scholarly journals Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus–Host Interactions

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 747 ◽  
Author(s):  
Hulda R. Jonsdottir ◽  
Sabrina Marti ◽  
Dirk Geerts ◽  
Regulo Rodriguez ◽  
Volker Thiel ◽  
...  

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus–host interactions in human respiratory epithelium.

2019 ◽  
Author(s):  
Hulda R. Jonsdottir ◽  
Sabrina Marti ◽  
Dirk Geerts ◽  
Regulo Rodriguez ◽  
Volker Thiel ◽  
...  

AbstractPrimary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for FACS-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly(I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (RSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures thereby unlocking a unique potential for detailed molecular characterization of virus – host interactions in human respiratory epithelium.


2009 ◽  
Vol 156 (1-2) ◽  
pp. 19-26 ◽  
Author(s):  
Bridget S. Banach ◽  
Jan M. Orenstein ◽  
Linda M. Fox ◽  
Scott H. Randell ◽  
Anne H. Rowley ◽  
...  

2001 ◽  
Vol 280 (1) ◽  
pp. L127-L133 ◽  
Author(s):  
S. Tavakoli ◽  
M. J. Cowan ◽  
T. Benfield ◽  
C. Logun ◽  
J. H. Shelhamer

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E2(10−7M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 ± 306 vs. 1,051 ± 154 pg/ml (PGE2-treated cells vs. control cells). PGE2(10−7to 10−10M) induced a dose-related increase in IL-6 release at 24 h. PGF2α(10−6M) treatment caused a similar effect to that of PGE2(10−7M). PGE2analogs with relative selectivity for PGE2receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE2, an EP-2/4 agonist, and 17-phenyl trinor PGE2, an agonist selective for the EP-1 > EP-3 receptor subtype (10−6to 10−8M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE2treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE2increases airway epithelial cell IL-6 production and release.


2009 ◽  
Vol 149 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Takahito Chiba ◽  
Shigeharu Ueki ◽  
Wataru Ito ◽  
Hikari Kato ◽  
Masahide Takeda ◽  
...  

2004 ◽  
Vol 3 (2) ◽  
pp. 99-117 ◽  
Author(s):  
Gavin M Braunstein ◽  
Akos Zsembery ◽  
Torry A Tucker ◽  
Erik M Schwiebert

Sign in / Sign up

Export Citation Format

Share Document