Project description:Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.
Project description:Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR). Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e127; doi:10.1038/mtna.2013.55; published online 8 October 2013.
Project description:Human conducting airways contain two anatomically distinct epithelial cell compartments: surface epithelium and submucosal glands (SMG). Surface epithelial cells interface directly with the environment and function in pathogen detection, fluid and electrolyte transport, and mucus elevation. SMG secrete antimicrobial molecules and most of the airway surface fluid. Despite the unique functional roles of surface epithelia and SMG, little is known about the differences in gene expression and cellular metabolism that orchestrate the specialized functions of these epithelial compartments. To approach this problem, we performed large-scale transcript profiling using epithelial cell samples obtained by laser capture microdissection (LCM) of human bronchus specimens. We found that SMG expressed high levels of many transcripts encoding known or putative innate immune factors, including lactoferrin, zinc alpha-2 glycoprotein, and proline-rich protein 4. By contrast, surface epithelial cells expressed high levels of genes involved in basic nutrient catabolism, xenobiotic clearance, and ciliated structure assembly. Selected confirmation of differentially expressed genes in surface and SMG epithelia demonstrated the predictive power of this approach in identifying genes with localized tissue expression. To characterize metabolic differences between surface epithelial cells and SMG, immunostaining for a mitochondrial marker (isocitrate dehydrogenase) was performed. Because greater staining was observed in the surface compartment, we predict that these cells use significantly more energy than SMG cells. This study illustrates the power of LCM in defining the roles of specific anatomic features in airway biology and may be useful in examining how disease states alter transcriptional programs in the conducting airways.
Project description:The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.
Project description:Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.
Project description:BackgroundThe coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic with over 627 million cases and over 6.5 million deaths. It was reported that smoking-related chronic obstructive pulmonary disease (COPD) might be a crucial risk for COVID-19 patients to develop severe condition. As cigarette smoke (CS) is the major risk factor for COPD, we hypothesize that barrier dysfunction and an altered cytokine response in CS-exposed airway epithelial cells may contribute to increased SARS-CoV-2-induced immune response that may result in increased susceptibility to severe disease. The aim of this study was to evaluate the role of CS on SARS-CoV-2-induced immune and inflammatory responses, and epithelial barrier integrity leading to airway epithelial damage.MethodsPrimary human airway epithelial cells were differentiated under air-liquid interface culture. Cells were then exposed to cigarette smoke medium (CSM) before infection with SARS-CoV-2 isolated from a local patient. The infection susceptibility, morphology, and the expression of genes related to host immune response, airway inflammation and damages were evaluated.ResultsCells pre-treated with CSM significantly caused higher replication of SARS-CoV-2 and more severe SARS-CoV-2-induced cellular morphological alteration. CSM exposure caused significant upregulation of long form angiotensin converting enzyme (ACE)2, a functional receptor for SARS-CoV-2 viral entry, transmembrane serine protease (TMPRSS)2 and TMPRSS4, which cleave the spike protein of SARS-CoV-2 to allow viral entry, leading to an aggravated immune response via inhibition of type I interferon pathway. In addition, CSM worsened SARS-CoV-2-induced airway epithelial cell damage, resulting in severe motile ciliary disorder, junctional disruption and mucus hypersecretion.ConclusionSmoking led to dysregulation of host immune response and cell damage as seen in SARS-CoV-2-infected primary human airway epithelia. These findings may contribute to increased disease susceptibility with severe condition and provide a better understanding of the pathogenesis of SARS-CoV-2 infection in smokers.
Project description:Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.
Project description:Cilia are microscopic projections that extend from eukaryotic cells. There are two general types of cilia; primary cilia serve as sensory organelles, whereas motile cilia exert mechanical force. The motile cilia emerging from human airway epithelial cells propel harmful inhaled material out of the lung. We found that these cells express sensory bitter taste receptors, which localized on motile cilia. Bitter compounds increased the intracellular calcium ion concentration and stimulated ciliary beat frequency. Thus, airway epithelia contain a cell-autonomous system in which motile cilia both sense noxious substances entering airways and initiate a defensive mechanical mechanism to eliminate the offending compound. Hence, like primary cilia, classical motile cilia also contain sensors to detect the external environment.
Project description:scRNA-seq of air-liquid interface airway epithelial cultures derived from a human fluorescent reporter hPSC line labeling MUC5B-expressing cells
Project description:The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate 'off-the shelf' CAR-T cells.