RESUMO
Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With Type-I interferon (IFN) signalling being an important first line cellular defence mechanism against VZV infection by the peripheral tissues, we here investigated the triggering of innate immune responses in a human neural-like environment. For this, we established and characterised 5-month matured hiPSC-derived neurospheroids (NSPHs) containing neurons and astrocytes. Subsequently, NSPHs were infected with reporter strains of VZV (VZVeGFP-ORF23) or Sendai virus (SeVeGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZVeGFP-ORF23 infection throughout the NSPHs, while SeVeGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeVeGFP-infected NSPHs activated a clear Type-I IFN response, while this was not the case in VZVeGFP-ORF23-infected NSPHs. Moreover, the latter displayed a strong suppression of genes related to IFN signalling and antigen presentation, as further demonstrated by suppression of IL-6 and CXCL10 production, failure to upregulate Type-I IFN activated anti-viral proteins (Mx1, IFIT2 and ISG15), as well as reduced expression of CD74, a key-protein in the MHC class II antigen presentation pathway. Finally, even though VZVeGFP-ORF23-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules upon long-term infection, as well as disruption of cellular integrity within the infected NSPHs. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, and have the capacity to recapitulate the strong immune evasive behaviour towards VZV.
Assuntos
Herpesvirus Humano 3 , Células-Tronco Pluripotentes Induzidas , Humanos , Herpesvirus Humano 3/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/virologia , Imunidade Inata , Neurônios/imunologia , Neurônios/virologia , Infecção pelo Vírus da Varicela-Zoster/imunologia , Infecção pelo Vírus da Varicela-Zoster/virologia , Células Cultivadas , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Evasão da Resposta Imune , Citocinas/metabolismo , Citocinas/imunologia , Astrócitos/imunologia , Astrócitos/virologia , Astrócitos/metabolismo , Transdução de Sinais/imunologiaAssuntos
Astrócitos , Camundongos Knockout , Neurônios , Phlebovirus , Receptor de Interferon alfa e beta , Transdução de Sinais , Animais , Camundongos , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/deficiência , Neurônios/virologia , Neurônios/patologia , Astrócitos/virologia , Phlebovirus/genética , Morte Celular , Febre Grave com Síndrome de Trombocitopenia/virologia , Modelos Animais de DoençasRESUMO
The Zika virus (ZIKV) epidemic declared in Brazil between 2015 and 2016 was associated with an increased prevalence of severe congenital malformations, including microcephaly. The distribution of microcephaly cases was not uniform across the country, with a disproportionately higher incidence in the Northeast region (NE). Our previous work demonstrated that saxitoxin (STX), a toxin present in the drinking water reservoirs of the NE, exacerbated the damaging effects of ZIKV on the developing brain. We hypothesized that the impact of STX might vary among different neural cell types. While ZIKV infection caused severe damages on astrocytes and neural stem cells (NSCs), the addition of STX did not exacerbate these effects. We observed that neurons subjected to STX exposure were more prone to apoptosis and displayed higher ZIKV infection rate. These findings suggest that STX exacerbates the harmful effects of ZIKV on neurons, thereby providing a plausible explanation for the heightened severity of ZIKV-induced congenital malformations observed in Brazil's NE. This study highlights the importance of understanding the interactive effects of environmental toxins and infectious pathogens on neural development, with potential implications for public health policies.
Assuntos
Astrócitos , Células-Tronco Neurais , Neurônios , Saxitoxina , Infecção por Zika virus , Zika virus , Células-Tronco Neurais/virologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Humanos , Zika virus/fisiologia , Astrócitos/virologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Neurônios/virologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Infecção por Zika virus/virologia , Infecção por Zika virus/patologia , Saxitoxina/toxicidade , Apoptose/efeitos dos fármacos , Microcefalia/virologia , Morte Celular/efeitos dos fármacos , Brasil , Células CultivadasRESUMO
BACKGROUND: Infection of astrocytes by Human Immunodeficiency Virus (HIV-1) remains a topic of debate, with conflicting data, yet instances of astrocytes containing viral DNA have been observed in vivo. In this study, we aimed to elucidate potential routes through which astrocytes could be infected and their ability to produce infectious particles using primary human astrocytes. METHODS: We infected primary astrocytes derived from either neuroprogenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) that express both C-X-C chemokine receptor type 4 (CXCR4) and the C-C chemokine receptor type 5 (CCR5) coreceptors, using either cell-free HIV-1 virus directly or cell-associated virus indirectly through infected macrophages and microglia. RESULTS: Low-level infectivity by cell-free viruses was primarily attributed to a defect in the entry process. Bypassing HIV-specific receptor-mediated entry using pseudotyped viruses resulted in productive infection and the release of infectious particles. CONCLUSIONS: These findings suggest that astrocytes may be one of the potential sources of neurotoxicity in HIV-associated neurocognitive disorders (HAND) and could possibly act as reservoirs for HIV in the central nervous system (CNS).
Assuntos
Astrócitos , HIV-1 , Astrócitos/virologia , Astrócitos/metabolismo , Humanos , HIV-1/fisiologia , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/virologia , Células-Tronco Neurais/virologia , Células-Tronco Neurais/metabolismo , Receptores CXCR4/metabolismo , Receptores CCR5/metabolismo , Infecções por HIVRESUMO
The COVID-19, caused by SARS-CoV-2, first affects the respiratory tract but evidence is emerging that the virus, reaching the central nervous system (CNS), can lead to severe neurological disorders. In particular, CoV infection could cause an acceleration of the neurodegenerative process. On the other hand, patients diagnosed with Alzheimer's disease (AD) develop more serious forms of COVID-19 with worse relapses. Therefore, understanding the connection between the two pathologies, AD and infection by coronavirus, could help in the development of new therapeutic approaches to counter them. We used the SH-SY5Y cell line differentiated into neurons, as widely used in studies of AD if supplemented with exogenous fibrillary ß-amyloid (Aß). As a glial counterpart, human microglia (HMC3) and astrocytic (D54MG) cell lines were used to create co-cultures with neurons via transwell systems. In these experimental models, we generated infection with the Human Coronavirus OC43 (HCoV-OC43), a low-risk model of SARS-CoV-2. Our results suggest that the infection by HCoV-OC43 leads to a neurotoxic effect not depending on an already present event of Aß deposition. Indeed, unlike microglia, neurons and even more astrocytes are susceptible to CoV infection and, although the infection does not show a cytotoxic effect in the neurons in the first few days, significant alterations at a biochemical and morphological level have been observed, suggesting that the neurons are reacting to a stressful condition, including the prodromal and neurodegenerative features of AD. Interestingly, the interaction of infected astrocytes with the neurons resulted in the manifestation of signs of neurodegeneration, such as amyloid-beta deposition. By using exogenous fibrillary Aß, as an AD in vitro model, our data suggest that there is an aggravating effect both on the infection itself and on the neurological disease progression. In conclusion, the results of this study suggest a causal interplay between HCoV-OC43 and neurological diseases and demonstrate that the co-presence of different CNS cell populations is the necessary condition to study the pathogenic effects in vitro as a whole.
Assuntos
Doença de Alzheimer , Astrócitos , COVID-19 , Microglia , Neurônios , Humanos , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/virologia , COVID-19/patologia , Microglia/metabolismo , Microglia/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Neurônios/patologia , Neurônios/metabolismo , Neurônios/virologia , Técnicas de Cocultura , Coronavirus Humano OC43 , Peptídeos beta-Amiloides/metabolismo , Progressão da Doença , SARS-CoV-2/patogenicidade , Linhagem Celular Tumoral , Linhagem CelularRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) has a high mortality rate compared to other infectious diseases. SFTS is particularly associated with a high risk of mortality in immunocompromised individuals, while most patients who die of SFTS exhibit symptoms of severe encephalitis before death. However, the region of brain damage and mechanisms by which the SFTS virus (SFTSV) causes encephalitis remains unknown. Here, we revealed that SFTSV infects the brainstem and spinal cord, which are regions of the brain associated with respiratory function, and motor nerves in IFNAR1-/- mice. Further, we show that A1-reactive astrocytes are activated, causing nerve cell death, in infected mice. Primary astrocytes of SFTSV-infected IFNAR1-/- mice also induced neuronal cell death through the activation of A1-reactive astrocytes. Herein, we showed that SFTSV induces fatal neuroinflammation in the brain regions important for respiratory function and motor nerve, which may underlie mortality in SFTS patients. This study provides new insights for the treatment of SFTS, for which there is currently no therapeutic approach.
Assuntos
Astrócitos , Infecções por Bunyaviridae , Camundongos Knockout , Phlebovirus , Receptor de Interferon alfa e beta , Animais , Astrócitos/virologia , Astrócitos/patologia , Camundongos , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/deficiência , Phlebovirus/genética , Phlebovirus/fisiologia , Phlebovirus/patogenicidade , Infecções por Bunyaviridae/virologia , Infecções por Bunyaviridae/patologia , Infecções por Bunyaviridae/imunologia , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/imunologia , Medula Espinal/virologia , Medula Espinal/patologia , Modelos Animais de Doenças , Neurônios/virologia , Neurônios/patologia , Camundongos Endogâmicos C57BL , Tronco Encefálico/virologia , Tronco Encefálico/patologia , Morte CelularRESUMO
Zika virus (ZIKV) infection was first reported in 2015 in Brazil as causing microcephaly and other developmental abnormalities in newborns, leading to the identification of Congenital Zika Syndrome (CZS). Viral infections have been considered an environmental risk factor for neurodevelopmental disorders outcome, such as Autism Spectrum Disorder (ASD). Moreover, not only the infection per se, but maternal immune system activation during pregnancy, has been linked to fetal neurodevelopmental disorders. To understand the impact of ZIKV vertical infection on brain development, we derived induced pluripotent stem cells (iPSC) from Brazilian children born with CZS, some of the patients also being diagnosed with ASD. Comparing iPSC-derived neurons from CZS with a control group, we found lower levels of pre- and postsynaptic proteins and reduced functional synapses by puncta co-localization. Furthermore, neurons and astrocytes derived from the CZS group showed decreased glutamate levels. Additionally, the CZS group exhibited elevated levels of cytokine production, one of which being IL-6, already associated with the ASD phenotype. These preliminary findings suggest that ZIKV vertical infection may cause long-lasting disruptions in brain development during fetal stages, even in the absence of the virus after birth. These disruptions could contribute to neurodevelopmental disorders manifestations such as ASD. Our study contributes with novel knowledge of the CZS outcomes and paves the way for clinical validation and the development of potential interventions to mitigate the impact of ZIKV vertical infection on neurodevelopment.
Assuntos
Encéfalo , Células-Tronco Pluripotentes Induzidas , Transmissão Vertical de Doenças Infecciosas , Sinapses , Infecção por Zika virus , Zika virus , Humanos , Infecção por Zika virus/virologia , Infecção por Zika virus/patologia , Feminino , Zika virus/patogenicidade , Sinapses/patologia , Sinapses/metabolismo , Encéfalo/virologia , Encéfalo/patologia , Gravidez , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/virologia , Neurônios/virologia , Neurônios/metabolismo , Neurônios/patologia , Masculino , Astrócitos/virologia , Astrócitos/metabolismo , Doenças Neuroinflamatórias/virologia , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/metabolismo , Complicações Infecciosas na Gravidez/virologia , Complicações Infecciosas na Gravidez/patologia , Brasil , Recém-Nascido , Transtorno do Espectro Autista/virologia , CriançaRESUMO
Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb, whereas later, oligodendrocytes were the main producers. IFNb+ cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection.
Assuntos
Astrócitos , Animais , Camundongos , Astrócitos/virologia , Astrócitos/metabolismo , Replicação Viral , Microglia/virologia , Microglia/metabolismo , Microglia/imunologia , Neurônios/virologia , Neurônios/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Vesiculovirus/fisiologia , Vesiculovirus/imunologia , Vesiculovirus/genética , Oligodendroglia/virologia , Oligodendroglia/metabolismo , Estomatite Vesicular/virologia , Estomatite Vesicular/imunologia , Imunidade Inata , Camundongos Endogâmicos C57BL , Encéfalo/virologia , Encéfalo/metabolismo , Encéfalo/imunologia , Neuroglia/virologia , Neuroglia/metabolismoRESUMO
Tick-borne encephalitis virus (TBEV) is a neurotropic member of the genus Orthoflavivirus (former Flavivirus) and is of significant health concern in Europe and Asia. TBEV pathogenesis may occur directly via virus-induced damage to neurons or through immunopathology due to excessive inflammation. While primary cells isolated from the host can be used to study the immune response to TBEV, it is still unclear how well these reflect the immune response elicited in vivo. Here, we compared the transcriptional response to TBEV and the less pathogenic tick-borne flavivirus, Langat virus (LGTV), in primary monocultures of neurons, astrocytes and microglia in vitro, with the transcriptional response in vivo captured by single-nuclei RNA sequencing (snRNA-seq) of a whole mouse cortex. We detected similar transcriptional changes induced by both LGTV and TBEV infection in vitro, with the lower response to LGTV likely resulting from slower viral kinetics. Gene set enrichment analysis showed a stronger transcriptional response in vivo than in vitro for astrocytes and microglia, with a limited overlap mainly dominated by interferon signaling. Together, this adds to our understanding of neurotropic flavivirus pathogenesis and the strengths and limitations of available model systems.
Assuntos
Astrócitos , Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Microglia , Neurônios , Animais , Astrócitos/virologia , Microglia/virologia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Camundongos , Neurônios/virologia , Encefalite Transmitida por Carrapatos/virologia , Infecções por Flavivirus/virologia , Infecções por Flavivirus/imunologia , Células Cultivadas , Flavivirus/fisiologia , Flavivirus/genética , Camundongos Endogâmicos C57BL , Transcrição GênicaRESUMO
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.
Assuntos
Enzima de Conversão de Angiotensina 2 , Astrócitos , COVID-19 , Plexo Corióideo , Células Epiteliais , Neurônios , Pericitos , SARS-CoV-2 , Serina Endopeptidases , Humanos , Pericitos/virologia , SARS-CoV-2/fisiologia , Astrócitos/virologia , Plexo Corióideo/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Neurônios/virologia , COVID-19/virologia , COVID-19/patologia , Células Epiteliais/virologia , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Células Cultivadas , Encéfalo/virologia , Encéfalo/patologia , Sistema Nervoso Central/virologiaRESUMO
HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.
Assuntos
Encéfalo , Infecções por HIV , HIV-1 , Humanos , Encéfalo/virologia , Encéfalo/patologia , HIV-1/fisiologia , Infecções por HIV/virologia , Infecções por HIV/patologia , Adulto , Neurônios/virologia , Neurônios/metabolismo , Macrófagos/virologia , Macrófagos/metabolismo , Astrócitos/virologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cultura de TecidosRESUMO
The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment.
Assuntos
Barreira Hematoencefálica , Plexo Corióideo , Vírus JC , Humanos , Barreira Hematoencefálica/virologia , Barreira Hematoencefálica/metabolismo , Plexo Corióideo/virologia , Plexo Corióideo/metabolismo , Vírus JC/fisiologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Animais , Astrócitos/virologia , Astrócitos/metabolismo , Linhagem Celular , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
Caffeine is one of the most popular consumed psychostimulants that mitigates several neurodegenerative diseases. Nevertheless, the roles and molecular mechanisms of caffeine in HIV-associated neurocognitive disorders (HAND) remain largely unclear. Transactivator of transcription (Tat) is a major contributor to the neuropathogenesis of HAND in the central nervous system. In the present study, we determined that caffeine (100 µM) treatment significantly ameliorated Tat-induced decreased astrocytic viability, oxidative stress, inflammatory response and excessive glutamate and ATP release, thereby protecting neurons from apoptosis. Subsequently, SIRT3 was demonstrated to display neuroprotective effects against Tat during caffeine treatment. In addition, Tat downregulated SIRT3 expression via activation of EGR1 signaling, which was reversed by caffeine treatment in astrocytes. Overexpression of EGR1 entirely abolished the neuroprotective effects of caffeine against Tat. Furthermore, counteracting Tat or caffeine-induced differential expression of SIRT3 abrogated the neuroprotection of caffeine against Tat-triggered astrocytic dysfunction and neuronal apoptosis. Taken together, our study establishes that caffeine ameliorates astrocytes-mediated Tat neurotoxicity by targeting EGR1/SIRT3 signaling pathway. Our findings highlight the beneficial effects of caffeine on Tat-induced astrocytic dysfunction and neuronal death and propose that caffeine might be a novel therapeutic drug for relief of HAND.
Assuntos
Apoptose , Astrócitos , Cafeína , Proteína 1 de Resposta de Crescimento Precoce , HIV-1 , Neurônios , Transdução de Sinais , Sirtuína 3 , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Astrócitos/patologia , Sirtuína 3/genética , Sirtuína 3/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Cafeína/farmacologia , Humanos , Apoptose/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Regulação para Cima/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Complexo AIDS Demência/tratamento farmacológico , Complexo AIDS Demência/genética , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/patologia , Estimulantes do Sistema Nervoso Central/farmacologia , Estimulantes do Sistema Nervoso Central/toxicidadeRESUMO
Although previous studies have suggested that subtype B HIV-1 proviruses in the brain are associated with physiological changes and immune activation accompanied with microgliosis and astrogliosis, and indicated that both HIV-1 subtype variation and geographical location might influence the neuropathogenicity of HIV-1 in the brain. The natural course of neuropathogenesis of the most widespread subtype C HIV-1 has not been adequately investigated, especially for people living with HIV (PLWH) in sub-Saharan Africa. To characterize the natural neuropathology of subtype C HIV-1, postmortem frontal lobe and basal ganglia tissues were collected from nine ART-naïve individuals who died of late-stage AIDS with subtype C HIV-1 infection, and eight uninfected deceased individuals as controls. Histological staining was performed on all brain tissues to assess brain pathologies. Immunohistochemistry (IHC) against CD4, p24, Iba-1, GFAP, and CD8 in all brain tissues was conducted to evaluate potential viral production and immune activation. Histological results showed mild perivascular cuffs of lymphocytes only in a minority of the infected individuals. Viral capsid p24 protein was only detected in circulating immune cells of one infected individual, suggesting a lack of productive HIV-1 infection of the brain even at the late-stage of AIDS. Notably, similar levels of Iba-1 or GFAP between HIV + and HIV- brain tissues indicated a lack of microgliosis and astrogliosis, respectively. Similar levels of CD8 + cytotoxic T lymphocyte (CTL) infiltration between HIV + and HIV- brain tissues indicated CTL were not likely to be involved within subtype C HIV-1 infected participants of this cohort. Results from this subtype C HIV-1 study suggest that there is a lack of productive infection and limited neuropathogenesis by subtype C HIV-1 even at late-stage disease, which is in contrast to what was reported for subtype B HIV-1 by other investigators.
Assuntos
Proteína Glial Fibrilar Ácida , HIV-1 , Humanos , HIV-1/imunologia , HIV-1/patogenicidade , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , África Subsaariana , Proteína Glial Fibrilar Ácida/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/patologia , Gânglios da Base/imunologia , Gânglios da Base/patologia , Gânglios da Base/virologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/genética , Lobo Frontal/imunologia , Lobo Frontal/patologia , Lobo Frontal/virologia , Proteína do Núcleo p24 do HIV/imunologia , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Antígenos CD4/imunologia , Linfócitos T CD8-Positivos/imunologia , Gliose/imunologia , Gliose/patologia , Gliose/virologia , Astrócitos/imunologia , Astrócitos/patologia , Astrócitos/virologia , Encéfalo/patologia , Encéfalo/imunologia , Encéfalo/virologia , Proteínas dos MicrofilamentosRESUMO
Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.
Assuntos
Astrócitos , Infecções por Citomegalovirus , Citomegalovirus , Proteínas Hedgehog , Placenta , Proteínas Tirosina Quinases , Transdução de Sinais , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Citomegalovirus/fisiologia , Gravidez , Placenta/virologia , Placenta/metabolismo , Astrócitos/virologia , Astrócitos/metabolismo , Feminino , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosforilação , Trofoblastos/virologia , Trofoblastos/metabolismo , Quinases Dyrk , Linhagem Celular , Células CultivadasRESUMO
SARS-CoV-2 spike proteins have been shown to cross the blood-brain barrier (BBB) in mice and affect the integrity of human BBB cell models. However, the effects of SARS-CoV-2 spike proteins in relation to sporadic, late onset, Alzheimer's disease (AD) risk have not been extensively investigated. Here we characterized the individual and combined effects of SARS-CoV-2 spike protein subunits S1 RBD, S1 and S2 on BBB cell types (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) generated from induced pluripotent stem cells (iPSCs) harboring low (APOE3 carrier) or high (APOE4 carrier) relative Alzheimer's risk. We found that treatment with spike proteins did not alter iBEC integrity, although they induced the expression of several inflammatory cytokines. iAstrocytes exhibited a robust inflammatory response to SARS-CoV-2 spike protein treatment, with differences found in the levels of cytokine secretion between spike protein-treated APOE3 and APOE4 iAstrocytes. Finally, we tested the effects of potentially anti-inflammatory drugs during SARS-CoV-2 spike protein exposure in iAstrocytes, and discovered different responses between spike protein treated APOE4 iAstrocytes and APOE3 iAstrocytes, specifically in relation to IL-6, IL-8 and CCL2 secretion. Overall, our results indicate that APOE3 and APOE4 iAstrocytes respond differently to anti-inflammatory drug treatment during SARS-CoV-2 spike protein exposure with potential implications to therapeutic responses.
Assuntos
Apolipoproteína E3 , Apolipoproteína E4 , Astrócitos , Barreira Hematoencefálica , Citocinas , Glicoproteína da Espícula de Coronavírus , Barreira Hematoencefálica/metabolismo , Humanos , Citocinas/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Astrócitos/metabolismo , Astrócitos/virologia , Astrócitos/efeitos dos fármacos , Apolipoproteína E3/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , SARS-CoV-2 , COVID-19/metabolismo , COVID-19/imunologia , Células CultivadasRESUMO
Introduction: While astrocytes participate in the CNS innate immunity against herpes simplex virus type 1 (HSV-1) infection, they are the major target for the virus. Therefore, it is of importance to understand the interplay between the astrocyte-mediated immunity and HSV-1 infection. Methods: Both primary human astrocytes and the astrocyte line (U373) were used in this study. RT-qPCR and Western blot assay were used to measure IFNs, the antiviral IFN-stimulated genes (ISGs), IFN regulatory factors (IRFs) and HSV-1 DNA. IRF1 knockout or knockdown was performed with CRISPR/Cas9 and siRNA transfection techniques. Results: Poly(dA:dT) could inhibit HSV-1 replication and induce IFN-ß/IFN-λs production in human astrocytes. Poly(dA:dT) treatment of astrocytes also induced the expression of the antiviral ISGs (Viperin, ISG56 and MxA). Among IRFs members examined, poly(dA:dT) selectively unregulated IRF1 and IRF9, particularly IRF1 in human astrocytes. The inductive effects of poly(dA:dT) on IFNs and ISGs were diminished in the IRF1 knockout cells. In addition, IRF1 knockout attenuated poly(dA:dT)-mediated HSV-1 inhibition in the cells. Conclusion: The DNA sensors activation induces astrocyte intracellular innate immunity against HSV-1. Therefore, targeting the DNA sensors has potential for immune activation-based HSV-1 therapy.
Assuntos
Astrócitos , Herpesvirus Humano 1 , Fator Regulador 1 de Interferon , Replicação Viral , Humanos , Astrócitos/virologia , Astrócitos/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Imunidade Inata , Poli dA-dT , Herpes Simples/imunologia , Herpes Simples/virologia , Citosol/metabolismo , Linhagem Celular , Células Cultivadas , DNA Viral/genética , Técnicas de Inativação de GenesRESUMO
Objective To clarify the relationship between astrocyte activation patterns and disease progression in epidemic encephalitis B (Japanese encephalitis). Methods First, a mouse model of epidemic encephalitis B was constructed by foot-pad injection of Japanese encephalitis virus (JEV), and the expression of viral protein NS3 in different brain regions was detected by immunofluorescence assay (IFA). Next, IFA, RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) were used to clarify the changes in the astrocyte activation patterns at different stages of epidemic encephalitis B. Finally, intracerebroventricular administration of irisin was conducted to regulate the proportion of activation in complement C3-positive A1 astrocytes and S100A10-positive A2 astrocytes, investigating whether it could improve the body mass, behavioral scores, and brain tissue damage in a mouse model. Results NS3 protein was detected by IFA predominantly in the M1/M2 region of the motor cortex and the hippocampus. The number and volume of GFAP-positive astrocytes significantly increased in JEV-infected brain regions, in which the expression of multiple genes associated with A1/A2 astrocyte activation was significantly enhanced. Although intracerebroventricular or intraperitoneal injection of irisin did not improve the prognosis of epidemic encephalitis B, it inhibited the activation of A1 astrocytes and ameliorate neuroinflammation. Conclusion Neurons in the M1/M2 motor cortex and hippocampus are susceptible to JEV infection, in which the abnormal astrocyte activation contributes to the neuroinflammatory injury. Irisin administration may restrain A1 astrocyte activation and alleviate neuroinflammation following JEV infection.
Assuntos
Astrócitos , Modelos Animais de Doenças , Progressão da Doença , Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Animais , Astrócitos/metabolismo , Astrócitos/virologia , Camundongos , Encefalite Japonesa/imunologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encéfalo/metabolismo , Encéfalo/virologia , Encéfalo/patologia , Masculino , Fibronectinas/metabolismo , Fibronectinas/genéticaRESUMO
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.
Assuntos
Enzima de Conversão de Angiotensina 2 , Astrócitos , Barreira Hematoencefálica , COVID-19 , Dipeptidil Peptidase 4 , Pericitos , SARS-CoV-2 , Transcitose , Internalização do Vírus , Barreira Hematoencefálica/virologia , Barreira Hematoencefálica/metabolismo , Humanos , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Pericitos/virologia , Pericitos/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Astrócitos/virologia , Astrócitos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Encéfalo/virologia , Encéfalo/metabolismo , Endocitose , Células Endoteliais da Veia Umbilical Humana/virologia , PermeabilidadeRESUMO
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.