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Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

ABSTRACT: BACKGROUND:Robust methods to culture primary airway epithelial cells were developed several decades ago and these cells provide the model of choice to investigate many diseases of the human lung. However, the molecular signature of cells from different regions of the airway epithelium has not been well characterized. METHODS:We utilize DNase-seq and RNA-seq to examine the molecular signatures of primary cells derived from human tracheal and bronchial tissues, as well as healthy and diseased (cystic fibrosis (CF)) donor lung tissue. RESULTS:Our data reveal an airway cell signature that is divergent from other epithelial cell types and from common airway epithelial cell lines. The differences between tracheal and bronchial cells are clearly evident as are common regulatory features. Only minor variation is seen between bronchial cells from healthy or CF donors. CONCLUSIONS:These data are a valuable resource for functional genomics analysis of airway epithelial tissues in human disease.

SUBMITTER: Gillen AE

PROVIDER: S-EPMC6026051 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

Gillen Austin E AE Yang Rui R Cotton Calvin U CU Perez Aura A Randell Scott H SH Leir Shih-Hsing SH Harris Ann A

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 20180217 4

<h4>Background</h4>Robust methods to culture primary airway epithelial cells were developed several decades ago and these cells provide the model of choice to investigate many diseases of the human lung. However, the molecular signature of cells from different regions of the airway epithelium has not been well characterized.<h4>Methods</h4>We utilize DNase-seq and RNA-seq to examine the molecular signatures of primary cells derived from human tracheal and bronchial tissues, as well as healthy an ...[more]

PMID: 29459038

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Project description:Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis.ImportanceAdaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

Project description:BackgroundAn increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized.MethodsTo determine if expression by primary BECs from asthmatic and non-asthmatic children of selected genes associated with airway remodeling, innate immune response, immunomodulatory factors, and markers of differentiated airway epithelium, are stable over increasing cell passage number, we studied gene expression patterns in passages 1, 2, 3, 4, and 5 BECs from asthmatic (n?=?6) and healthy (n?=?6) subjects that were differentiated at an air-liquid interface. RNA was harvested from BECs and RT-PCR was performed for TGF?1, TGF?2, activin A, FSTL3, MUC5AC, TSLP, IL-33, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1.ResultsExpression of TGF?1, TGF?2, activin A, FSTL3, MUC5AC, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1 by primary BECs from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3; however, gene expression at cell passages 4 and 5 was significantly greater and more variable compared to passage 1 BECs for many of these genes. IL-33 and FOXJ1 expression was also stable between passages 1 through 3, however, expression at passages 4 and 5 was significantly lower than by passage 1 BECs. TSLP, p63, and KRT5 expression was stable across BEC passages 1 through 5 for both asthmatic and healthy BECs.ConclusionsThese observations illustrate the importance of using BECs from passage ?3 when studying gene expression by asthmatic and non-asthmatic primary BECs and characterizing the expression pattern across increasing cell passage number for each new gene studied, as beyond passage 3 genes expressed by primary BECs appear to less accurately model in vivo airway epithelial gene expression.

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Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

Publications

Molecular characterization of gene regulatory networks in primary human tracheal and bronchial epithelial cells.

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