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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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Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF, and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ΔF508-CF epithelia with TNF-α and IL-17, two inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl- (Na+/K+/2Cl- cotransport) and HCO3- (carbonic anhydrases and Na+/HCO3- cotransport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions but prevented the rise in viscosity in epithelia treated with TNF-α+IL-17. Finally, antiinflammatory drugs limited CFTR modulator responses in TNF-α+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits and limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.
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Antiinflamatorios , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Interleucina-17 , Factor de Necrosis Tumoral alfa , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-17/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismoRESUMEN
Asthma affects 25 million Americans, and recent advances in treatment are effective for only a portion of severe asthma patients. TREM-1, an innate receptor that canonically amplifies inflammatory signaling in neutrophils and monocytes, plays a central role in regulating lung inflammation. It is unknown how TREM-1 contributes to allergic asthma pathology. Utilizing a murine model of asthma, flow cytometry revealed TREM-1+ eosinophils in the lung tissue and airway during allergic airway inflammation. TREM-1 expression was restricted to recruited, inflammatory eosinophils. Expression was induced on bone marrow-derived eosinophils by incubation with interleukin 33, lipopolysaccharide, or granulocyte-macrophage colony-stimulating factor. Compared to TREM-1- airway eosinophils, TREM-1+ eosinophils were enriched for proinflammatory gene sets, including migration, respiratory burst, and cytokine production. Unexpectedly, eosinophil-specific ablation of TREM-1 exacerbated airway interleukin (IL) 5 production, airway MUC5AC production, and lung tissue eosinophil accumulation. Further investigation of transcriptional data revealed apoptosis and superoxide generation-related gene sets were enriched in TREM-1+ eosinophils. Consistent with these findings, annexin V and caspase-3/7 staining demonstrated higher rates of apoptosis among TREM-1+ eosinophils compared to TREM-1- eosinophils in the inflammatory airway. In vitro, Trem1/3-/- bone marrow-derived eosinophils consumed less oxygen than wild-type in response to phorbol myristate acetate, suggesting that TREM-1 promotes superoxide generation in eosinophils. These data reveal protein-level expression of TREM-1 by eosinophils, define a population of TREM-1+ inflammatory eosinophils, and demonstrate that eosinophil TREM-1 restricts key features of type 2 lung inflammation.
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Eosinófilos , Neumonía , Receptor Activador Expresado en Células Mieloides 1 , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Receptor Activador Expresado en Células Mieloides 1/genética , Animales , Eosinófilos/metabolismo , Eosinófilos/inmunología , Eosinófilos/patología , Ratones , Neumonía/metabolismo , Neumonía/patología , Ratones Endogámicos C57BL , Asma/metabolismo , Asma/patología , Asma/inmunología , Pulmón/patología , Pulmón/metabolismo , Pulmón/inmunologíaRESUMEN
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
Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.
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Amilorida , Deficiencia de Vitamina D , Humanos , Vitamina D , Calcitriol/farmacología , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Pulmón/metabolismo , Vitaminas , ARN Mensajero/genéticaRESUMEN
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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Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced FXYD3 expression using siRNA. After permeabilizing the apical membrane with nystatin, epithelia pretreated with FXYD3-targeting siRNA had lower ouabain-sensitive short-circuit currents than control epithelia. FXYD3-targeting siRNA also reduced amiloride-sensitive short-circuit currents and liquid absorption across intact epithelia. These data are consistent with FXYD3 facilitating Na+ and liquid absorption. FXYD3 may be needed to maintain the high rates of Na+ and fluid absorption observed for airway and other FXYD3-expressing epithelia.
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Amilorida , Ouabaína , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Nistatina , ARN Interferente Pequeño/genética , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.
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Fibrosis Quística , Alanina , Bumetanida , Humanos , Concentración de Iones de Hidrógeno , Prolina , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
INTRODUCTION: Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune mechanisms in vitro. METHODS: Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs. RESULTS: Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1. CONCLUSION: While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.
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Calcitriol , Vitamina D , Antivirales/metabolismo , Calcitriol/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inmunidad Innata , Vitamina D/metabolismo , Vitamina D/farmacología , Vitaminas/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.
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COVID-19 , SARS-CoV-2 , Animales , Eicosanoides , Leucocitos Mononucleares , Ratones , Compuestos Orgánicos , Oxazoles , Piperazinas , Poliésteres , Prostaglandinas , Glicoproteína de la Espiga del Coronavirus , SulfonamidasRESUMEN
Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Animales , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Humanos , Inflamación/metabolismo , Transporte Iónico , Sistema Respiratorio/metabolismo , PorcinosRESUMEN
Background The long-term effects of SARS-CoV-2 infection on pulmonary structure and function remain incompletely characterized. Purpose To test whether SARS-CoV-2 infection leads to small airways disease in patients with persistent symptoms. Materials and Methods In this single-center study at a university teaching hospital, adults with confirmed COVID-19 who remained symptomatic more than 30 days following diagnosis were prospectively enrolled from June to December 2020 and compared with healthy participants (controls) prospectively enrolled from March to August 2018. Participants with post-acute sequelae of COVID-19 (PASC) were classified as ambulatory, hospitalized, or having required the intensive care unit (ICU) based on the highest level of care received during acute infection. Symptoms, pulmonary function tests, and chest CT images were collected. Quantitative CT analysis was performed using supervised machine learning to measure regional ground-glass opacity (GGO) and using inspiratory and expiratory image-matching to measure regional air trapping. Univariable analyses and multivariable linear regression were used to compare groups. Results Overall, 100 participants with PASC (median age, 48 years; 66 women) were evaluated and compared with 106 matched healthy controls; 67% (67 of 100) of the participants with PASC were classified as ambulatory, 17% (17 of 100) were hospitalized, and 16% (16 of 100) required the ICU. In the hospitalized and ICU groups, the mean percentage of total lung classified as GGO was 13.2% and 28.7%, respectively, and was higher than that in the ambulatory group (3.7%, P < .001 for both comparisons). The mean percentage of total lung affected by air trapping was 25.4%, 34.6%, and 27.3% in the ambulatory, hospitalized, and ICU groups, respectively, and 7.2% in healthy controls (P < .001). Air trapping correlated with the residual volume-to-total lung capacity ratio (ρ = 0.6, P < .001). Conclusion In survivors of COVID-19, small airways disease occurred independently of initial infection severity. The long-term consequences are unknown. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Elicker in this issue.
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COVID-19/complicaciones , Enfermedades Pulmonares , COVID-19/diagnóstico por imagen , Femenino , Humanos , Enfermedades Pulmonares/diagnóstico por imagen , Enfermedades Pulmonares/virología , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos X/métodos , Síndrome Post Agudo de COVID-19RESUMEN
Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Mutación , Mucosa Respiratoria/metabolismo , Células Acinares/metabolismo , Animales , Biomarcadores , Fibrosis Quística/etiología , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Mucinas/metabolismo , Depuración Mucociliar , Moco/metabolismo , Mucosa Respiratoria/patología , PorcinosRESUMEN
Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The common ΔF508-CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508-CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508-CFTR trafficking and function, including low-temperature, small molecules, overexpression of miR-138, or knockdown of SIN3A. The challenge remains to translate such interventions into therapies and to understand their mechanisms. One approach for connecting such interventions to small molecule therapies that has previously succeeded for CF and other diseases is via mRNA expression profiling and iterative searches of small molecules with similar expression signatures. Here, we query the Library of Integrated Network-based Cellular Signatures using transcriptomic signatures from previously generated CF expression data, including RNAi- and low temperature-based rescue signatures. This LINCS in silico screen prioritized 135 small molecules that mimicked our rescue interventions based on their genomewide transcriptional perturbations. Functional screens of these small molecules identified eight compounds that partially restored ΔF508-CFTR function, as assessed by cAMP-activated chloride conductance. Of these, XL147 rescued ΔF508-CFTR function in primary CF airway epithelia, while also showing cooperativity when administered with C18. Improved CF corrector therapies are needed and this integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508-CFTR function and identify gene networks underlying such rescue.
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Fibrosis Quística , MicroARNs , Línea Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Descubrimiento de Drogas , Humanos , MicroARNs/genética , MutaciónRESUMEN
RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.
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Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Células Cultivadas , ADN Complementario/síntesis química , Fibroblastos , Fludrocortisona , Humanos , ImidazolesRESUMEN
Life-long expression of a gene therapy agent likely requires targeting stem cells. Here we ask the question: does viral vector transduction or ectopic expression of a therapeutic transgene preclude airway stem cell function? We used a lentiviral vector containing a GFP or cystic fibrosis transmembrane conductance regulator (CFTR) transgene to transduce primary airway basal cells from human cystic fibrosis (CF) or non-CF lung donors and monitored expression and function after differentiation. Ussing chamber measurements confirmed CFTR-dependent chloride channel activity in CF donor cells. Immunostaining, quantitative real-time PCR, and single-cell sequencing analysis of cell-type markers indicated that vector transduction or CFTR expression does not alter the formation of pseudostratified, fully differentiated epithelial cell cultures or cell type distribution. These results have important implications for use of gene addition or gene editing strategies as life-long curative approaches for lung genetic diseases.
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Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2'-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV's strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.
Asunto(s)
Células Epiteliales/virología , Macrófagos/virología , Virus del Sarampión/patogenicidad , Sarampión/virología , Sistema Respiratorio/virología , Internalización del Virus , Replicación Viral , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Macrófagos/metabolismo , Sarampión/genética , Sarampión/metabolismo , RNA-Seq , Sistema Respiratorio/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
RATIONALE: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. OBJECTIVES: To identify MRSA genes associated with low lung function and potential MRSA transmission in CF. METHODS: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies, classified isolates by accessory gene content, and correlated the accessory genome to clinical outcomes. RESULTS: The most prevalent ST were ST5 (N = 55), ST8 (N = 15), and ST105 (N = 14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three clusters of MRSA were identified by accessory genome content. Cluster A, including ST5 and ST105, was highly prevalent at all ages. Cluster B, including ST8, was more limited to younger patients. Cluster C included 6 distantly related strains. Patients 20 years old and younger infected with Cluster A had lower forced expiratory volume in the first second (FEV1 ) and higher sputum biomass compared to similar-aged patients with Cluster B. CONCLUSIONS: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. The most prevalent cluster of MRSA, including ST5 and ST105, was associated with lower FEV1 . ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.