RESUMO
Modeling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated via injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting single or multiple tumor suppressor genes. What has been lacking so far is the possibility to propagate and characterize the induced cancers via transplantation. Here, we describe the generation of a rag2 knockout line in Xenopus tropicalis that is deficient in functional T and B cells. This line was validated by means of allografting experiments with primary tp53-/- and apc+/-/tp53-/- donor tumors. In addition, we optimized available protocols for the sub-lethal irradiation of wild-type X. tropicalis froglets. Irradiated animals also allowed the stable, albeit transient, engraftment of transplanted X. tropicalis tumor cells. The novel rag2-/- line and the irradiated wild-type froglets will further expand the experimental toolbox in the diploid amphibian X. tropicalis and help to establish it as a versatile and relevant model for exploring human cancer.
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Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.
Assuntos
Complexo Mediador , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Proteínas não Estruturais Virais , Células A549 , Humanos , Interferons/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. OBJECTIVE: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. METHODS: Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. RESULTS: Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. CONCLUSIONS: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.
Assuntos
Asma , Hiper-Reatividade Brônquica , Alérgenos , Animais , Hiper-Reatividade Brônquica/patologia , Citocinas , Dermatophagoides pteronyssinus , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interleucina-33 , Pulmão , Linfócitos , Camundongos , Proteínas Serina-Treonina Quinases , Pyroglyphidae , Células Th2RESUMO
Antigen-presenting conventional dendritic cells (cDCs) are broadly divided into type 1 and type 2 subsets that further adapt their phenotype and function to perform specialized tasks in the immune system. The precise signals controlling tissue-specific adaptation and differentiation of cDCs are currently poorly understood. We found that mice deficient in the Ste20 kinase Thousand and One Kinase 3 (TAOK3) lacked terminally differentiated ESAM+ CD4+ cDC2s in the spleen and failed to prime CD4+ T cells in response to allogeneic red-blood-cell transfusion. These NOTCH2- and ADAM10-dependent cDC2s were absent selectively in the spleen, but not in the intestine of Taok3-/- and CD11c-cre Taok3fl/fl mice. The loss of splenic ESAM+ cDC2s was cell-intrinsic and could be rescued by conditional overexpression of the constitutively active NOTCH intracellular domain in CD11c-expressing cells. Therefore, TAOK3 controls the terminal differentiation of NOTCH2-dependent splenic cDC2s.
Assuntos
Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Proteínas Quinases/metabolismo , Receptor Notch2/metabolismo , Baço/citologia , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Domínios Proteicos , Proteínas Quinases/deficiência , Receptor Notch2/química , Transdução de SinaisRESUMO
AIMS: Therapies to prevent vein graft disease, a major problem in cardiovascular and lower extremity bypass surgeries, are currently lacking. Short-term preoperative protein restriction holds promise as an effective preconditioning method against surgical stress in rodent models, but whether it can improve vein graft patency after bypass surgery is undetermined. Here, we hypothesized that short-term protein restriction would limit vein graft disease via up-regulation of cystathionine γ-lyase and increased endogenous production of the cytoprotective gaseous signalling molecule hydrogen sulfide. METHODS AND RESULTS: Low-density lipoprotein receptor knockout mice were preconditioned for 1 week on a high-fat high-cholesterol (HFHC) diet with or without protein prior to left common carotid interposition vein graft surgery with caval veins from donor mice on corresponding diets. Both groups were returned to a complete HFHC diet post-operatively, and vein grafts analysed 4 or 28 days later. A novel global transgenic cystathionine γ-lyase overexpressing mouse model was also employed to study effects of genetic overexpression on graft patency. Protein restriction decreased vein graft intimal/media+adventitia area and thickness ratios and intimal smooth muscle cell infiltration 28 days post-operatively, and neutrophil transmigration 4 days post-operatively. Protein restriction increased cystathionine γ-lyase protein expression in aortic and caval vein endothelial cells (ECs) and frequency of lung EC producing hydrogen sulfide. The cystathionine γ-lyase inhibitor propargylglycine abrogated protein restriction-mediated protection from graft failure and the increase in hydrogen sulfide-producing ECs, while cystathionine γ-lyase transgenic mice displayed increased hydrogen sulfide production capacity and were protected from vein graft disease independent of diet. CONCLUSION: One week of protein restriction attenuates vein graft disease via increased cystathionine γ-lyase expression and hydrogen sulfide production, and decreased early inflammation. Dietary or pharmacological interventions to increase cystathionine γ-lyase or hydrogen sulfide may thus serve as new and practical strategies to improve vein graft durability.
Assuntos
Cistationina gama-Liase/biossíntese , Dieta com Restrição de Proteínas , Oclusão de Enxerto Vascular/prevenção & controle , Veia Cava Inferior/transplante , Animais , Artéria Carótida Primitiva/cirurgia , Colesterol na Dieta , Cistationina gama-Liase/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Indução Enzimática , Oclusão de Enxerto Vascular/enzimologia , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/fisiopatologia , Sulfeto de Hidrogênio/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neointima , Estado Nutricional , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Grau de Desobstrução Vascular , Veia Cava Inferior/enzimologia , Veia Cava Inferior/patologia , Veia Cava Inferior/fisiopatologiaRESUMO
Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.
Assuntos
Células Endoteliais/fisiologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/fisiologia , Células de Kupffer/fisiologia , Fígado/citologia , Macrófagos/fisiologia , Monócitos/fisiologia , Animais , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Feminino , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.
Assuntos
Células de Kupffer/citologia , Receptores X do Fígado/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Animais , Linhagem da Célula/imunologia , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Células de Kupffer/imunologia , Fígado/citologia , Receptores X do Fígado/metabolismo , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The generation of allergen-specific TCR transgenic animals allows for the characterization of allergen-specific T-cell responses in vivo and in vitro and is a powerful tool to study adaptive immunity to allergens. Here we describe an approach starting from the isolation of antigen-specific T-cell hybridomas and using PCR, flow cytometric, and co-culture methods to obtain antigen-specific MHC class II-restricted CD4+ TCR transgenic mice on the Rag2-/- background.
Assuntos
Alérgenos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular/imunologia , Linhagem Celular , Clonagem Molecular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Linfonodos/inervação , Linfonodos/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T/genéticaRESUMO
Type III collagen is a major fibrillar collagen consisting of three identical α1(III)-chains that is particularly present in tissues exhibiting elastic properties, such as the skin and the arterial wall. Heterozygous mutations in the COL3A1 gene result in vascular Ehlers-Danlos syndrome (vEDS), a severe, life-threatening disorder, characterized by thin, translucent skin and propensity to arterial, intestinal and uterine rupture. Most human vEDS cases result from a missense mutation substituting a crucial glycine residue in the triple helical domain of the α1(III)-chains. The mechanisms by which these mutant type III collagen molecules cause dermal and vascular fragility are not well understood. We generated a transgenic mouse line expressing mutant type III collagen, containing a typical helical glycine substitution (p.(Gly182Ser)). This Col3a1Tg-G182S mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1Tg-G182S mice develop severe transdermal skin wounds, resulting in early demise at 13-14weeks of age. We found that this phenotype was associated with a reduced total collagen content and an abnormal collagen III:I ratio, leading to the production of severely malformed collagen fibrils in the extracellular matrix of dermal and arterial tissues. These results indicate that expression of the glycine substitution in the α1(III)-chain disturbs formation of heterotypic type III:I collagen fibrils, and thereby demonstrate a key role for type III collagen in collagen fibrillogenesis in dermal and arterial tissues.
Assuntos
Substituição de Aminoácidos , Artérias/metabolismo , Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/genética , Mutação , Pele/metabolismo , Animais , Artérias/patologia , Colágeno Tipo III/química , Colágeno Tipo III/deficiência , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/mortalidade , Síndrome de Ehlers-Danlos/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Glicina/química , Glicina/metabolismo , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Serina/química , Serina/metabolismo , Fatores Sexuais , Pele/patologia , Técnicas de Cultura de TecidosRESUMO
TNF has as detrimental role in multiple sclerosis (MS), however, anti-TNF medication is not working. Selective TNF/TNFR1 inhibition whilst sparing TNFR2 signaling reduces the pro-inflammatory effects of TNF but preserves the important neuroprotective signals via TNFR2. We previously reported the generation of a Nanobody-based selective inhibitor of human TNFR1, TROS that will be tested in experimental autoimmune encephalomyelitis (EAE). We specifically antagonized TNF/TNFR1 signaling using TROS in a murine model of MS, namely MOG35-55-induced EAE. Because TROS does not cross-react with mouse TNFR1, we generated mice expressing human TNFR1 in a mouse TNFR1-knockout background (hTNFR1 Tg), and we determined biodistribution of 99mTc-TROS and effectiveness of TROS in EAE in those mice. Biodistribution analysis demonstrated that intraperitoneally injected TROS is retained more in organs of hTNFR1 Tg mice compared to wild type mice. TROS was also detected in the cerebrospinal fluid (CSF) of hTNFR1 Tg mice. Prophylactic TROS administration significantly delayed disease onset and ameliorated its symptoms. Moreover, treatment initiated early after disease onset prevented further disease development. TROS reduced spinal cord inflammation and neuroinflammation, and preserved myelin and neurons. Collectively, our data illustrate that TNFR1 is a promising therapeutic target in MS.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Fatores Imunológicos/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Animais , Encefalomielite Autoimune Experimental/patologia , Humanos , Fatores Imunológicos/farmacocinética , Masculino , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , Fármacos Neuroprotetores/farmacocinética , Fragmentos de Peptídeos , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Tecnécio , Fator de Necrose Tumoral alfa/metabolismo , Imagem Corporal TotalRESUMO
Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.
Assuntos
Proteína ADAM10/metabolismo , Imunidade Adaptativa , Secretases da Proteína Precursora do Amiloide/metabolismo , Linfócitos B/fisiologia , Diferenciação Celular , Linhagem da Célula , Centro Germinativo/imunologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células Cultivadas , Seleção Clonal Mediada por Antígeno , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Receptor Notch2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de SinaisRESUMO
Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Fatores Reguladores de Interferon/metabolismo , Fatores de Transcrição/metabolismo , Animais , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
ORMDL proteins are believed to be negative regulators of serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single-nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and total SL levels. Ormdl3-knockout (Ormdl3-/-) and transgenic (Ormdl3Tg/wt) mice were generated to study the effect of ORMDL3 on total SL levels in plasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormdl3-/- mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Furthermore, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population-based cohort of 971 individuals. Total C18-long chain bases were not significantly altered in the plasma and tissues of Ormdl3-/- mice, whereas C18-sphinganine showed a small and significant increase in plasma, lung, and liver tissues. Mouse embryonic fibroblast cells from Ormdl3-/- mice did not show an altered SPT activity compared with Ormdl3+/- and Ormdl3+/+ mice. Overexpression or knockdown of ORMDL3 in HEK293 cells did not alter SPT activity; however, parallel knockdown of all 3 ORMDL isoforms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long-chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seem not to be directly associated with changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.-Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Animais , Asma/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Proteínas de Membrana/genética , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Esfingolipídeos/sangueRESUMO
Cellular senescence suppresses cancer by halting the growth of premalignant cells, yet the accumulation of senescent cells is thought to drive age-related pathology through a senescence-associated secretory phenotype (SASP), the function of which is unclear. To understand the physiological role(s) of the complex senescent phenotype, we generated a mouse model in which senescent cells can be visualized and eliminated in living animals. We show that senescent fibroblasts and endothelial cells appear very early in response to a cutaneous wound, where they accelerate wound closure by inducing myofibroblast differentiation through the secretion of platelet-derived growth factor AA (PDGF-AA). In two mouse models, topical treatment of senescence-free wounds with recombinant PDGF-AA rescued the delayed wound closure and lack of myofibroblast differentiation. These findings define a beneficial role for the SASP in tissue repair and help to explain why the SASP evolved.
Assuntos
Senescência Celular , Células Endoteliais/citologia , Mesoderma/citologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Cicatrização , Animais , Apoptose , Diferenciação Celular , Feminino , Fibroblastos/metabolismo , Luminescência , Masculino , Camundongos , Camundongos Transgênicos , Miofibroblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/química , TransgenesRESUMO
Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the 26S proteasome. Rad23b-null mice are NER proficient, likely due to the redundant function of the Rad23b paralogue, Rad23a. However, Rad23b-null midgestation embryos are anemic, and most embryos die before birth. Using an unbiased proteomics approach, we found that the majority of Rad23b-interacting partners are associated with the ubiquitin-proteasome system (UPS). We tested the requirement for Rad23b-dependent UPS activity in cellular proliferation and more specifically in the process of erythropoiesis. In cultured fibroblasts derived from embryos lacking Rad23b, proliferation rates were reduced. In fetal livers of Rad23b-null embryos, we observed reduced proliferation, accumulation of early erythroid progenitors, and a block during erythroid maturation. In primary wild-type (WT) erythroid cells, knockdown of Rad23b or chemical inhibition of the proteasome reduced survival and differentiation capability. Finally, the defects linked to Rad23b loss specifically affected fetal definitive erythropoiesis and stress erythropoiesis in adult mice. Together, these data indicate a previously unappreciated requirement for Rad23b and the UPS in regulation of proliferation in different cell types.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/genética , Eritropoese/genética , Complexo de Endopeptidases do Proteassoma/genética , Animais , Western Blotting , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Interferência de RNARESUMO
Dendritic cells (DCs) are crucial for mounting allergic airway inflammation, but it is unclear which subset of DCs performs this task. By using CD64 and MAR-1 staining, we reliably separated CD11b(+) monocyte-derived DCs (moDCs) from conventional DCs (cDCs) and studied antigen uptake, migration, and presentation assays of lung and lymph node (LN) DCs in response to inhaled house dust mite (HDM). Mainly CD11b(+) cDCs but not CD103(+) cDCs induced T helper 2 (Th2) cell immunity in HDM-specific T cells in vitro and asthma in vivo. Studies in Flt3l(-/-) mice, lacking all cDCs, revealed that moDCs were also sufficient to induce Th2 cell-mediated immunity but only when high-dose HDM was given. The main function of moDCs was the production of proinflammatory chemokines and allergen presentation in the lung during challenge. Thus, we have identified migratory CD11b(+) cDCs as the principal subset inducing Th2 cell-mediated immunity in the LN, whereas moDCs orchestrate allergic inflammation in the lung.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Asma/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Pyroglyphidae/imunologia , Células Th2/imunologia , Administração por Inalação , Transferência Adotiva , Alérgenos/isolamento & purificação , Animais , Antígenos de Dermatophagoides/administração & dosagem , Antígenos de Dermatophagoides/isolamento & purificação , Antígenos Ly/genética , Antígenos Ly/imunologia , Asma/patologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Movimento Celular , Proliferação de Células , Células Dendríticas/transplante , Expressão Gênica , Inflamação/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Transgênicos , Monócitos/imunologia , Monócitos/transplante , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Especificidade de Órgãos , Receptores de IgG/genética , Receptores de IgG/imunologiaRESUMO
The recognition of helix-distorting deoxyribonucleic acid (DNA) lesions by the global genome nucleotide excision repair subpathway is performed by the XPC-RAD23-CEN2 complex. Although it has been established that Rad23 homologs are essential to protect XPC from proteasomal degradation, it is unclear whether RAD23 proteins have a direct role in the recognition of DNA damage. In this paper, we show that the association of XPC with ultraviolet-induced lesions was impaired in the absence of RAD23 proteins. Furthermore, we show that RAD23 proteins rapidly dissociated from XPC upon binding to damaged DNA. Our data suggest that RAD23 proteins facilitate lesion recognition by XPC but do not participate in the downstream DNA repair process.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Humanos , CamundongosRESUMO
Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of "null" alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals.
Assuntos
Alelos , Transtornos do Crescimento/genética , Doenças do Cabelo/genética , Homozigoto , Ictiose/genética , Progéria/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Animais , Dano ao DNA , Genes Letais , Genes Recessivos , Transtornos do Crescimento/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Progéria/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Raios UltravioletaRESUMO
Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.
Assuntos
Síndrome de Cockayne/patologia , Progéria/patologia , Neoplasias Cutâneas/patologia , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Transformada , Síndrome de Cockayne/complicações , Síndrome de Cockayne/metabolismo , Reparo do DNA , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , Mutação , Papiloma/etiologia , Papiloma/metabolismo , Papiloma/patologia , Fenótipo , Progéria/complicações , Progéria/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genéticaRESUMO
The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.