RESUMO
We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.
Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Peptidil Dipeptidase A/farmacologia , Pneumonia Viral/tratamento farmacológico , Proteínas Recombinantes/farmacologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/ultraestrutura , Vasos Sanguíneos/virologia , COVID-19 , Chlorocebus aethiops , Humanos , Rim/citologia , Rim/virologia , Camundongos , Organoides/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.
Assuntos
Encéfalo/citologia , Técnicas de Cultura de Células , Modelos Biológicos , Organoides , Zika virus/fisiologia , Reatores Biológicos , Técnicas de Cultura de Células/economia , Embrião de Mamíferos , Desenvolvimento Embrionário , Humanos , Células-Tronco Pluripotentes Induzidas , Neurogênese , Neurônios/citologia , Organoides/virologia , Infecção por Zika virus/fisiopatologia , Infecção por Zika virus/virologiaRESUMO
There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.
Assuntos
Antivirais/farmacologia , COVID-19/virologia , Colo/citologia , Avaliação Pré-Clínica de Medicamentos/métodos , Pulmão/citologia , Organoides/efeitos dos fármacos , Organoides/virologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/prevenção & controle , Colo/efeitos dos fármacos , Colo/virologia , Aprovação de Drogas , Feminino , Xenoenxertos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/virologia , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , SARS-CoV-2/genética , Estados Unidos , United States Food and Drug Administration , Tropismo Viral , Internalização do Vírus/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.
Assuntos
COVID-19 , Pulmão , Organoides , SARS-CoV-2 , Internalização do Vírus , Humanos , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologia , Organoides/virologia , Tratamento Farmacológico da COVID-19 , Células-Tronco Pluripotentes Induzidas/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Inflamação , Citocinas/metabolismo , ApoptoseRESUMO
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Assuntos
COVID-19/virologia , Pulmão/citologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , COVID-19/metabolismo , COVID-19/patologia , Diferenciação Celular , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Células Clonais/virologia , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrina alfa6/análise , Integrina beta4/análise , Queratina-5/análise , Organoides/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Receptor de TWEAK/análiseRESUMO
COVID-19 is increasingly understood as a systemic disease with pathogenic manifestations beyond the respiratory tract. Recent work by Ramani et al (2020) dissects the cellular and molecular mechanisms of SARS-CoV-2's neurotrophic properties, using viral exposure of human brain organoids. Their findings highlight neurons as primary target of cerebral SARS-CoV-2 infection and uncover its Tau-related neurotoxicity.
Assuntos
Encéfalo/patologia , Encéfalo/virologia , COVID-19/patologia , Organoides/patologia , Proteínas tau/metabolismo , Humanos , Neurônios/patologia , Neurônios/virologia , Organoides/virologia , SARS-CoV-2/patogenicidadeRESUMO
Defining the pulmonary cell types infected by SARS-CoV-2 and finding ways to prevent subsequent tissue damage are key goals for controlling COVID-19. Recent work establishing a human lung organoid-derived air-liquid interface model permissive to SARS-CoV-2 infection identifies alveolar type II cells as the primary cell type infected, reports an infection-induced interferon response and demonstrates the effectiveness of interferon lambda 1 treatment in dampening lung infection.
Assuntos
Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Modelos Biológicos , Organoides/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , COVID-19/patologia , Humanos , Organoides/patologia , Organoides/virologia , Tratamento Farmacológico da COVID-19RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.
Assuntos
Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Modelos Biológicos , Organoides/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , COVID-19/virologia , Chlorocebus aethiops , Regulação da Expressão Gênica , Humanos , Interferon Tipo I/biossíntese , Interferons/biossíntese , Organoides/patologia , Organoides/virologia , Células Vero , Interferon lambdaRESUMO
Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.
Assuntos
Norovirus , Humanos , Antivirais/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Gastroenterite/tratamento farmacológico , Gastroenterite/virologia , Intestinos/virologia , Norovirus/efeitos dos fármacos , Norovirus/fisiologia , Animais , Organoides/efeitos dos fármacos , Organoides/virologia , Cultura de VírusRESUMO
Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.
Assuntos
Gastroenterite Suína Transmissível , Glicólise , Inflamação , Organoides , Vírus da Gastroenterite Transmissível , Animais , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Gastroenterite Suína Transmissível/virologia , Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/metabolismo , Inflamação/virologia , Intestinos/virologia , Intestinos/patologia , NF-kappa B/metabolismo , Organoides/virologia , Organoides/metabolismo , Organoides/patologia , Transdução de Sinais , Suínos , Vírus da Gastroenterite Transmissível/fisiologiaRESUMO
Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.
Assuntos
Ácidos e Sais Biliares , Norovirus , Receptores de Esfingosina-1-Fosfato , Replicação Viral , Humanos , Norovirus/efeitos dos fármacos , Norovirus/fisiologia , Norovirus/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Ácidos e Sais Biliares/metabolismo , Infecções por Caliciviridae/virologia , Infecções por Caliciviridae/metabolismo , Piridinas/farmacologia , Gastroenterite/virologia , Jejuno/virologia , Jejuno/metabolismo , Organoides/virologia , Organoides/metabolismo , PirazóisRESUMO
With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.
Assuntos
Enzima de Conversão de Angiotensina 2 , Rim , Organoides , SARS-CoV-2 , Internalização do Vírus , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/complicações , COVID-19/virologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/virologia , Lisinopril/farmacologia , Lisinopril/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/virologia , Pandemias , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/virologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/virologia , Receptores de Coronavírus/metabolismo , Modelos Biológicos , Serina Endopeptidases/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco/citologiaRESUMO
Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.
Assuntos
Anticorpos Neutralizantes , Receptor 1 de Quimiocina CX3C , Testes de Neutralização , Organoides , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Humanos , Vírus Sincicial Respiratório Humano/imunologia , Anticorpos Neutralizantes/imunologia , Organoides/metabolismo , Organoides/imunologia , Organoides/virologia , Organoides/citologia , Animais , Testes de Neutralização/métodos , Chlorocebus aethiops , Células Vero , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/imunologia , Anticorpos Antivirais/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Lactente , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/virologia , Anticorpos Monoclonais/imunologiaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.
Assuntos
Astrócitos , Córtex Cerebral , SARS-CoV-2 , Tropismo Viral , Enzima de Conversão de Angiotensina 2/metabolismo , Astrócitos/enzimologia , Astrócitos/virologia , Córtex Cerebral/virologia , Humanos , Organoides/virologia , Cultura Primária de Células , SARS-CoV-2/fisiologiaRESUMO
Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has spread across the globe. SARS-CoV-2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS-CoV-2. SARS-CoV-2 is a new member of the betacoronavirus genus like other closely related viruses including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Both SARS-CoV and MERS-CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon-stimulated genes (ISGs), cholesterol 25-hydroxylase (CH25H), is induced by SARS-CoV-2 infection in vitro and in COVID-19-infected patients. CH25H converts cholesterol to 25-hydrocholesterol (25HC) and 25HC shows broad anti-coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA-WA1/2020 SARS-CoV-2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus-cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID-19 and emerging viral diseases in the future.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Colesterol/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Mucosa Respiratória/virologia , Esteroide Hidroxilases/farmacologia , Internalização do Vírus/efeitos dos fármacos , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , COVID-19 , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Organoides/virologia , Pandemias , Mucosa Respiratória/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , SARS-CoV-2 , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.
Assuntos
Anticorpos Neutralizantes , Infecções por Citomegalovirus , Citomegalovirus , Expressão Gênica , Fatores de Crescimento Neural , Humanos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/uso terapêutico , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Organoides/citologia , Organoides/metabolismo , Organoides/virologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Tapasin, a crucial molecular chaperone involved viral antigen processing and presentation, plays an important role in antivirus immunity. However, its impact on T cell differentiation in the context of virus clearance remains unclear. In this study, we employed induced pluripotent stem cells to differentiate into hepatocyte-like cell, which were subsequently inserted to the inverted colloidal crystal scaffolds, thus establishing a hepatocyte organoid (HO). By inoculating hepatitis B virus (HBV) particles in the system, we successfully engineered a robust in vitro HBV infection model for at least 3 weeks. Furthermore, we aimed to explore the effects of lentivirus-mediated short hairpin RNA (shRNA) targeting human Tapasin on the differentiation and antiviral function of CD8+ T cells. Specifically, we transfected dendritic cells (DCs) with Tapasin-shRNA and cocultured with T cells. The results demonstrated that Tapasin-shRNA transfected DCs effectively suppressed T cell proliferation and impeded HBV-specific cytotoxic T lymphocyte responses. Our investigation also revealed the role of mTOR pathway activation in reducing autophagy activity within CD8+ T cells. Expressions of autophagy-related proteins, beclin-1, LC3II/LC3I were decreased and PI3K/AKT/mTOR activity was increased in Tapasin-shRNA group. Collectively, our findings elucidate that shRNA targeting the Tapasin gene within DCs inhibits T cell differentiation by reducing autophagy activity to hamper viral clearance in the HBV-infected HO.
Assuntos
Células Dendríticas , Hepatite B , Proteínas de Membrana Transportadoras , Humanos , Autofagia/genética , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Regulação para Baixo , Hepatite B/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B , Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Organoides/metabolismo , Organoides/virologiaRESUMO
The escalation of antibiotic resistance, pandemics, and nosocomial infections underscores the importance of research in both animal and human infectious diseases. Recent advancements in three-dimensional tissue cultures, or "organoids", have revolutionized the development of in vitro models for infectious diseases. Our study conducts a bibliometric analysis on the use of organoids in modeling infectious diseases, offering an in-depth overview of this field's current landscape. We examined scientific contributions from 2009 onward that focused on organoids in hostâpathogen interactions using the Web of Science Core Collection and OpenAlex database. Our analysis included temporal trends, reference aging, author, and institutional productivity, collaborative networks, citation metrics, keyword cluster dynamics, and disruptiveness of organoid models. VOSviewer, CiteSpace, and Python facilitated this analytical assessment. The findings reveal significant growth and advancements in organoid-based infectious disease research. Analysis of keywords and impactful publications identified three distinct developmental phases in this area that were significantly influenced by outbreaks of Zika and SARS-CoV-2 viruses. The research also highlights the synergistic efforts between academia and publishers in tackling global pandemic challenges. Through mostly consolidating research efforts, organoids are proving to be a promising tool in infectious disease research for both human and animal infectious disease. Their integration into the field necessitates methodological refinements for better physiological emulation and the establishment of extensive organoid biobanks. These improvements are crucial for fully harnessing the potential of organoids in understanding infectious diseases and advancing the development of targeted treatments and vaccines.
Assuntos
Bibliometria , Organoides , Organoides/virologia , Animais , Humanos , Doenças Transmissíveis/veterinária , Doenças Transmissíveis/epidemiologia , Modelos Animais de Doenças , COVID-19/epidemiologia , COVID-19/virologiaRESUMO
Recent advancements in stem cell biology and tissue engineering have revolutionized the field of neurodegeneration research by enabling the development of sophisticated in vitro human brain models. These models, including 2D monolayer cultures, 3D organoids, organ-on-chips, and bioengineered 3D tissue models, aim to recapitulate the cellular diversity, structural organization, and functional properties of the native human brain. This review highlights how these in vitro brain models have been used to investigate the effects of various pathogens, including viruses, bacteria, fungi, and parasites infection, particularly in the human brain cand their subsequent impacts on neurodegenerative diseases. Traditional studies have demonstrated the susceptibility of different 2D brain cell types to infection, elucidated the mechanisms underlying pathogen-induced neuroinflammation, and identified potential therapeutic targets. Therefore, current methodological improvement brought the technology of 3D models to overcome the challenges of 2D cells, such as the limited cellular diversity, incomplete microenvironment, and lack of morphological structures by highlighting the need for further technological advancements. This review underscored the significance of in vitro human brain cell from 2D monolayer to bioengineered 3D tissue model for elucidating the intricate dynamics for pathogen infection modeling. These in vitro human brain cell enabled researchers to unravel human specific mechanisms underlying various pathogen infections such as SARS-CoV-2 to alter blood-brain-barrier function and Toxoplasma gondii impacting neural cell morphology and its function. Ultimately, these in vitro human brain models hold promise as personalized platforms for development of drug compound, gene therapy, and vaccine. Overall, we discussed the recent progress in in vitro human brain models, their applications in studying pathogen infection-related neurodegeneration, and future directions.
Assuntos
Encéfalo , Doenças Neurodegenerativas , Humanos , Encéfalo/patologia , Encéfalo/virologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/virologia , COVID-19/virologia , SARS-CoV-2/fisiologia , Organoides/virologia , Organoides/patologia , Modelos Biológicos , Engenharia Tecidual/métodos , Barreira Hematoencefálica/metabolismoRESUMO
Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that is asymptomatic in healthy individuals but can cause serious disease in immunocompromised patients. HCMV is also the leading cause of virus-mediated birth defects. Many of these defects manifest within the central nervous system and include microcephaly, sensorineural hearing loss, and cognitive developmental delays. Nitric oxide is a critical effector molecule produced as a component of the innate immune response during infection. Congenitally infected fetal brains show regions of brain damage, including necrotic foci with infiltrating macrophages and microglia, cell types that produce nitric oxide during infection. Using a 3-dimensional cortical organoid model, we demonstrate that nitric oxide inhibits HCMV spread and simultaneously disrupts neural rosette structures, resulting in tissue disorganization. Nitric oxide also attenuates HCMV replication in 2-dimensional cultures of neural progenitor cells (NPCs), a prominent cell type in cortical organoids that differentiate into neurons and glial cells. The multipotency factor SOX2 was decreased during nitric oxide exposure, suggesting that early neural differentiation is affected. Nitric oxide also reduced maximal mitochondrial respiration in both uninfected and infected NPCs. We determined that this reduction likely influences neural differentiation, as neurons (Tuj1+ GFAP- Nestin-) and glial populations (Tuj1- GFAP+ Nestin-) were reduced following differentiation. Our studies indicate a prominent, immunopathogenic role of nitric oxide in promoting developmental defects within the brain despite its antiviral activity during congenital HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of virus-mediated congenital birth defects. Congenitally infected infants can have a variety of symptoms manifesting within the central nervous system. The use of 3-dimensional (3-D) cortical organoids to model infection of the fetal brain has advanced the current understanding of development and allowed broader investigation of the mechanisms behind disease. However, the impact of the innate immune molecule nitric oxide during HCMV infection has not been explored in neural cells or cortical 3-D models. Here, we investigated the effect of nitric oxide on cortical development during HCMV infection. We demonstrate that nitric oxide plays an antiviral role during infection yet results in disorganized cortical tissue. Nitric oxide contributes to differentiation defects of neuron and glial cells from neural progenitor cells despite inhibiting viral replication. Our results indicate that immunopathogenic consequences of nitric oxide during congenital infection promote developmental defects that undermine its antiviral activity.