RESUMO
Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic ß-amyloid (Aß)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.
Assuntos
Apolipoproteínas E/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Transcriptoma , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apoptose/genética , Apoptose/imunologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Análise por Conglomerados , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Marcação de Genes , Humanos , Tolerância Imunológica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Doenças Neurodegenerativas/imunologia , Neurônios/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Fenótipo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.
Assuntos
Ciclinas , Hipertensão Arterial Pulmonar , Humanos , Camundongos , Animais , Ciclinas/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Cisteína/metabolismo , Células Endoteliais/metabolismo , Proliferação de Células , Artéria Pulmonar/metabolismo , Fosforilação , Pontos de Checagem do Ciclo Celular , Ciclina D/metabolismo , Células Cultivadas , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismoRESUMO
The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+-macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients.
Assuntos
Hospedeiro Imunocomprometido , Monkeypox virus , Mpox , Músculo Esquelético , Humanos , Músculo Esquelético/virologia , Músculo Esquelético/patologia , Músculo Esquelético/imunologia , Mpox/virologia , Mpox/imunologia , Monkeypox virus/imunologia , Masculino , Macrófagos/imunologia , Macrófagos/virologia , Fibroblastos/virologia , Fibroblastos/imunologia , Imuno-Histoquímica , Abscesso/imunologia , Abscesso/virologia , Abscesso/patologia , Pessoa de Meia-IdadeRESUMO
Chronic hypoxia causes pulmonary hypertension (PH), vascular remodeling, right ventricular (RV) hypertrophy, and cardiac failure. Protein kinase G Iα (PKGIα) is susceptible to oxidation, forming an interprotein disulfide homodimer associated with kinase targeting involved in vasodilation. Here we report increased disulfide PKGIα in pulmonary arteries from mice with hypoxic PH or lungs from patients with pulmonary arterial hypertension. This oxidation is likely caused by oxidants derived from NADPH oxidase-4, superoxide dismutase 3, and cystathionine γ-lyase, enzymes that were concomitantly increased in these samples. Indeed, products that may arise from these enzymes, including hydrogen peroxide, glutathione disulfide, and protein-bound persulfides, were increased in the plasma of hypoxic mice. Furthermore, low-molecular-weight hydropersulfides, which can serve as "superreductants" were attenuated in hypoxic tissues, consistent with systemic oxidative stress and the oxidation of PKGIα observed. Inhibiting cystathionine γ-lyase resulted in decreased hypoxia-induced disulfide PKGIα and more severe PH phenotype in wild-type mice, but not in Cys42Ser PKGIα knock-in (KI) mice that are resistant to oxidation. In addition, KI mice also developed potentiated PH during hypoxia alone. Thus, oxidation of PKGIα is an adaptive mechanism that limits PH, a concept further supported by polysulfide treatment abrogating hypoxia-induced RV hypertrophy in wild-type, but not in the KI, mice. Unbiased transcriptomic analysis of hypoxic lungs before structural remodeling identified up-regulation of endothelial-to-mesenchymal transition pathways in the KI compared with wild-type mice. Thus, disulfide PKGIα is an intrinsic adaptive mechanism that attenuates PH progression not only by promoting vasodilation but also by limiting maladaptive growth and fibrosis signaling.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Artéria Pulmonar/patologia , Adulto , Animais , Linhagem Celular , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dissulfetos/química , Feminino , Fibrose , Técnicas de Introdução de Genes , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/sangue , Hipóxia/tratamento farmacológico , Pulmão/irrigação sanguínea , Pulmão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Oxidantes/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sulfetos/administração & dosagem , Sulfetos/sangue , Sulfetos/metabolismo , Regulação para Cima , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF.IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Surtos de Doenças , Mucosa Intestinal/imunologia , Febre Lassa/imunologia , Vírus Lassa/patogenicidade , Ativação Linfocitária , Adolescente , Adulto , Idoso , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Lactente , Recém-Nascido , Integrina beta1/genética , Integrina beta1/imunologia , Interferon gama/genética , Interferon gama/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Febre Lassa/genética , Febre Lassa/mortalidade , Febre Lassa/virologia , Vírus Lassa/crescimento & desenvolvimento , Vírus Lassa/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Nigéria/epidemiologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Pele/imunologia , Pele/patologia , Pele/virologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Strategically located in mucosal barriers, innate lymphoid cells (ILCs) are relevant in local containment and tolerance of commensal microflora. ILCs have been recently described at the fetomaternal interface, where the development of a semi-allogeneic fetus can only succeed in a well-controlled immune environment. We postulate that ILCs adapt their antigen presentation capacity to protect pregnancy from excessive immune responses. Human ILCs were studied in deciduae of term pregnancies, peripheral blood and in in vitro generated ILCs. Fresh isolated lymphocytes or cells treated with pregnancy-related factors were investigated. The fetal antigen rejection-based CBA/J × DBA/2J mouse model (poor outcome pregnant mice; POPM) was used to characterize ILC antigen presentation potential in normal and immunologically disturbed pregnancies. ILC antigen presentation potential was characterized by flow cytometry and qPCR. We discovered that the distribution of ILC subsets changed during both human and murine pregnancy. Moreover, the pregnancy was accompanied by reduced MHCII expression in splenic ILCs during normal pregnancy (CBA/J × BALB/c; good outcome pregnant mice; GOPM) but increased in splenic and intestinal ILCs of CBA/J × DBA/2J mice. In vitro, splenic ILCs from pregnant mice increased MHCII expression after stimulation with IL-1ß and IL-23. In contrast, uterine ILCs displayed lower MHCII expression, which remained unchanged after stimulation. Finally, pregnancy-related factors and hormones present in the uterine environment reduced antigen presentation potential of human ILCs in vitro. Together, these data indicate that, during pregnancy, peripheral and especially uterine ILCs adapt their antigen presenting potential to maintain a level of tolerance and support pregnancy.
Assuntos
Apresentação de Antígeno/imunologia , Feto/imunologia , Hormônios/farmacologia , Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Feminino , Feto/efeitos dos fármacos , Humanos , Tolerância Imunológica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , GravidezRESUMO
Severe human adenovirus (HAdV) infections are an increasing threat for immunosuppressed individuals, particularly those who have received stem cell transplants. It has been previously hypothesized that severe infections might be due to reactivation of a persistent infection, but this hypothesis has been difficult to test owing to the lack of a permissive in vivo model of HAdV infection. Here we established a humanized mouse model that reproduces features of acute and persistent HAdV infection. In this model, acute infection correlated with high mortality, weight loss, liver pathology, and expression of viral proteins in several organs. In contrast, persistent infection was asymptomatic and led to establishment of HAdV-specific adaptive immunity and expression of early viral genes exclusively in the bone marrow. These findings validate the use of humanized mice to study acute and persistent HAdV infection and strongly suggest the presence of cellular reservoirs in the bone marrow.
Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Infecções Assintomáticas , Modelos Animais de Doenças , Doença Aguda , Imunidade Adaptativa , Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Animais , Medula Óssea/virologia , DNA Viral/genética , Humanos , Hospedeiro Imunocomprometido , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Transgênicos , Carga Viral , ViremiaRESUMO
Rickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhiGFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhiGFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhiGFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhiGFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Rickettsia typhi/patogenicidade , Tifo Endêmico Transmitido por Pulgas/imunologia , Animais , Proteínas de Fluorescência Verde/genética , Fígado/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neutrófilos/microbiologia , Plasmídeos , Rickettsia typhi/genética , Baço/microbiologia , Transformação Bacteriana , Tifo Endêmico Transmitido por Pulgas/microbiologiaRESUMO
We have previously reported that the hormone calcitonin (CT) negatively regulates bone formation by inhibiting the release of sphingosine-1-phosphate from bone-resorbing osteoclasts. In the context of this study we additionally observed that CT repressed the expression of Pate4, encoding the secreted protein caltrin/Svs7, in osteoclasts from wildtype mice. To assess a possible function of Pate4 in bone remodeling, we utilized commercially available embryonic stem cells with a targeted Pate4 allele to generate Pate4-deficient mice. These were born at the expected Mendelian ratio and did not display obvious abnormalities until the age of 6 months. A bone-specific histomorphometric analysis further revealed that bone remodeling is unaffected in male and female Pate4-deficient mice. Since a subsequently performed multi-tissue expression analysis confirmed that Pate4 is primarily expressed in prostate and seminal vesicles, we additionally analyzed the respective tissues of Pate4-deficient mice, but failed to detect histological abnormalities. Most importantly, as assessed by mating with female wildtype mice, we did not observe reduced fertility associated with Pate4-deficiency. Taken together, our study was the first to generate and analyze a mouse model lacking Pate4, a gene with strong expression in prostate and seminal vesicles, yet without major function for fertility.
Assuntos
Anormalidades Múltiplas/genética , Reabsorção Óssea/genética , Proteínas de Transporte/genética , Camundongos/genética , Proteínas Secretadas pela Vesícula Seminal/genética , Animais , Predisposição Genética para Doença/genética , Camundongos Knockout , FenótipoRESUMO
We characterized the world's second case with ascertained extreme resilience to autosomal dominant Alzheimer's disease (ADAD). Side-by-side comparisons of this male case and the previously reported female case with ADAD homozygote for the APOE3 Christchurch (APOECh) variant allowed us to discern common features. The male remained cognitively intact until 67 years of age despite carrying a PSEN1-E280A mutation. Like the APOECh carrier, he had extremely elevated amyloid plaque burden and limited entorhinal Tau tangle burden. He did not carry the APOECh variant but was heterozygous for a rare variant in RELN (H3447R, termed COLBOS after the Colombia-Boston biomarker research study), a ligand that like apolipoprotein E binds to the VLDLr and APOEr2 receptors. RELN-COLBOS is a gain-of-function variant showing stronger ability to activate its canonical protein target Dab1 and reduce human Tau phosphorylation in a knockin mouse. A genetic variant in a case protected from ADAD suggests a role for RELN signaling in resilience to dementia.
Assuntos
Doença de Alzheimer , Animais , Feminino , Humanos , Masculino , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Heterozigoto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de SinaisRESUMO
Prion diseases are neurodegenerative diseases that affect humans and animals. They are always fatal and, to date, no treatment exists. The hallmark of prion disease pathophysiology is the misfolding of an endogenous protein, the cellular prion protein (PrPC), into its disease-associated isoform PrPSc. Besides the aggregation and deposition of misfolded PrPSc, prion diseases are characterized by spongiform lesions and the activation of astrocytes and microglia. Microglia are the innate immune cells of the brain. Activated microglia and astrocytes represent a common pathological feature in neurodegenerative disorders. The role of activated microglia has already been studied in prion disease mouse models; however, it is still not fully clear how they contribute to disease progression. Moreover, the role of microglia in human prion diseases has not been thoroughly investigated thus far, and specific molecular pathways are still undetermined. Here, we review the current knowledge on the different roles of microglia in prion pathophysiology. We discuss microglia markers that are also dysregulated in other neurodegenerative diseases including microglia homeostasis markers. Data on murine and human brain tissues show that microglia are highly dysregulated in prion diseases. We highlight here that the loss of homeostatic markers may especially stand out.
Assuntos
Doenças Neurodegenerativas , Doenças Priônicas , Príons , Animais , Homeostase , Humanos , Camundongos , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
OBJECTIVE: Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. RESULTS: Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. CONCLUSION: We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. SIGNIFICANCE: Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Animais , Reatores Biológicos , DNA , Matriz Extracelular , Músculo Esquelético , Perfusão , Ratos , Engenharia Tecidual/métodosRESUMO
Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFß-1). EVTs contracted in response to TGFß-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.
RESUMO
Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.
Assuntos
Cardiomiopatia Hipertrófica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Homeodomínio LIM , Proteínas Musculares , Miócitos Cardíacos , Fatores de Transcrição , Animais , Cardiomiopatia Hipertrófica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.
Assuntos
Barreira Hematoencefálica/virologia , Sistema Nervoso Central/virologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Anticorpos/farmacologia , Benzamidinas/farmacologia , COVID-19/patologia , COVID-19/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Guanidinas/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.
Assuntos
COVID-19 , Autopsia , Humanos , RNA Viral/análise , Reprodutibilidade dos Testes , SARS-CoV-2 , Proteínas ViraisRESUMO
Nowadays, the beneficial role of a healthy lifestyle, particularly emphasizing the quality of foods and cancer management, is accepted worldwide. Polyphenols and oleic acid play a key role in this context, but are still scarcely used as anti-cancer agents due to their bio-accessibility limits. Therefore, we aimed to synthesize a set of new oleoyl-hybrids of quercetin, morin, pinocembrin, and catechin to overcome the low bioavailability of polyphenols, throughout a bio-catalytic approach using pancreatic porcine lipase as a catalyst. The in vitro assays, using a wide panel of human cancer cell lines showed, mainly for two novel regioisomer oleoyl-hybrids of quercetin, a remarkable increase in apoptotic cell populations. We suggested that the DNA damage shown as ɣH2AX signals might be the major cause of apoptotic cell death. Finally, we demonstrated convincing data about two novel polyphenol-based hybrids displaying a highly selective anti-cancer cytotoxicity and being superior compared to their reference/parental compounds.
RESUMO
Astrogliosis and activation of microglia are hallmarks of prion diseases in humans and animals. Both were viewed to be rather independent events in disease pathophysiology, with proinflammatory microglia considered to be the potential neurotoxic species at late disease stages. Recent investigations have provided substantial evidence that a proinflammatory microglial cytokine cocktail containing TNF-α, IL-1α and C1qa reprograms a subset of astrocytes to change their expression profile and phenotype, thus becoming neurotoxic (designated as A1-astrocytes). Knockout or antibody blockage of the three cytokines abolish formation of A1-astrocytes, therefore, this pathway is of high therapeutic interest in neurodegenerative diseases. Since astrocyte polarization profiles have never been investigated in prion diseases, we performed several analyses and could show that C3+-PrPSc-reactive-astrocytes, which may represent a subtype of A1-astrocytes, are highly abundant in prion disease mouse models and human prion diseases. To investigate their impact on prion disease pathophysiology and to evaluate their potential therapeutic targeting, we infected TNF-α, IL-1α, and C1qa Triple-KO mice (TKO-mice), which do not transit astrocytes into A1, with prions. Although formation of C3+-astrocytes was significantly reduced in prion infected Triple-KO-mice, this did not affect the amount of PrPSc deposition or titers of infectious prions. Detailed characterization of the astrocyte activation signature in thalamus tissue showed that astrocytes in prion diseases are highly activated, showing a mixed phenotype that is distinct from other neurodegenerative diseases and were therefore termed C3+-PrPSc-reactive-astrocytes. Unexpectedly, Triple-KO led to a significant acceleration of prion disease course. While pan-astrocyte and -microglia marker upregulation was unchanged compared to WT-brains, microglial homeostatic markers were lost early in disease in TKO-mice, pointing towards important functions of different glia cell types in prion diseases.
Assuntos
Astrócitos/patologia , Complemento C3/metabolismo , Microglia/metabolismo , Microglia/patologia , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Idoso , Animais , Astrócitos/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Citocinas/genética , Citocinas/metabolismo , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas PrPSc/metabolismoRESUMO
Filoviruses of the genus Ebolavirus include 6 species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus, case fatality rates can range between 0% and 90%. In order to understand the molecular basis of these differences, it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Nonhuman primates (NHPs) are the gold-standard models for filovirus pathogenesis, but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHPs. Here we used HLA-A2-transgenic, NOD-scid-IL-2γ receptor-knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While markedly less pathogenic than Ebola virus, Reston virus killed 20% of infected mice, a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition, the case fatality ratios of different Ebolavirus species in humans were recapitulated in the humanized mice. Our findings point to humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses, and suggest that further investigations on Reston virus pathogenesis in humans are warranted.
Assuntos
Doença pelo Vírus Ebola/patologia , Animais , Modelos Animais de Doenças , Ebolavirus/patogenicidade , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mucosa/virologia , Carga Viral , Replicação ViralRESUMO
Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrPC ) into a misfolded isoform (PrPSc ) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrPSc -clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion-only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.