RESUMEN
All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19/prevención & control , Citidina/análogos & derivados , Hidroxilaminas/administración & dosificación , Hidroxilaminas/uso terapéutico , Administración Oral , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , COVID-19/inmunología , Quimioprevención , Quirópteros/virología , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Citidina/administración & dosificación , Citidina/uso terapéutico , Citocinas/inmunología , Células Epiteliales/virología , Femenino , Xenoinjertos , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Trasplante de Pulmón , Masculino , Ratones , Profilaxis Posexposición , Profilaxis Pre-Exposición , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Replicación ViralRESUMEN
The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.
Asunto(s)
Amino Alcoholes/farmacología , Enzima Convertidora de Angiotensina 2/química , Antivirales/farmacología , Éteres Fenílicos/farmacología , Receptores Virales/química , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/química , Compuestos de Sulfhidrilo/farmacología , Regulación Alostérica , Amino Alcoholes/química , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Sitios de Unión , COVID-19/virología , Línea Celular , Disulfuros/antagonistas & inhibidores , Disulfuros/química , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , Oxidación-Reducción , Éteres Fenílicos/química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptores Virales/antagonistas & inhibidores , Receptores Virales/genética , Receptores Virales/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Compuestos de Sulfhidrilo/química , Tratamiento Farmacológico de COVID-19RESUMEN
Cell therapy is a potential treatment for cystic fibrosis (CF). However, cell engraftment into the airway epithelium is challenging. Here, we model cell engraftment in vitro using the air-liquid interface (ALI) culture system by injuring well-differentiated CF ALI cultures and delivering non-CF cells at the time of peak injury. Engraftment efficiency was quantified by measuring chimerism by droplet digital PCR and functional ion transport in Ussing chambers. Using this model, we found that human bronchial epithelial cells (HBECs) engraft more efficiently when they are cultured by conditionally reprogrammed cell (CRC) culture methods. Cell engraftment into the airway epithelium requires airway injury, but the extent of injury needed is unknown. We compared three injury models and determined that severe injury with partial epithelial denudation facilitates long-term cell engraftment and functional CFTR recovery up to 20% of wildtype function. The airway epithelium promptly regenerates in response to injury, creating competition for space and posing a barrier to effective engraftment. We examined competition dynamics by time-lapse confocal imaging and found that delivered cells accelerate airway regeneration by incorporating into the epithelium. Irradiating the repairing epithelium granted engrafting cells a competitive advantage by diminishing resident stem cell proliferation. Intentionally, causing severe injury to the lungs of people with CF would be dangerous. However, naturally occurring events like viral infection can induce similar epithelial damage with patches of denuded epithelium. We found that viral preconditioning promoted effective engraftment of cells primed for viral resistance.NEW & NOTEWORTHY Cell therapy is a potential treatment for cystic fibrosis (CF). Here, we model cell engraftment by injuring CF air-liquid interface cultures and delivering non-CF cells. Successful engraftment required severe epithelial injury. Intentionally injuring the lungs to this extent would be dangerous. However, naturally occurring events like viral infection induce similar epithelial damage. We found that viral preconditioning promoted the engraftment of cells primed for viral resistance leading to CFTR functional recovery to 20% of the wildtype.
Asunto(s)
Fibrosis Quística , Virosis , Humanos , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Epitelio , Células Epiteliales , Tratamiento Basado en Trasplante de Células y Tejidos , Células CultivadasRESUMEN
The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Proteínas Mitocondriales/inmunología , Infecciones por Orthomyxoviridae/inmunología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Interferón beta/biosíntesis , Interferón beta/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , FN-kappa B/inmunología , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular , Factor 6 Asociado a Receptor de TNF/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.
Asunto(s)
Quirópteros/virología , Enfermedades Transmisibles Emergentes/virología , Infecciones por Coronaviridae/virología , Coronaviridae/patogenicidad , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Células Cultivadas , Chlorocebus aethiops , Coronaviridae/genética , Coronaviridae/inmunología , Coronaviridae/aislamiento & purificación , Coronaviridae/fisiología , Infecciones por Coronaviridae/prevención & control , Infecciones por Coronaviridae/transmisión , Infecciones por Coronaviridae/veterinaria , Reacciones Cruzadas , Encefalitis Viral/virología , Células Epiteliales/virología , Especificidad del Huésped , Humanos , Pulmón/citología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Moleculares , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/fisiología , Mutación Puntual , Conformación Proteica , Receptores Virales/genética , Receptores Virales/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Especificidad de la Especie , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/fisiología , Células Vero , Replicación Viral , ZoonosisRESUMEN
The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules mediate host immunity to various pathogenic stimuli. However, in vivo evidence for the involvement of NLR proteins in viral sensing has not been widely investigated and remains controversial. As a test of the physiologic role of the NLR molecule NLRP3 during RNA viral infection, we explored the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, Pycard, or caspase-1, but not Nlrc4, exhibited dramatically increased mortality and a reduced immune response after exposure to the influenza virus. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrated that an NLRP3-mediated response could be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by the influenza virus was dependent on lysosomal maturation and reactive oxygen species (ROS). Inhibition of ROS induction eliminated IL-1beta production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.
Asunto(s)
Proteínas Portadoras/fisiología , Exosomas/inmunología , Inmunidad Innata , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , ARN Viral , Virosis/inmunología , Animales , Proteínas Portadoras/genética , Línea Celular , Humanos , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/inmunología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Infants and young children with acute onset of wheezing and reduced respiratory airflows are often diagnosed with obstruction and inflammation of the small bronchiolar airways, ie bronchiolitis. The most common aetological agents causing bronchiolitis in young children are the respiratory viruses, and of the commonly encountered respiratory viruses, respiratory syncytial virus (RSV) has a propensity for causing bronchiolitis. Indeed, RSV bronchiolitis remains the major reason why previously healthy infants are admitted to hospital. Why RSV infection is such a predominant cause of bronchiolitis is the subject of this review. By reviewing the available histopathology of RSV bronchiolitis, both in humans and relevant animal models, we identify hallmark features of RSV infection of the distal airways and focus attention on the consequences of columnar cell cytopathology occurring in the bronchioles, which directly impacts the development of bronchiolar obstruction, inflammation and disease.
Asunto(s)
Bronquiolos/virología , Bronquiolitis/virología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/patogenicidad , Infecciones del Sistema Respiratorio/virología , Animales , Biopsia , Bronquiolos/patología , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Humanos , Patología Molecular/métodos , Valor Predictivo de las Pruebas , Infecciones por Virus Sincitial Respiratorio/patología , Infecciones del Sistema Respiratorio/patología , Factores de Riesgo , Índice de Severidad de la Enfermedad , Virología/métodos , VirulenciaRESUMEN
MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.
Asunto(s)
Modelos Animales de Enfermedad , Subtipo H1N1 del Virus de la Influenza A/inmunología , Pulmón/inmunología , Mucina 5AC/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Secuencia de Bases , Western Blotting , Líquido del Lavado Bronquioalveolar/química , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Pulmón/metabolismo , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Datos de Secuencia Molecular , Mucina 5AC/genética , Mucina 5AC/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.
Asunto(s)
Cilios/patología , Células Epiteliales/patología , Mucosa Respiratoria/patología , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitial Respiratorio Humano/fisiología , Animales , Polaridad Celular , Células Cultivadas , Cilios/inmunología , Cilios/virología , Citocinas/biosíntesis , Citocinas/inmunología , Células Epiteliales/inmunología , Células Epiteliales/virología , Especificidad del Huésped , Interacciones Huésped-Patógeno , Humanos , Modelos Biológicos , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Replicación ViralRESUMEN
Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable "muco-trapping" variant of motavizumab (Mota-MT), a potent neutralizing mAb against RSV F is engineered. Mota-MT traps RSV in AM via polyvalent Fc-mucin bonds, reducing the fraction of fast-moving RSV particles in both fresh pediatric and adult AM by ≈20-30-fold in a Fc-glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota-MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota-MT to RSV-infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000-fold and 100 000-fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota-MT-treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco-trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.
Asunto(s)
Anticuerpos Monoclonales , Infecciones por Virus Sincitial Respiratorio , Humanos , Lactante , Niño , Animales , Ovinos , Ratones , Anciano , Anticuerpos Monoclonales/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Palivizumab/uso terapéutico , Virus Sincitiales Respiratorios , PulmónRESUMEN
In vitro models play a major role in studying airway physiology and disease. However, the native lung's complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.
RESUMEN
Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, ß-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Fibrosis Quística/metabolismo , Dependovirus/metabolismo , Dependovirus/patogenicidad , Retículo Endoplásmico/metabolismo , Infecciones por Parvoviridae/metabolismo , Animales , Línea Celular , Cricetinae , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Susceptibilidad a Enfermedades , Citometría de Flujo , Células HeLa , Humanos , Inflamación , Pulmón , Mesocricetus , Centro Organizador de los Microtúbulos/metabolismo , Mutación , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Estrés FisiológicoRESUMEN
Respiratory syncytial virus (RSV) infection causes significant morbidity and mortality in infants, immunocompromised individuals, and older individuals. There is an urgent need for effective antivirals and vaccines for high-risk individuals. We used 2 complementary in vivo models to analyze RSV-associated human lung pathology and human immune correlates of protection. RSV infection resulted in widespread human lung epithelial damage, a proinflammatory innate immune response, and elicited a natural adaptive human immune response that conferred protective immunity. We demonstrated a key role for human T cells in controlling RSV infection. Specifically, primed human CD8+ T cells or CD4+ T cells effectively and independently control RSV replication in human lung tissue in the absence of an RSV-specific antibody response. These preclinical data support the development of RSV vaccines, which also elicit effective T cell responses to improve RSV vaccine efficacy.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Lactante , Humanos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Pulmón/patología , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Linfocitos T CD4-PositivosRESUMEN
The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Virales/metabolismo , Animales , Aves/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Cobayas , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/clasificación , Gripe Aviar/virología , Ácido N-Acetilneuramínico/química , Pandemias , Valor Predictivo de las Pruebas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tráquea/citologíaRESUMEN
Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(-) and Na(+) epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Moco/metabolismo , Mucosa Respiratoria/metabolismo , Análisis de Varianza , Transporte Biológico/fisiología , Células Cultivadas , Cloruros/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Virus de la Parainfluenza 1 Humana/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Mucosa Respiratoria/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismoRESUMEN
BACKGROUND: The nose is the portal for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, suggesting the nose as a target for topical antiviral therapies. The purpose of this study was to assess both the in vivo and in vitro efficacy of a detergent-based virucidal agent, Johnson and Johnson's Baby Shampoo (J&J), in SARS-CoV-2-infected subjects. METHODS: Subjects were randomized into three treatment groups: (1) twice daily nasal irrigation with J&J in hypertonic saline, (2) hypertonic saline alone, and (3) no intervention. Complementary in vitro experiments were performed in cultured human nasal epithelia. The primary outcome measure in the clinical trial was change in SARS-CoV-2 viral load over 21 days. Secondary outcomes included symptom scores and change in daily temperature. Outcome measures for in vitro studies included change in viral titers. RESULTS: Seventy-two subjects completed the clinical study (n = 24 per group). Despite demonstrated safety and robust efficacy in in vitro virucidal assays, J&J irrigations had no impact on viral titers or symptom scores in treated subjects relative to controls. Similar findings were observed administering J&J to infected cultured human airway epithelia using protocols mimicking the clinical trial regimen. Additional studies of cultured human nasal epithelia demonstrated that lack of efficacy reflected pharmacokinetic failure, with the most virucidal J&J detergent components rapidly absorbed from nasal surfaces. CONCLUSION: In this randomized clinical trial of subjects with SARS-CoV-2 infection, a topical detergent-based virucidal agent had no effect on viral load or symptom scores. Complementary in vitro studies confirmed a lack of efficacy, reflective of pharmacokinetic failure and rapid absorption from nasal surfaces.
Asunto(s)
COVID-19 , Resfriado Común , Antivirales , Detergentes , Humanos , SARS-CoV-2 , Carga ViralRESUMEN
Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell-associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bronquios/metabolismo , Epitelio/metabolismo , Células Caliciformes/metabolismo , Células Caliciformes/patología , Metaplasia/metabolismo , Mucinas/metabolismo , Western Blotting , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/virología , Calcio/metabolismo , Células Cultivadas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ensayo de Inmunoadsorción Enzimática , Epitelio/efectos de los fármacos , Epitelio/virología , Etilmaleimida/farmacología , Exocitosis , Células Caliciformes/virología , Humanos , Técnicas para Inmunoenzimas , Interleucina-13/farmacología , Metaplasia/patología , Metaplasia/virología , ARN Mensajero/genética , Receptores Purinérgicos P2Y2/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/patogenicidad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.
Asunto(s)
Toxina de Adenilato Ciclasa/metabolismo , Bronquios/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Células Epiteliales/metabolismo , Tráquea/metabolismo , Acilación , Animales , Transporte Biológico , Bronquios/citología , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Polaridad Celular , Células Cultivadas , Cloruros/metabolismo , Electrofisiología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Cinética , Potenciales de la Membrana , Transporte de Proteínas , Tráquea/citologíaRESUMEN
Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/terapia , Células Epiteliales/fisiología , Terapia Genética/métodos , Vectores Genéticos , Virus Sincitial Respiratorio Humano/genética , Línea Celular , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , HumanosRESUMEN
Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32 degrees C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40 degrees C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32 degrees C and 37 degrees C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32 degrees C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32 degrees C may represent a critical evolutionary step enabling human-to-human transmission.