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1.
Nat Commun ; 11(1): 4964, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009394

RESUMO

Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.


Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Megacariócitos/citologia , MicroRNAs/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Endocitose , Perfilação da Expressão Gênica , Humanos , Fígado/lesões , Fígado/patologia , Masculino , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Poliploidia , Proteína rhoB de Ligação ao GTP/metabolismo
2.
J Hematol Oncol ; 13(1): 120, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887634

RESUMO

BACKGROUND: Critically ill patients diagnosed with COVID-19 may develop a pro-thrombotic state that places them at a dramatically increased lethal risk. Although platelet activation is critical for thrombosis and is responsible for the thrombotic events and cardiovascular complications, the role of platelets in the pathogenesis of COVID-19 remains unclear. METHODS: Using platelets from healthy volunteers, non-COVID-19 and COVID-19 patients, as well as wild-type and hACE2 transgenic mice, we evaluated the changes in platelet and coagulation parameters in COVID-19 patients. We investigated ACE2 expression and direct effect of SARS-CoV-2 virus on platelets by RT-PCR, flow cytometry, Western blot, immunofluorescence, and platelet functional studies in vitro, FeCl3-induced thrombus formation in vivo, and thrombus formation under flow conditions ex vivo. RESULTS: We demonstrated that COVID-19 patients present with increased mean platelet volume (MPV) and platelet hyperactivity, which correlated with a decrease in overall platelet count. Detectable SARS-CoV-2 RNA in the blood stream was associated with platelet hyperactivity in critically ill patients. Platelets expressed ACE2, a host cell receptor for SARS-CoV-2, and TMPRSS2, a serine protease for Spike protein priming. SARS-CoV-2 and its Spike protein directly enhanced platelet activation such as platelet aggregation, PAC-1 binding, CD62P expression, α granule secretion, dense granule release, platelet spreading, and clot retraction in vitro, and thereby Spike protein enhanced thrombosis formation in wild-type mice transfused with hACE2 transgenic platelets, but this was not observed in animals transfused with wild-type platelets in vivo. Further, we provided evidence suggesting that the MAPK pathway, downstream of ACE2, mediates the potentiating role of SARS-CoV-2 on platelet activation, and that platelet ACE2 expression decreases following SARS-COV-2 stimulation. SARS-CoV-2 and its Spike protein directly stimulated platelets to facilitate the release of coagulation factors, the secretion of inflammatory factors, and the formation of leukocyte-platelet aggregates. Recombinant human ACE2 protein and anti-Spike monoclonal antibody could inhibit SARS-CoV-2 Spike protein-induced platelet activation. CONCLUSIONS: Our findings uncovered a novel function of SARS-CoV-2 on platelet activation via binding of Spike to ACE2. SARS-CoV-2-induced platelet activation may participate in thrombus formation and inflammatory responses in COVID-19 patients.


Assuntos
Betacoronavirus/metabolismo , Plaquetas/metabolismo , Infecções por Coronavirus/metabolismo , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Trombose/metabolismo , Adulto , Idoso , Animais , Betacoronavirus/genética , Células CACO-2 , Infecções por Coronavirus/virologia , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Células PC-3 , Pandemias , Peptidil Dipeptidase A/genética , Agregação Plaquetária/imunologia , Contagem de Plaquetas , Pneumonia Viral/virologia , RNA Viral/sangue , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Trombose/virologia
3.
Nat Commun ; 11(1): 4454, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901001

RESUMO

Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/virologia , Doença Crônica , Citotoxicidade Imunológica , Modelos Animais de Doenças , Regulação para Baixo , Tolerância Imunológica , Imunidade Celular , Técnicas In Vitro , Ativação Linfocitária , Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Receptor de Morte Celular Programada 1/imunologia , RNA-Seq , Transdução de Sinais/imunologia
4.
Nat Commun ; 11(1): 4457, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901017

RESUMO

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis.


Assuntos
Ácidos Graxos Voláteis/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade Inata , Interleucinas/biossíntese , Animais , Butiratos/imunologia , Butiratos/metabolismo , Butiratos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Citrobacter rodentium , Colite/imunologia , Colite/microbiologia , Colite/prevenção & controle , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/prevenção & controle , Ácidos Graxos Voláteis/metabolismo , Ácidos Graxos Voláteis/farmacologia , Microbioma Gastrointestinal/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Interleucinas/deficiência , Interleucinas/genética , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
5.
PLoS Pathog ; 16(8): e1008775, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866218

RESUMO

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.


Assuntos
Vírus da Influenza A/metabolismo , Mutação de Sentido Incorreto , Proteínas da Matriz Viral/metabolismo , Células A549 , Substituição de Aminoácidos , Animais , Cães , Feminino , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas da Matriz Viral/genética
6.
Nat Commun ; 11(1): 4364, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868781

RESUMO

Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.


Assuntos
Arritmias Cardíacas/etiologia , Insuficiência Cardíaca , Miócitos Cardíacos/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Arritmias Cardíacas/prevenção & controle , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Receptores de Dopamina D1/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/prevenção & controle
7.
Nat Commun ; 11(1): 4217, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868778

RESUMO

The cerebellum plays a crucial role in sensorimotor and associative learning. However, the contribution of molecular layer interneurons (MLIs) to these processes is not well understood. We used two-photon microscopy to study the role of ensembles of cerebellar MLIs in a go-no go task where mice obtain a sugar water reward if they lick a spout in the presence of the rewarded odorant and avoid a timeout when they refrain from licking for the unrewarded odorant. In naive animals the MLI responses did not differ between the odorants. With learning, the rewarded odorant elicited a large increase in MLI calcium responses, and the identity of the odorant could be decoded from the differential response. Importantly, MLIs switched odorant responses when the valence of the stimuli was reversed. Finally, mice took a longer time to refrain from licking in the presence of the unrewarded odorant and had difficulty becoming proficient when MLIs were inhibited by chemogenetic intervention. Our findings support a role for MLIs in learning valence in the cerebellum.


Assuntos
Cerebelo/fisiologia , Condicionamento Operante/fisiologia , Interneurônios/fisiologia , Aprendizagem/fisiologia , Células de Purkinje/fisiologia , Algoritmos , Animais , Cerebelo/citologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Odorantes , Recompensa , Fatores de Tempo
8.
Nat Commun ; 11(1): 4819, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968048

RESUMO

In many parts of the nervous system, experience-dependent refinement of neuronal circuits predominantly involves synapse elimination. The role of sleep in this process remains unknown. We investigated the role of sleep in experience-dependent dendritic spine elimination of layer 5 pyramidal neurons in the visual (V1) and frontal association cortex (FrA) of 1-month-old mice. We found that monocular deprivation (MD) or auditory-cued fear conditioning (FC) caused rapid spine elimination in V1 or FrA, respectively. MD- or FC-induced spine elimination was significantly reduced after total sleep or REM sleep deprivation. Total sleep or REM sleep deprivation also prevented MD- and FC-induced reduction of neuronal activity in response to visual or conditioned auditory stimuli. Furthermore, dendritic calcium spikes increased substantially during REM sleep, and the blockade of these calcium spikes prevented MD- and FC-induced spine elimination. These findings reveal an important role of REM sleep in experience-dependent synapse elimination and neuronal activity reduction.


Assuntos
Córtex Cerebral/fisiologia , Espinhas Dendríticas/fisiologia , Sono REM/fisiologia , Animais , Condicionamento Clássico , Medo/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Animais , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Células Piramidais/fisiologia , Privação Sensorial/fisiologia , Privação do Sono , Sinapses , Córtex Visual/fisiologia
9.
Nat Commun ; 11(1): 4810, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968061

RESUMO

Chimeric antigen receptor (CAR) therapy is a promising immunotherapeutic strategy for treating multiple refractory blood cancers, but further advances are required for solid tumor CAR therapy. One challenge is identifying a safe and effective tumor antigen. Here, we devise a strategy for targeting hepatocellular carcinoma (HCC, one of the deadliest malignancies). We report that T and NK cells transduced with a CAR that recognizes the surface marker, CD147, also known as Basigin, can effectively kill various malignant HCC cell lines in vitro, and HCC tumors in xenograft and patient-derived xenograft mouse models. To minimize any on-target/off-tumor toxicity, we use logic-gated (log) GPC3-synNotch-inducible CD147-CAR to target HCC. LogCD147-CAR selectively kills dual antigen (GPC3+CD147+), but not single antigen (GPC3-CD147+) positive HCC cells and does not cause severe on-target/off-tumor toxicity in a human CD147 transgenic mouse model. In conclusion, these findings support the therapeutic potential of CD147-CAR-modified immune cells for HCC patients.


Assuntos
Basigina/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Imunoterapia Adotiva/métodos , Neoplasias Hepáticas/tratamento farmacológico , Receptores de Antígenos Quiméricos/efeitos dos fármacos , Animais , Basigina/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Células Matadoras Naturais , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 11(1): 4520, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908154

RESUMO

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Colágeno/metabolismo , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Receptores Imunológicos/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA-Seq , Receptores Imunológicos/genética
11.
Nat Commun ; 11(1): 4527, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913197

RESUMO

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Evolução Clonal , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Cultura Primária de Células , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA-Seq , Estudos Retrospectivos , Esferoides Celulares , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína Supressora de Tumor p53/genética , Microtomografia por Raio-X
12.
Nat Commun ; 11(1): 4571, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917871

RESUMO

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APPSw/Ind) J20 and wild-type mice with VX-765 delays both APPSw/Ind- and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aß) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Disfunção Cognitiva/metabolismo , Serpinas/metabolismo , Proteínas Virais/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Comportamento Animal , Disfunção Cognitiva/tratamento farmacológico , Citocinas/metabolismo , Dipeptídeos/sangue , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Encefalite/metabolismo , Encefalite/patologia , Feminino , Humanos , Inflamação/metabolismo , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Serpinas/sangue , Serpinas/farmacologia , Memória Espacial/fisiologia , Proteínas Virais/sangue , Proteínas Virais/farmacologia , para-Aminobenzoatos/sangue , para-Aminobenzoatos/farmacologia
13.
Nat Commun ; 11(1): 4549, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917889

RESUMO

Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.


Assuntos
Artérias/citologia , Arterite/imunologia , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Macrófagos/fisiologia , Envelhecimento/fisiologia , Angiotensina II/administração & dosagem , Angiotensina II/imunologia , Animais , Artérias/fisiologia , Medula Óssea/fisiologia , Transplante de Medula Óssea , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Regeneração/fisiologia , Análise de Célula Única , Quimeras de Transplante
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(4): 519-524, 2020 Apr 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895127

RESUMO

OBJECTIVE: To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout. METHODS: Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM+/+, VIM-/-, VIM+/+/gp120 Tg and VIM-/-/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting. RESULTS: The results of PCR showed the presence of the target bands in VIM+/+, VIM-/-, VIM+/+/gp120 Tg and VIM-/-/gp120 Tg mice. In VIM-/-/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected. CONCLUSIONS: We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.


Assuntos
HIV-1 , Animais , Encéfalo , Modelos Animais de Doenças , Feminino , Proteína gp120 do Envelope de HIV , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Vimentina
15.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(8): 1184-1191, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895175

RESUMO

OBJECTIVE: To construct a HIV-1 gp120 transgenic mouse model (gp120+) with α7 nicotinic acetylcholine receptor (α7nAChR) gene knockout. METHODS: The α7nAChR gene knockout mice (α7R-/-) were crossed with HIV-1gp120 transgenic mice (gp120+) to generate F1 generation mice. We selected the F1 mice with the genotype of α7R+/-/gp120+ to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and α7nAChR inhibitor, and the expressions of IL-1ß and TNF-α were detected using ELISA. RESULTS: The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (α7R-/-/gp120+) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express α7 nAChR but with high gp120 protein expression. In the in vitro cell experiment, treatment with gp120 promoted the secretion of IL-1ß and TNF-α in BV2 cells, while inhibition of α7nAChR significantly decreased the expression of IL-1ß and TNF-α (P < 0.001). CONCLUSIONS: By mating gp120 Tg mice with α7R-/- mice, we obtained gp120 transgenic mice with α7nAChR gene deletion, which serve as a new animal model for exploring the role of α7nAChR in gp120-induced neurotoxicity.


Assuntos
Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Modelos Animais de Doenças , Glicoproteínas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Necrose Tumoral alfa
16.
Nat Commun ; 11(1): 4416, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887881

RESUMO

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Dilatada/metabolismo , Infarto do Miocárdio/fisiopatologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/fisiopatologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/cirurgia , Transdução de Sinais
17.
PLoS Pathog ; 16(9): e1008875, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898162

RESUMO

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.


Assuntos
Encéfalo/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Arvicolinae , Encéfalo/patologia , Cricetinae , Mesocricetus , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Domínios Proteicos
18.
Nat Commun ; 11(1): 4752, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958754

RESUMO

Growth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Gigantismo/metabolismo , Hormônio do Crescimento/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Acromegalia/metabolismo , Acromegalia/patologia , Animais , Composição Corporal , Linhagem Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Gigantismo/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Hipófise/metabolismo , Proteína Quinase C/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/genética
19.
PLoS Pathog ; 16(9): e1008857, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936838

RESUMO

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.


Assuntos
Antígenos Virais/genética , Enterovirus Humano A/genética , Variação Genética , Genoma Viral , Genótipo , Doença de Mão, Pé e Boca/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Enterovirus Humano A/imunologia , Enterovirus Humano A/isolamento & purificação , Feminino , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Taiwan
20.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
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