RESUMEN
Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.
Asunto(s)
Encéfalo , Quimiocina CCL5 , Quimiocina CXCL10 , Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Pericitos , Pericitos/virología , Pericitos/metabolismo , Humanos , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Encéfalo/virología , Encéfalo/metabolismo , Encéfalo/patología , Quimiocina CXCL10/metabolismo , Encefalitis Transmitida por Garrapatas/virología , Encefalitis Transmitida por Garrapatas/metabolismo , Quimiocina CCL5/metabolismo , Células Cultivadas , Replicación Viral , Citocinas/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Astrocitos , COVID-19 , Plexo Coroideo , Células Epiteliales , Neuronas , Pericitos , SARS-CoV-2 , Serina Endopeptidasas , Humanos , Pericitos/virología , SARS-CoV-2/fisiología , Astrocitos/virología , Plexo Coroideo/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Neuronas/virología , COVID-19/virología , COVID-19/patología , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Células Cultivadas , Encéfalo/virología , Encéfalo/patología , Sistema Nervioso Central/virologíaRESUMEN
Coronavirus disease 2019 (COVID-19) was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice and inflammation induced by bacterial lipopolysaccharide (LPS), we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.
Asunto(s)
COVID-19 , Células Endoteliales , Pericitos , SARS-CoV-2 , Pericitos/virología , Pericitos/metabolismo , Pericitos/patología , Humanos , Animales , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , COVID-19/virología , COVID-19/patología , Ratones , Células Endoteliales/virología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Factores de Riesgo , Lipopolisacáridos/farmacología , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Inflamación/virología , Inflamación/patologíaRESUMEN
Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Enterovirus Humano A , Infecciones por Enterovirus , Barrera Hematoencefálica/virología , Humanos , Enterovirus Humano A/genética , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/inmunología , Células Endoteliales/virología , Replicación Viral , Monocitos/virología , Monocitos/inmunología , Pericitos/virología , Leucocitos/virología , Leucocitos/inmunología , Encéfalo/virología , Células Asesinas Naturales/inmunología , Neutrófilos/inmunología , Neutrófilos/virologíaRESUMEN
The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery of whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to neurological complications both acutely and in long COVID.
Asunto(s)
Barrera Hematoencefálica , COVID-19 , Plexo Coroideo , SARS-CoV-2 , Barrera Hematoencefálica/virología , Animales , Plexo Coroideo/virología , Plexo Coroideo/patología , COVID-19/virología , COVID-19/patología , COVID-19/complicaciones , COVID-19/fisiopatología , Ratones , Uniones Estrechas/virología , Modelos Animales de Enfermedad , Enzima Convertidora de Angiotensina 2/metabolismo , Inflamación/virología , Humanos , Pericitos/virología , Pericitos/patologíaRESUMEN
The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.
Asunto(s)
Astrocitos , Barrera Hematoencefálica , Células Endoteliales , Infecciones por VIH , VIH-1 , Barrera Hematoencefálica/virología , Barrera Hematoencefálica/metabolismo , Humanos , Astrocitos/virología , Astrocitos/metabolismo , Astrocitos/inmunología , Células Endoteliales/virología , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , VIH-1/inmunología , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Pericitos/virología , Pericitos/metabolismo , Pericitos/inmunología , Enfermedades Neuroinflamatorias/virología , Enfermedades Neuroinflamatorias/inmunología , Técnicas de Cocultivo/métodos , Células Cultivadas , Encéfalo/virología , Encéfalo/inmunología , Encéfalo/metabolismoRESUMEN
SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Astrocitos , Barrera Hematoencefálica , COVID-19 , Dipeptidil Peptidasa 4 , Pericitos , SARS-CoV-2 , Transcitosis , Internalización del Virus , Barrera Hematoencefálica/virología , Barrera Hematoencefálica/metabolismo , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Pericitos/virología , Pericitos/metabolismo , COVID-19/virología , COVID-19/metabolismo , Astrocitos/virología , Astrocitos/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Encéfalo/virología , Encéfalo/metabolismo , Endocitosis , Células Endoteliales de la Vena Umbilical Humana/virología , PermeabilidadRESUMEN
Despite antiretroviral therapy (ART), chronic forms of HIV-associated neurocognitive disorders (HAND) affect an estimated 50% of individuals living with HIV, greatly impacting their quality of life. The prevailing theory of HAND progression posits that chronic inflammation arising from the activation of latent viral reservoirs leads to progressive damage in the central nervous system (CNS). Recent evidence indicates that blood-brain barrier (BBB) pericytes are capable of active HIV-1 infection; however, their latent infection has not been defined. Given their location and function, BBB pericytes are poised to be a key viral reservoir in the development of HAND. We present the first transcriptional analysis of uninfected, active, and latent human BBB pericytes, revealing distinct transcriptional phenotypes. In addition, we demonstrate that latent infection of BBB pericytes relies on AKT signaling for reservoir survival. These findings provide insight into the state of reservoir maintenance in the CNS during HIV-1 infection and provide novel targets for reservoir clearance.
Asunto(s)
Barrera Hematoencefálica , Reservorios de Enfermedades , Infecciones por VIH , VIH-1 , Infección Latente , Pericitos , Humanos , Barrera Hematoencefálica/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/transmisión , Infecciones por VIH/virología , Infección Latente/virología , Pericitos/virología , Proteínas Proto-Oncogénicas c-akt/genética , Calidad de Vida , Latencia del Virus , Reservorios de Enfermedades/virologíaRESUMEN
Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.
Asunto(s)
Vectores de Enfermedades , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Encefalitis Transmitida por Garrapatas/virología , Ixodes/virología , Animales , Células Cultivadas , Virus de la Encefalitis Transmitidos por Garrapatas/clasificación , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/aislamiento & purificación , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/transmisión , Células Endoteliales , Orden Génico , Genoma Viral , Interacciones Huésped-Patógeno/inmunología , Humanos , Interferones/farmacología , Pericitos/virología , Filogenia , Replicación Viral/efectos de los fármacosRESUMEN
A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRß in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood-brain barrier.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Encéfalo/virología , COVID-19/fisiopatología , Encefalitis Viral/virología , Pericitos/virología , Enzima Convertidora de Angiotensina 2/genética , Animales , Barrera Hematoencefálica , Encéfalo/patología , COVID-19/etiología , Estudios de Casos y Controles , Encefalitis Viral/patología , Fibrinógeno/metabolismo , Humanos , Inmunohistoquímica/métodos , Ratones , Pericitos/metabolismo , Pericitos/patología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/líquido cefalorraquídeoRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Basigina/metabolismo , Miocardio/enzimología , Pericitos/enzimología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/sangre , Células CACO-2 , Muerte Celular , Niño , Preescolar , Citocinas/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Miocardio/citología , Pericitos/virología , Cultivo Primario de Células , Adulto JovenRESUMEN
Viruses may exploit the cardiovascular system to facilitate transmission or within-host dissemination, and the symptoms of many viral diseases stem at least in part from a loss of vascular integrity. The microvascular architecture is comprised of an endothelial cell barrier ensheathed by perivascular cells (pericytes). Pericytes are antigen-presenting cells (APCs) and play crucial roles in angiogenesis and the maintenance of microvascular integrity through complex reciprocal contact-mediated and paracrine crosstalk with endothelial cells. We here review the emerging ways that viruses interact with pericytes and pay consideration to how these interactions influence microvascular function and viral pathogenesis. Major outcomes of virus-pericyte interactions include vascular leakage or haemorrhage, organ tropism facilitated by barrier disruption, including viral penetration of the blood-brain barrier and placenta, as well as inflammatory, neurological, cognitive and developmental sequelae. The underlying pathogenic mechanisms may include direct infection of pericytes, pericyte modulation by secreted viral gene products and/or the dysregulation of paracrine signalling from or to pericytes. Viruses we cover include the herpesvirus human cytomegalovirus (HCMV, Human betaherpesvirus 5), the retrovirus human immunodeficiency virus (HIV; causative agent of acquired immunodeficiency syndrome, AIDS, and HIV-associated neurocognitive disorder, HAND), the flaviviruses dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV), and the coronavirus severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2; causative agent of coronavirus disease 2019, COVID-19). We touch on promising pericyte-focussed therapies for treating the diseases caused by these important human pathogens, many of which are emerging viruses or are causing new or long-standing global pandemics.
Asunto(s)
Fenómenos Fisiológicos Celulares , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno , Pericitos/virología , Virosis/metabolismo , Virosis/virología , Animales , Comunicación Celular , Virus del Dengue/fisiología , Manejo de la Enfermedad , Células Endoteliales/virología , Endotelio/metabolismo , Endotelio/virología , VIH/fisiología , Humanos , Comunicación Paracrina , SARS-CoV-2/fisiología , Virosis/diagnóstico , Virosis/terapia , Fenómenos Fisiológicos de los VirusAsunto(s)
Encéfalo/fisiopatología , Encéfalo/virología , COVID-19/fisiopatología , COVID-19/virología , SARS-CoV-2/patogenicidad , Animales , Anticuerpos Antivirales/efectos adversos , Anticuerpos Antivirales/inmunología , Astrocitos/virología , Autoanticuerpos/inmunología , Autopsia , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , COVID-19/inmunología , COVID-19/patología , Capilares/virología , Cricetinae , Fatiga/complicaciones , Fatiga/virología , Sustancia Gris/patología , Humanos , Inmunoglobulinas Intravenosas/inmunología , Inmunoglobulinas Intravenosas/uso terapéutico , Losartán/uso terapéutico , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/virología , Neuronas/inmunología , Organoides/virología , Pericitos/virología , Preimpresos como Asunto , SARS-CoV-2/inmunología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/virología , VasoconstricciónRESUMEN
Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.
Asunto(s)
Astrocitos/virología , Encéfalo/virología , COVID-19/patología , Pericitos/virología , Tropismo Viral/fisiología , Astrocitos/citología , Encéfalo/patología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Interferón Tipo I/inmunología , SARS-CoV-2 , Replicación Viral/fisiologíaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.
Asunto(s)
Alphavirus/genética , Biosíntesis de Proteínas , Proteínas no Estructurales Virales/genética , eIF-2 Quinasa/genética , Alphavirus/clasificación , Alphavirus/fisiología , Astrocitos/metabolismo , Astrocitos/virología , Muerte Celular , Células Cultivadas , Virus de la Encefalitis Equina Venezolana/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células HEK293 , Humanos , Pericitos/metabolismo , Pericitos/virología , ARN Viral/metabolismo , Respuesta de Proteína Desplegada , Proteínas no Estructurales Virales/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Retinopatía Diabética/enzimología , Receptores Virales/metabolismo , Retina/enzimología , SARS-CoV-2/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Sitios de Unión , Western Blotting , Células Cultivadas , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/virología , Retinopatía Diabética/patología , Retinopatía Diabética/virología , Endotelio Vascular/enzimología , Endotelio Vascular/virología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pericitos/enzimología , Pericitos/virología , Vasos Retinianos/enzimología , Vasos Retinianos/patología , Vasos Retinianos/virología , Serina Endopeptidasas/metabolismoRESUMEN
Mayaro virus (MAYV) and chikungunya virus (CHIKV) are known for their arthrotropism, but accumulating evidence shows that CHIKV infections are occasionally associated with serious neurological complications. However, little is known about the capacity of MAYV to invade the central nervous system (CNS). We show that human neural progenitors (hNPCs), pericytes and astrocytes are susceptible to MAYV infection, resulting in the production of infectious viral particles. In primary astrocytes, MAYV, and to a lesser extent CHIKV, elicited a strong antiviral response, as demonstrated by an increased expression of several interferon-stimulated genes, including ISG15, MX1 and OAS2. Infection with either virus led to an enhanced expression of inflammatory chemokines, such as CCL5, CXCL10 and CXCL11, whereas MAYV induced higher levels of IL-6, IL-12 and IL-15 in these cells. Moreover, MAYV was more susceptible than CHIKV to the antiviral effects of both type I and type II interferons. Taken together, this study shows that although MAYV and CHIKV are phylogenetically related, they induce different types of antiviral responses in astrocytes. This work is the first to evaluate the potential neurotropism of MAYV and shows that brain cells and particularly astrocytes and hNPCs are permissive to MAYV, which, consequently, could lead to MAYV-induced neuropathology.
Asunto(s)
Infecciones por Alphavirus/inmunología , Alphavirus/inmunología , Astrocitos/inmunología , Astrocitos/virología , Encéfalo/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Infecciones por Alphavirus/patología , Animales , Encéfalo/virología , Línea Celular , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Fiebre Chikungunya/inmunología , Virus Chikungunya/inmunología , Chlorocebus aethiops , Citocinas/metabolismo , Humanos , Interferón Tipo I/inmunología , Interferón gamma/inmunología , Proteínas de Resistencia a Mixovirus/metabolismo , Células-Madre Neurales/virología , Pericitos/virología , Ubiquitinas/metabolismo , Células VeroAsunto(s)
Enzima Convertidora de Angiotensina 2/genética , Perfilación de la Expresión Génica/métodos , Insuficiencia Cardíaca/genética , Miocardio/metabolismo , Análisis de la Célula Individual/métodos , Adulto , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/epidemiología , COVID-19/virología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/virología , Ontología de Genes , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/virología , Humanos , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Pandemias , Pericitos/citología , Pericitos/metabolismo , Pericitos/virología , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologíaRESUMEN
Cancer-associated fibroblasts (CAFs) are activated fibroblasts constituting the major stromal components in many types of cancer. CAFs contribute to hallmarks of cancer such as proliferation, invasion and immunosuppressive tumor microenvironment, and are associated with poor prognosis of patients with cancer. However, in glioblastoma (GBM), the most common and aggressive primary malignant brain tumor, our knowledge about CAFs or CAF-like stromal cells is limited. Here, using commonly accepted CAF markers, we characterized CAF-like cell populations in clinical glioma specimens and datasets along with mouse models of GBM. We found that tumor-associated pericytes marked by co-expression of fibroblast activation protein α (FAP) and PDGFRß represent major stromal cell subsets in both human GBM and mouse GBM models, while a fraction of mesenchymal neoplastic cells also express FAP in patient tumors. Since oncolytic viruses can kill cancer cells and simultaneously modulate the tumor microenvironment by impacting non-neoplastic populations such as immune cells and tumor vasculature, we further investigated the ability of oncolytic viruses to target GBM-associated stromal cells. An oncolytic adenovirus, ICOVIR15, carrying ∆24-E1A and an RGD-fiber, infects and depletes FAP+ pericytes as well as GBM cells in murine GBM. Our study thus identifies FAP+/PDGFRß+ pericytes as a major CAF-like stromal cell population in GBM, and highlights the unique property of this oncolytic adenovirus to target both GBM cells and GBM-associated stromal FAP+ cells.