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The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms1. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal2, most experimental infections provide insights into mild disease3. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies4, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.
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Anosmia/virología , COVID-19/fisiopatología , COVID-19/terapia , Modelos Animales de Enfermedad , SARS-CoV-2/patogenicidad , Animales , Anosmia/fisiopatología , Anosmia/terapia , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , COVID-19/inmunología , COVID-19/virología , Epitelio/inmunología , Epitelio/virología , Femenino , Humanos , Inmunización Pasiva , Inflamación/patología , Inflamación/terapia , Inflamación/virología , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/terapia , Enfermedades Pulmonares/virología , Masculino , Ratones , Senos Paranasales/inmunología , Senos Paranasales/virología , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Resultado del Tratamiento , Sueroterapia para COVID-19RESUMEN
The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Ratones , Regiones no Traducidas 5' , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Nucleótidos , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
BACKGROUND: Chronic pulmonary conditions such as asthma and COPD increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). METHODS: We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine IL-13, examining how this impacted DPP4 protein levels along with MERS-CoV entry and replication. RESULTS: IL-13 exposure for 3 days led to increased DPP4 protein abundance, while a 21-day treatment increased DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly impacted by IL-13 treatment. CONCLUSIONS: Our results suggest that increased DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13 induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.
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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , Receptores de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/análisis , Variación Biológica Poblacional , Bronquios/citología , Bronquios/patología , Bronquios/virología , COVID-19/virología , Células Epiteliales , Femenino , Humanos , Masculino , Cultivo Primario de Células , Receptores de Coronavirus/análisis , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Factores Sexuales , Internalización del VirusRESUMEN
The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with â¼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.
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Infecciones por Coronavirus/complicaciones , Dipeptidil Peptidasa 4/genética , Modelos Animales de Enfermedad , Enfermedades Pulmonares/etiología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Infecciones por Coronavirus/virología , Dipeptidil Peptidasa 4/metabolismo , Femenino , Humanos , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Replicación ViralRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.
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Bradiquinina/análogos & derivados , Lipopolisacáridos/toxicidad , Infiltración Neutrófila/inmunología , Peptidil-Dipeptidasa A/fisiología , Neumonía/inmunología , Receptor de Bradiquinina B1/metabolismo , Tráquea/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , Antiinflamatorios , Bradiquinina/farmacología , Células Cultivadas , Quimiocina CXCL5/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neumonía/inducido químicamente , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Tráquea/efectos de los fármacos , Tráquea/patologíaRESUMEN
Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.
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Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Pulmón/metabolismo , Pulmón/microbiología , Viabilidad Microbiana , Sistema Respiratorio/metabolismo , Animales , Animales Recién Nacidos , Antiinfecciosos/farmacología , Bicarbonatos/metabolismo , Líquidos Corporales/efectos de los fármacos , Líquidos Corporales/metabolismo , Fibrosis Quística/patología , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Femenino , Concentración de Iones de Hidrógeno/efectos de los fármacos , Transporte Iónico , Pulmón/patología , Masculino , Viabilidad Microbiana/efectos de los fármacos , Sus scrofa/microbiologíaRESUMEN
RATIONALE: Studies suggest that inappropriate responses to proinflammatory stimuli might contribute to inflammation in cystic fibrosis (CF) lungs. However, technical challenges have made it difficult to distinguish whether altered responses in CF airways are an intrinsic defect or a secondary effect of chronic disease in their tissue of origin. The CF pig model provides an opportunity to study the inflammatory responses of CF airways at birth, before the onset of infection and inflammation. OBJECTIVES: To test the hypothesis that acute inflammatory responses are perturbed in porcine CF airways. METHODS: We investigated the inflammatory responses of newborn CF and non-CF pig airways following a 4-hour exposure to heat-killed Staphylococcus aureus, in vivo and in vitro. MEASUREMENTS AND MAIN RESULTS: Following an in vivo S. aureus challenge, markers of inflammation were similar between CF and littermate control animals through evaluation of bronchoalveolar lavage and tissues. However, transcriptome analysis revealed genotype-dependent differences as CF pigs showed a diminished host defense response compared with their non-CF counterparts. Furthermore, CF pig airways exhibited an increase in apoptotic pathways and a suppression of ciliary and flagellar biosynthetic pathways. Similar differences were observed in cultured airway epithelia from CF and non-CF pigs exposed to the stimulus. CONCLUSIONS: Transcriptome profiling suggests that acute inflammatory responses are dysregulated in the airways of newborn CF pigs.
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Fibrosis Quística/inmunología , Pulmón/inmunología , Staphylococcus aureus/inmunología , Animales , Animales Recién Nacidos , Apoptosis/genética , Proliferación Celular/genética , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Progresión de la Enfermedad , Epitelio/inmunología , Genotipo , Técnicas In Vitro , Inflamación/genética , Inflamación/inmunología , Modelos Animales , Mucosa Respiratoria/inmunología , Transducción de Señal/genética , Porcinos , Transcriptoma/genéticaRESUMEN
In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6-8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections.
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Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Infecciones del Sistema Respiratorio/virología , Animales , Anticuerpos Antivirales/inmunología , Linfocitos T CD8-positivos/virología , Coronavirus/inmunología , Coronavirus/fisiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Reacciones Cruzadas/inmunología , Humanos , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Medio Oriente , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Síndrome Respiratorio Agudo Grave/inmunología , Transducción de Señal/inmunologíaRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV) causes life-threatening disease. Dipeptidyl peptidase 4 (DPP4) is the receptor for cell binding and entry. There is a need for small-animal models of MERS, but mice are not susceptible to MERS because murine dpp4 does not serve as a receptor. We developed transgenic mice expressing human DPP4 (hDPP4) under the control of the surfactant protein C promoter or cytokeratin 18 promoter that are susceptible to infection with MERS-CoV. Notably, mice expressing hDPP4 with the cytokeratin 18 promoter developed progressive, uniformly fatal disease following intranasal inoculation. High virus titers were present in lung and brain tissues 2 and 6 days after infection, respectively. MERS-CoV-infected lungs revealed mononuclear cell infiltration, alveolar edema, and microvascular thrombosis, with airways generally unaffected. Brain disease was observed, with the greatest involvement noted in the thalamus and brain stem. Animals immunized with a vaccine candidate were uniformly protected from lethal infection. These new mouse models of MERS-CoV should be useful for investigation of early disease mechanisms and therapeutic interventions.
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Enfermedades Virales del Sistema Nervioso Central/virología , Infecciones por Coronavirus/virología , Dipeptidil Peptidasa 4/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Animales , Enfermedades Virales del Sistema Nervioso Central/mortalidad , Enfermedades Virales del Sistema Nervioso Central/patología , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/patología , Dipeptidil Peptidasa 4/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , ARN Viral/aislamiento & purificaciónRESUMEN
RATIONALE: Cathepsin S (CTSS) activity is increased in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF). This activity contributes to lung inflammation via degradation of antimicrobial proteins, such as lactoferrin and members of the ß-defensin family. OBJECTIVES: In this study, we investigated the hypothesis that airway epithelial cells are a source of CTSS, and mechanisms underlying CTSS expression in the CF lung. METHODS: Protease activity was determined using fluorogenic activity assays. Protein and mRNA expression were analyzed by ELISA, Western blotting, and reverse-transcriptase polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: In contrast to neutrophil elastase, CTSS activity was detectable in 100% of CF BAL fluid samples from patients without Pseudomonas aeruginosa infection. In this study, we identified epithelial cells as a source of pulmonary CTSS activity with the demonstration that CF airway epithelial cells express and secrete significantly more CTSS than non-CF control cells in the absence of proinflammatory stimulation. Furthermore, levels of the transcription factor IRF-1 correlated with increased levels of its target gene CTSS. We discovered that miR-31, which is decreased in the CF airways, regulates IRF-1 in CF epithelial cells. Treating CF bronchial epithelial cells with a miR-31 mimic decreased IRF-1 protein levels with concomitant knockdown of CTSS expression and secretion. CONCLUSIONS: The miR-31/IRF-1/CTSS pathway may play a functional role in the pathogenesis of CF lung disease and may open up new avenues for exploration in the search for an effective therapeutic target.
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Líquido del Lavado Bronquioalveolar/química , Catepsinas/metabolismo , Fibrosis Quística/genética , Factor 1 Regulador del Interferón/metabolismo , MicroARNs/metabolismo , Mucosa Respiratoria/metabolismo , Adolescente , Biomarcadores/metabolismo , Western Blotting , Líquido del Lavado Bronquioalveolar/microbiología , Línea Celular , Niño , Preescolar , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactante , Péptido Hidrolasas/metabolismo , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/aislamiento & purificación , Mucosa Respiratoria/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signaling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. We investigated the role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2-expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe respiratory virus-induced lung disease.
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Evidence from in vitro studies and observational human disease data suggest the complement system plays a significant role in SARS-CoV-2 pathogenesis, although how complement dysregulation develops in patients with severe COVID-19 is unknown. Here, using a mouse-adapted SARS-CoV-2 virus (SARS2-N501YMA30) and a mouse model of severe COVID-19, we identify significant serologic and pulmonary complement activation following infection. We observed C3 activation in airway and alveolar epithelia, and in pulmonary vascular endothelia. Our evidence suggests that while the alternative pathway is the primary route of complement activation, components of both the alternative and classical pathways are produced locally by respiratory epithelial cells following infection, and increased in primary cultures of human airway epithelia in response to cytokine exposure. This locally generated complement response appears to precede and subsequently drive lung injury and inflammation. Results from this mouse model recapitulate findings in humans, which suggest sex-specific variance in complement activation, with predilection for increased C3 activity in males, a finding that may correlate with more severe disease. Our findings indicate that complement activation is a defining feature of severe COVID-19 in mice and lay the foundation for further investigation into the role of complement in COVID-19.
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MicroRNAs (miRNAs) are increasingly recognized as important posttranscriptional regulators of gene expression, and changes in their actions can contribute to disease states. Little is understood regarding miRNA functions in the airway epithelium under normal or diseased conditions. We profiled miRNA expression in well-differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia, and discovered that miR-509-3p and miR-494 concentrations were increased in CF epithelia. Human non-CF airway epithelia, transfected with the mimics of miR-509-3p or miR-494, showed decreased cystic fibrosis transmembrane conductance regulator (CFTR) expression, whereas their respective anti-miRs exerted the opposite effect. Interestingly, the two miRNAs acted cooperatively in regulating CFTR expression. Upon infecting non-CF airway epithelial cells with Staphylococcus aureus, or upon stimulating them with the proinflammatory cytokines TNF-α or IL-1ß, we observed an increased expression of both miRNAs and a concurrent decrease in CFTR expression and function, suggesting that inflammatory mediators may regulate these miRNAs. Transfecting epithelia with anti-miRs for miR-509-3p and miR-494, or inhibiting NF-κB signaling before stimulating cells with TNFα or IL-1ß, suppressed these responses, suggesting that the expression of both miRNAs was responsive to NF-κB signaling. Thus, miR-509-3p and miR-494 are dynamic regulators of CFTR abundance and function in normal, non-CF airway epithelia.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Línea Celular , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , MicroARNs/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Mucosa Respiratoria/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Clâ» conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses.
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Polaridad Celular , Neoplasias del Colon/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Oligonucleótidos/farmacología , ARN Interferente Pequeño/genética , Mucosa Respiratoria/efectos de los fármacos , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Western Blotting , Células Cultivadas , Neoplasias del Colon/genética , Electrofisiología , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Interleucina-8/metabolismo , Masculino , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
The airway mucosa and the alveolar surface form dynamic interfaces between the lung and the external environment. The epithelial cells lining these barriers elaborate a thin liquid layer containing secreted peptides and proteins that contribute to host defense and other functions. The goal of this study was to develop and apply methods to define the proteome of porcine lung lining liquid, in part, by leveraging the wealth of information in the Sus scrofa database of Ensembl gene, transcript, and protein model predictions. We developed an optimized workflow for detection of secreted proteins in porcine bronchoalveolar lavage (BAL) fluid and in methacholine-induced tracheal secretions [airway surface liquid (ASL)]. We detected 674 and 3,858 unique porcine-specific proteins in BAL and ASL, respectively. This proteome was composed of proteins representing a diverse range of molecular classes and biological processes, including host defense, molecular transport, cell communication, cytoskeletal, and metabolic functions. Specifically, we detected a significant number of secreted proteins with known or predicted roles in innate and adaptive immunity, microbial killing, or other aspects of host defense. In greatly expanding the known proteome of the lung lining fluid in the pig, this study provides a valuable resource for future studies using this important animal model of pulmonary physiology and disease.
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Proteoma/análisis , Mucosa Respiratoria/química , Animales , Animales Recién Nacidos , Líquidos Corporales , Bases de Datos de Proteínas , Células Epiteliales/química , Células Epiteliales/citología , Pulmón/metabolismo , Espectrometría de Masas , Cloruro de Metacolina , Mucosa Respiratoria/citología , Sus scrofa , Tráquea/metabolismoRESUMEN
In the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, no patients under 24 years of age died, while mortality was greater than 50% in those over 65 years. Greater than 90% of all deaths from influenza A virus (IAV) occur in the elderly (>65 years of age). To address this age-related susceptibility to SARS-CoV and IAV, we infected C57BL/6 (B6) mice with mouse-adapted SARS-CoV (MA15) or IAV (PR8), both of which cause severe disease in aged mice. Intranasal pretreatment of aged mice with poly(I·C) (a TLR3 agonist) and, to a lesser extent, CpG, R848, or lipopolysaccharide (TLR9, TLR7/8, or TLR4 agonists), provided a high level of protection [90% to 100% survival rate after poly(I·C) treatment] against lethal MA15 or IAV challenge and reduced pathological changes and virus loads in the lungs at early times after infection. Poly(I·C) pretreatment upregulated beta interferon (IFN-ß), IFN-γ, IL-1ß, and tumor necrosis factor (TNF) gene expression in the lungs. Intranasal pretreatment with IFN-ß or IFN-γ but not IL-1ß or TNF also protected aged mice, consistent with the notion that poly(I·C) pretreatment functioned, at least in part, by inducing IFN-ß and IFN-γ. We also identified a potential cellular target for poly(I·C) by showing that treatment inhibited virus replication in primary human airway epithelial cells. These results suggest that intranasal poly(I·C) should be evaluated as a prophylactic agent in aged individuals at high risk for contracting SARS-CoV or IAV infections.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Infecciones por Orthomyxoviridae/prevención & control , Poli I-C/administración & dosificación , Síndrome Respiratorio Agudo Grave/prevención & control , Administración Intranasal , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Virus de la Influenza A/patogenicidad , Lipopolisacáridos/administración & dosificación , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Infecciones por Orthomyxoviridae/mortalidad , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Síndrome Respiratorio Agudo Grave/mortalidad , Análisis de Supervivencia , Carga ViralRESUMEN
Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Células Epiteliales , Animales , Humanos , Ratones , Macaca mulatta/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Ribonucleoproteínas/metabolismo , Péptidos/genética , Sistemas CRISPR-CasRESUMEN
Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.
Asunto(s)
Adenoviridae/efectos de los fármacos , Antivirales/farmacología , Inmunidad Mucosa/efectos de los fármacos , Yoduro de Potasio/farmacología , Mucosa Respiratoria/efectos de los fármacos , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Yoduro de Sodio/farmacología , Adenoviridae/inmunología , Adenoviridae/patogenicidad , Animales , Antivirales/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Compuestos de Yodo/metabolismo , Lactoperoxidasa/metabolismo , Oxidación-Reducción , Yoduro de Potasio/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , Virus Sincitiales Respiratorios/efectos de los fármacos , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Yoduro de Sodio/metabolismo , Porcinos , Tiocianatos/metabolismo , Factores de Tiempo , Activación Viral/efectos de los fármacosRESUMEN
Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.