction protects against cancer invasion into the bone. Histological analyses of human lesions of head and neck squamous cell carcinomas (HNSCCs) show that periosteal thickening occurs in proximity to the tumour. We developed a genetically dissectible mouse model of HNSCC and demonstrate that inducible depletion of periosteal cells accelerates cancerous invasion of the bone. Single-cell RNA sequencing reveals that expression of the gene encoding the protease inhibitor TIMP1 is markedly increased in the periosteum at the pre-invasive stage. This increase is due to upregulation of HIF1α expression in the tumour microenvironment, and increased TIMP1 inactivates matrix-degrading proteases, promoting periosteal thickening to inhibit cancer invasion. Genetic deletion of Timp1 impairs periosteal expansion, exacerbating bone invasion and decreasing survival in tumour-bearing mice. Together, these data show that the periosteal reaction may act as a functional stromal barrier against tumour progression, representing a unique example of tissue immunity mediated by stromal cells.
RESUMEN
Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell-dependent antigen sampling and B cell-dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Inmunoglobulina A/inmunología , Tejido Linfoide/inmunología , Células Madre Mesenquimatosas/inmunología , Ligando RANK/inmunología , Animales , Linfocitos B/inmunología , Biodiversidad , Diferenciación Celular/inmunología , Quimiocina CCL20/inmunología , Células Dendríticas/inmunología , Citometría de Flujo , Microbioma Gastrointestinal/genética , Centro Germinal , Tejido Linfoide/citología , Células Madre Mesenquimatosas/ultraestructura , Ratones , Microscopía Electrónica , Ligando RANK/genética , ARN Ribosómico 16S/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.
Asunto(s)
Saponinas , Trehalosa , Saponinas/química , Trehalosa/química , Inmunoprecipitación/métodos , Animales , Detergentes/química , Unión Proteica , Humanos , Octoxinol/químicaRESUMEN
BACKGROUND: Cystinosis is a lysosomal storage disorder characterized by an autosomal recessive phenotype. Intermediate cystinosis, which progresses slowly and causes renal failure, accounts for approximately 5% of all cystinosis cases. Patients with intermediate cystinosis may not exhibit the typical symptoms of cystinosis, such as Fanconi syndrome and ocular symptoms. Because of its diverse clinical presentation and rarity, intermediate cystinosis can be difficult to diagnose. Additionally, few patients can tolerate cystine-depleting drugs, such as cysteamine, because of their complicated administration schedules and side effects. We report a case of intermediate cystinosis that was treated with cysteamine for 10 years. CASE PRESENTATION: Urinary abnormalities were first diagnosed when the patient was 3 years of age during a health examination specifically for 3-year-old children, which is unique to Japan. Cystinosis was diagnosed when the patient was 12 years of age. Cysteamine therapy was initiated and regular cystine concentration measurements were performed. Although proteinuria persisted, the patient's renal function progressed slowly. Two renal biopsies were performed, and multinucleated podocytes and cystine crystals without focal segmental glomerulosclerosis lesions were observed in the biopsy specimens. The patient's renal function remained stable. CONCLUSIONS: This case of intermediate cystinosis was treated with cysteamine over the course of 10 years. Intermediate cystinosis requires an appropriate diagnosis and long-term treatment.
Asunto(s)
Cisteamina , Depletores de Cistina , Cistinosis , Humanos , Cisteamina/uso terapéutico , Cistinosis/tratamiento farmacológico , Cistinosis/complicaciones , Depletores de Cistina/uso terapéutico , Masculino , Niño , Femenino , Resultado del Tratamiento , PreescolarRESUMEN
The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Macrófagos/fisiología , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animales , Diferenciación Celular , Línea Celular , Células Dendríticas/metabolismo , Desoxiglucosa/análogos & derivados , Desoxiglucosa/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Macrófagos/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Oligodesoxirribonucleótidos/farmacologíaRESUMEN
Patients with inflammatory bowel disease (IBD) who experience long-term chronic inflammation of the colon are at an increased risk of developing colorectal cancer (CRC). Mitotic spindle positioning (MISP), an actin-binding protein, plays a role in mitosis and spindle positioning. MISP is found on the apical membrane of the intestinal mucosa and helps stabilize and elongate microvilli, offering protection against colitis. This study explored the role of MISP in colorectal tumorigenesis using a database, human CRC cells, and a mouse model for colitis-induced colorectal tumors triggered by azoxymethane (AOM)/dextran sodium sulfate (DSS) treatment. We found that MISP was highly expressed in colon cancer patient tissues and that reduced MISP expression inhibited cell proliferation. Notably, MISP-deficient mice showed reduced colon tumor formation in the AOM/DSS-induced colitis model. Furthermore, MISP was found to form a complex with Opa interacting protein 5 (OIP5) in the cytoplasm, influencing the expression of OIP5 in a unidirectional manner. We also observed that MISP increased the levels of phosphorylated STAT3 in the JAK2-STAT3 signaling pathway, which is linked to tumorigenesis. These findings indicate that MISP could be a risk factor for CRC, and targeting MISP might provide insights into the mechanisms of colitis-induced colorectal tumorigenesis.
Asunto(s)
Colitis , Neoplasias Colorrectales , Animales , Humanos , Ratones , Azoximetano/efectos adversos , Carcinogénesis , Transformación Celular Neoplásica , Colitis/patología , Neoplasias Colorrectales/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Janus Quinasa 2/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal , Huso Acromático/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
A 44-year-old woman was diagnosed with acute myeloid leukemia (RUNX1::RUNX1T1 translocation) and received induction chemotherapy with idarubicin hydrochloride and cytosine arabinoside. The pneumonia that had been present since admission worsened, and a drug-induced skin rash appeared. On day 17, she presented with respiratory failure and shock, complicated by hemoconcentration and hypoalbuminemia. This was considered capillary leak syndrome due to pneumonia and drug allergy, so she was started on pulse steroid therapy and IVIG, and was intubated on the same day. On day 18, venovenous-extracorporeal membrane oxygenation (VV-ECMO) was started due to worsening blood gas parameters despite ventilatory management. Bronchoalveolar lavage fluid was serous, and both blood and sputum cultures yielded negative. The patient was weaned from VV-ECMO on day 26 as the pneumonia improved with recovery of hematopoiesis. She was disoriented, and a CT scan on day 28 revealed cerebral hemorrhage. Her strength recovered with rehabilitation. After induction chemotherapy, RUNX1::RUNX1T1 mRNA was not detected in bone marrow. The patient received consolidation chemotherapy, and has maintained complete remission. Severe respiratory failure during induction chemotherapy for acute leukemia can be fatal, but VV-ECMO may be lifesaving.
Asunto(s)
Síndrome de Fuga Capilar , Oxigenación por Membrana Extracorpórea , Leucemia Mieloide Aguda , Neumonía , Insuficiencia Respiratoria , Humanos , Femenino , Adulto , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Quimioterapia de Inducción , Síndrome de Fuga Capilar/complicaciones , Leucemia Mieloide Aguda/complicaciones , Leucemia Mieloide Aguda/terapiaRESUMEN
Docosahexaenoic acid (DHA), an omega-3 fatty acid, usually presents as a constituent of phospholipids in the cellular membrane. Lysophospholipid acyltransferase 3 (LPLAT3; AGPAT3) is the primary enzyme that incorporates DHA into phospholipids. LPLAT3-KO mice show male infertility and visual dysfunction accompanied by decreased phospholipids (PLs) containing DHA (PL-DHA) in the testis and retina, respectively. In this study, we evaluated the effect of diets consisting mainly of triacylglycerol-bound DHA (fish oil) and PL-bound DHA (salmon roe oil) on the amount of PL-DHA in a broad range of tissues and on reproductive functions. Both diets elevated phosphatidylcholines (PCs)-containing DHA in most tissues of wild type (WT) mice. Although LPLAT3-KO mice acquired a minimal amount of PC-DHA in the testes and sperm by eating either of the diets, reproductive function did not improve. The present study suggests that DHA-rich diets do not restore sufficient PL-DHA to improve male infertility in LPLAT3-KO mice. Alternatively, PL-DHA can be biosynthesized by LPLAT3 but not by external supplementation, which may be necessary for normal reproductive function.
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Ácidos Grasos Omega-3 , Infertilidad Masculina , Masculino , Ratones , Animales , Humanos , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Semen , Fosfolípidos , Dieta , Ácidos DocosahexaenoicosRESUMEN
Antibodies that block the interaction between PD-1 and PD-1 ligands (anti-PD-1) are in clinical use for the treatment of cancer, yet their efficacy is limited. Pre-approved therapies that enhance the effect of anti-PD-1 in combination are beneficial. Small-molecule inhibitors that attenuate T cell receptor signaling are reported to prevent T cell exhaustion and induce memory T cells with stem cell potential, resulting in a durable effector T cell response in combination with anti-PD-1. In search of such targets, we focused on protein kinase D (PKD), which is suggested to be suppressive in both tumor growth and TCR signaling. We report that CRT0066101, a PKD inhibitor (PKDi), suppressed the growth of mouse tumors at a sub-micromolar concentration in vitro. Despite its inhibitory effects on tumors, a single treatment of tumor-bearing mice with PKDi did not inhibit, but rather accelerated tumor growth, and reversed the therapeutic effect of anti-PD-1. Mice treated with PKDi showed reduced T cell infiltration and defects in the generation of effector T cells, compared to those treated with anti-PD-1, suggesting that PKDi inhibited ongoing antitumor responses. Mechanistically, PKDi inhibited phosphorylation of AKT, a primary checkpoint that is reactivated by anti-PD-1. In conclusion, PKD is fundamentally required for T cell reactivation by anti-PD-1; therefore, inhibition of PKD is not appropriate for combination therapy with anti-PD-1. On the other hand, a single dose of PKDi was shown to strongly suppress experimental autoimmunity in mice, indicating that PKDi could be useful for the treatment of immune-related adverse events that are frequently reported in anti-PD-1 therapy.
Asunto(s)
Neoplasias , Linfocitos T , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/farmacología , Inmunoterapia/métodos , Línea Celular Tumoral , Microambiente TumoralRESUMEN
SLC15A4 is a lysosome-resident, proton-coupled amino-acid transporter that moves histidine and oligopeptides from inside the lysosome to the cytosol of eukaryotic cells. SLC15A4 is required for Toll-like receptor 7 (TLR7)- and TLR9-mediated type I interferon (IFN-I) productions in plasmacytoid dendritic cells (pDCs) and is involved in the pathogenesis of certain diseases including lupus-like autoimmunity. How SLC15A4 contributes to diseases is largely unknown. Here we have shown that B cell SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Our findings revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production.
Asunto(s)
Formación de Anticuerpos/inmunología , Inflamación/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteínas de Transporte de Membrana/inmunología , Serina-Treonina Quinasas TOR/inmunología , Animales , Anticuerpos/inmunología , Autoanticuerpos/biosíntesis , Linfocitos B/inmunología , Células Cultivadas , Inmunoglobulina G/biosíntesis , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/biosíntesis , Lupus Eritematoso Sistémico/patología , Lisosomas/fisiología , Glicoproteínas de Membrana/inmunología , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 9/inmunología , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/inmunologíaRESUMEN
Acquired immune reaction is initiated by dendritic cells (DCs), which present Ags to a few naive Ag-specific T cells. Deregulation of gene expression in DCs may alter the outcome of the immune response toward immunodeficiency and/or autoimmune diseases. Expression of TRIM28, a nuclear protein that mediates gene silencing through heterochromatin, decreased in DCs from old mice, suggesting alteration of gene regulation. Mice specifically lacking TRIM28 in DCs show increased DC population in the spleen and enhanced T cell priming toward inflammatory effector T cells, leading to acceleration and exacerbation in experimental autoimmune encephalomyelitis. TRIM28-deficient DCs were found to ectopically transcribe endogenous retrovirus (ERV) elements. Combined genome-wide analysis revealed a strong colocalization among the decreased repressive histone mark H3K9me3-transcribed ERV elements and the derepressed host genes that were related to inflammation in TRIM28-deficient DCs. This suggests that TRIM28 occupancy of ERV elements critically represses expression of proximal inflammatory genes on the genome. We propose that gene silencing through repressive histone modification by TRIM28 plays a role in maintaining the integrity of precise gene regulation in DCs, which prevents aberrant T cell priming to inflammatory effector T cells.
Asunto(s)
Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Retrovirus Endógenos/fisiología , Inflamación/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T/inmunología , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Silenciador del Gen , Heterocromatina/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 28 que Contiene Motivos Tripartito/genéticaRESUMEN
AIM: Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. METHODS: Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. RESULTS: Liver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. CONCLUSIONS: Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.
RESUMEN
Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinß (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.
Asunto(s)
Distroglicanos/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Distrofina/metabolismo , Neuroglía/metabolismo , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Distroglicanos/genética , Distrofina/genética , Proteínas Asociadas a la Distrofina/genética , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismo , Neuropéptidos/genética , Unión ProteicaRESUMEN
Systemic autoimmune diseases frequently induce lupus nephritis, causing altered balance and expression of interleukin 36 receptor (IL-36R) ligands, including agonists (IL-36α, ß, γ) and antagonists (IL-36Ra, IL-38), in kidneys. Here, we established and analyzed a mouse model of lupus nephritis, MRL/MpJ-Faslpr/lpr with IL-36R-knockout (KO), compared to wild-type (WT) mice. In both genotypes, indices for immune abnormalities and renal functions were comparable, although female WT mice showed higher serum autoantibody levels than males. IL-36R ligand expression did not differ significantly between genotypes at the mRNA level or in IL-36α and IL-38 scores. However, glomerular lesions, especially mesangial matrix expansion, were significantly ameliorated in both sexes of IL-36R-KO mice compared to WT mice. Cell infiltration into the tubulointerstitium with the development of tertiary lymphoid structures was comparable between genotypes. However, the positive correlation with the IL-36α score in WT mice was not evident in IL-36R-KO mice. Fibrosis was less in female IL-36R-KO mice than in WT mice. Importantly, some IL-36α+ nuclei co-localized with acetylated lysine and GCN5 histone acetyltransferase, in both genotypes. Therefore, IL-36R ligands, especially IL-36α, contribute to the progression of renal pathology in lupus nephritis via IL-36R-dependent and IL-36R-independent pathways.
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Nefritis Lúpica , Receptores de Interleucina , Animales , Femenino , Masculino , Ratones , Núcleo Celular , Interleucinas , Riñón , Glomérulos Renales , Receptores de Interleucina/genéticaRESUMEN
Most mitochondrial diseases are hereditary and highly heterogeneous. Cattle born with the V79L mutation in the isoleucyl-tRNA synthetase 1 (IARS1) protein exhibit weak calf syndrome. Recent human genomic studies about pediatric mitochondrial diseases also identified mutations in the IARS1 gene. Although severe prenatal-onset growth retardation and infantile hepatopathy have been reported in such patients, the relationship between IARS mutations and the symptoms is unknown. In this study, we generated hypomorphic IARS1V79L mutant mice to develop an animal model of IARS mutation-related disorders. We found that compared to wild-type mice, IARSV79L mutant mice showed a significant increase in hepatic triglyceride and serum ornithine carbamoyltransferase levels, indicating that IARS1V79L mice suffer from mitochondrial hepatopathy. In addition, siRNA knockdown of the IARS1 gene decreased mitochondrial membrane potential and increased reactive oxygen species in the hepatocarcinoma-derived cell line HepG2. Furthermore, proteomic analysis revealed decreased levels of the mitochondrial function-associated protein NME4 (mitochondrial nucleoside diphosphate kinase). Concisely, our mutant mice model can be used to study IARS mutation-related disorders.
Asunto(s)
Hepatopatías , Enfermedades Mitocondriales , Embarazo , Femenino , Humanos , Niño , Animales , Bovinos , Ratones , Proteómica , Isoleucina-ARNt Ligasa/genética , Genoma , Hepatopatías/genética , Enfermedades Mitocondriales/genética , MutaciónRESUMEN
Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.
Asunto(s)
Sustitución de Aminoácidos , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Enfermedades Renales/inducido químicamente , Albuminuria/inducido químicamente , Albuminuria/complicaciones , Animales , Secuencia de Bases , Biomarcadores , Sistemas CRISPR-Cas , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Enfermedades Renales/complicaciones , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/complicaciones , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patologíaRESUMEN
Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.