RESUMEN
Rationale: The goal was to connect elements of idiopathic pulmonary fibrosis (IPF) pathogenesis, including chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling, distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B (mucin 5B), and associations between IPF risk and polymorphisms in the MUC5B promoter. Objectives: To test whether the endoplasmic reticulum (ER) stress sensor protein ERN2 (ER-to-nucleus signaling 2) and its downstream effector, the spliced form of XBP1S (X-box-binding protein 1), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia. Methods: Primary human airway epithelial (HAE) cells, transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE cells to explore therapeutic potential. Measurements and Main Results: ERN2 regulated MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially upregulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE cells was inhibited by KIRA6 and XBP1 CRISPR-Cas9. Conclusions: A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing an unfolded protein response-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.
Asunto(s)
Endorribonucleasas/genética , Fibrosis Pulmonar Idiopática/genética , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , Mucosa Respiratoria/metabolismo , Proteína 1 de Unión a la X-Box/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Polimorfismo Genético , Cultivo Primario de Células , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Understanding how expression of airway secretory mucins MUC5B and MUC5AC is regulated in health and disease is important to elucidating the pathogenesis of mucoobstructive respiratory diseases. The transcription factor SPDEF (sterile α-motif pointed domain epithelial specific transcription factor) is a key regulator of MUC5AC, but its role in regulating MUC5B in health and in mucoobstructive lung diseases is unknown. Characterization of Spdef-deficient mice upper and lower airways demonstrated region-specific, Spdef-dependent regulation of basal Muc5b expression. Neonatal Spdef-deficient mice exhibited reductions in BAL Muc5ac and Muc5b. Adult Spdef-deficient mice partially phenocopied Muc5b-deficient mice as they exhibited reduced Muc5b in nasopharyngeal and airway epithelia but not in olfactory Bowman glands, 75% incidence of nasopharyngeal hair/mucus plugs, and mild bacterial otitis media, without defective mucociliary clearance in the nasopharynx. In contrast, tracheal mucociliary clearance was reduced in Spdef-deficient mice in the absence of lung disease. To evaluate the role of Spdef in the development and persistence of Muc5b-predominant mucoobstructive lung disease, Spdef-deficient mice were crossed with Scnn1b-transgenic (Scnn1b-Tg) mice, which exhibit airway surface dehydration-induced airway mucus obstruction and inflammation. Spdef-deficient Scnn1b-Tg mice exhibited reduced Muc5ac, but not Muc5b, expression and BAL content. Airway mucus obstruction was not decreased in Spdef-deficient Scnn1b-Tg mice, consistent with Muc5b-dominant Scnn1b disease, but increased airway neutrophilia was observed compared with Spdef-sufficient Scnn1b-Tg mice. Collectively, these results indicate that Spdef regulates baseline Muc5b expression in respiratory epithelia but does not contribute to Muc5b regulation in a mouse model of Muc5b-predominant mucus obstruction caused by airway dehydration.
Asunto(s)
Enfermedades Pulmonares/metabolismo , Mucina 5B/metabolismo , Depuración Mucociliar/fisiología , Proteínas Proto-Oncogénicas c-ets/genética , Animales , Canales Epiteliales de Sodio/genética , Enfermedades Pulmonares/genética , Ratones Transgénicos , Mucina 5B/genéticaRESUMEN
The epithelial Na+ channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, ß, and γ, which are differentially expressed (α > ß > γ). Airway-targeted overexpression of the ß subunit results in Na+ hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na+ hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na+ transport and disease severity exceeding that of ßENaC-Tg mice, we generated double (αß, αγ, ßγ) and triple (αßγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double ßγENaC-Tg mice exhibited airway Na+ absorption greater than that of ßENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αßENaC-Tg mice exhibited Na+ transport rates comparable to those of ßENaC-Tg littermates. However, αßENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αßγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of ßENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of ß- and γENaC had additive effects on Na+ transport and disease severity, suggesting dose dependency of these two variables.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Enfermedades Pulmonares/patología , Neumonía/patología , Mucosa Respiratoria/patología , Animales , Canales Epiteliales de Sodio/genética , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fenotipo , Neumonía/etiología , Neumonía/metabolismo , Mucosa Respiratoria/metabolismo , Transducción de SeñalRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.
Asunto(s)
COVID-19 , Animales , COVID-19/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Epigénesis Genética , Humanos , Ratones , Mucinas/genética , SARS-CoV-2RESUMEN
Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1ß, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1ß alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1ß induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1ß are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1ß-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.
Asunto(s)
Fibrosis Quística/metabolismo , Interleucina-1beta/metabolismo , Moco/metabolismo , Animales , Estudios de Casos y Controles , Células Cultivadas , Fibrosis Quística/genética , Fibrosis Quística/patología , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucina 5AC/genética , Mucina 5AC/metabolismo , Mucina 5B/genética , Mucina 5B/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-ets/deficiencia , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Tipo I de Interleucina-1/deficiencia , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de SeñalRESUMEN
Airway diseases, including cigarette smoke-induced chronic bronchitis, cystic fibrosis, and primary ciliary dyskinesia are associated with decreased mucociliary clearance (MCC). However, it is not known whether a simple reduction in MCC or concentration-dependent mucus adhesion to airway surfaces dominates disease pathogenesis or whether decreasing the concentration of secreted mucins may be therapeutic. To address these questions, Scnn1b-Tg mice, which exhibit airway mucus dehydration/adhesion, were compared and crossed with Muc5b- and Muc5ac-deficient mice. Absence of Muc5b caused a 90% reduction in MCC, whereas Scnn1b-Tg mice exhibited an â¼50% reduction. However, the degree of MCC reduction did not correlate with bronchitic airway pathology, which was observed only in Scnn1b-Tg mice. Ablation of Muc5b significantly reduced the extent of mucus plugging in Scnn1b-Tg mice. However, complete absence of Muc5b in Scnn1b-Tg mice was associated with increased airway inflammation, suggesting that Muc5b is required to maintain immune homeostasis. Loss of Muc5ac had few phenotypic consequences in Scnn1b-Tg mice. These data suggest that: (i) mucus hyperconcentration dominates over MCC reduction alone to produce bronchitic airway pathology; (ii) Muc5b is the dominant contributor to the Scnn1b-Tg phenotype; and (iii) therapies that limit mucin secretion may reduce plugging, but complete Muc5b removal from airway surfaces may be detrimental.