Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

Bartman, Colleen M.; Stelzig, K.; Linden, David R.; Prakash, Y. S.; Chiarella, Sergio E.

doi:10.1152/ajplung.00122.2021

Cited by 12 publications

(8 citation statements)

References 42 publications

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“…This occurs via endothelial nitric oxide synthase (eNOS) localized to the base of the cilia. Confirming this, we used a published siRNA protocol for primary ALIs ( 86 ) and found that eNOS siRNA but not nNOS siRNA reduced T2R Ca 2+ responses ( Figure 3A ). We also noted that, despite nearly identical Ca 2+ responses, F508del/F508del CF ALI cultures produced less NO in response to T2R agonists PTC, 3oxoC12HSL, quinine, apigenin, and DPD ( Figures 3B–F ).…”

Section: Resultssupporting

confidence: 59%

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages

et al. 2023

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IntroductionBitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF.MethodsImmunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages.ResultsPrimary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation.ConclusionsTogether, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.

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Section: Resultssupporting

confidence: 59%

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages

et al. 2023

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“…This occurs via endothelial nitric oxide synthase (eNOS) localized to the base of the cilia. Confirming this, we used a published siRNA protocol for primary ALIs (83) and found that eNOS siRNA but not nNOS siRNA reduced T2R Ca 2+ responses ( Fig 3A ). We also noted that, despite nearly identical Ca 2+ responses, F508del/F508del CF ALI cultures produced less NO in response to T2R agonists PTC, 3oxoC12HSL, quinine, apigenin, and DPD ( Fig 3B-F ).…”

Section: Resultssupporting

confidence: 60%

Bitter taste receptor-stimulated nitric oxide innate immune responses are reduced by loss of CFTR function in nasal epithelial cells and macrophages

Carey¹,

Palmer²,

Adappa³

et al. 2022

Preprint

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Bitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses. Using qPCR, immunofluorescence, and live cell imaging of air-liquid interface cultures, we found primary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation. Together, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate this pathway.

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“…SiRNA transfection was performed on cells cultured submerged or cultured with ALI method as previously described with minor modified (5,(59)(60)(61). When BEAS-2B cells or submerged cultured HNECs reached 80% confluence, the cells were transfected with siMEX3B (100 nM, Thermo Fisher Scientific Company, Waltham, MA, USA), siTGFBR1 (100 nM, Viewsolid Biotech, Beijing, China), siTGFBR2 (100 nM, Viewsolid Biotech), siTGFBR3 (100 nM, Viewsolid Biotech), or the corresponding negative control siRNA (siNC, 100 nM) using Lipofectamine RNAimax (InvitroGen) for 8 hours according to the manufacturer's instructions.…”

Section: Cell Culture Stimulation and Transfectionmentioning

confidence: 99%

MEX3B inhibits collagen production in eosinophilic nasal polyps by downregulating epithelial cell TGFBR3 mRNA stability

Liu¹,

Chen²,

Yu³

et al. 2023

JCI Insight

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Although the expression of Mex3 RNA binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HENCs) predominately in the eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) subtype, its functions as an RNA binding protein in airway epithelial cells remain unknown. Here, we revealed the role of MEX3B based on different subtypes of CRS, and demonstrated that MEX3B decreased TGF-β receptor III (TGFBR3) mRNA level by binding to its 3' UTR and reducing its stability in HNECs. TGF-βR3 was found to be a TGF-β2 specific coreceptor in HNECs. Knocking down or overexpressing MEX3B promoted or inhibited TGF-β2-induced phosphorylation of Smad2 in HNECs, respectively. TGF-βR3 and p-Smad2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps (CRSsNP), with a more prominent downregulation in the eosinophilic CRSwNP. TGF-β2 promoted collagen production in HNECs. Collagen abundance decreased and edema scores increased in CRSwNP compared to control, again more prominently in the eosinophilic type. Collagen expression in eosinophilic CRSwNP was negatively correlated with MEX3B but positively correlated with TGF-βR3. These results suggest that MEX3B inhibits tissue fibrosis in eosinophilic CRSwNP by downregulating epithelial cell TGFBR3 expression; consequently, MEX3B might be a valuable therapeutic target against eosinophilic CRSwNP.

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Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

Cited by 12 publications

References 42 publications

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages

Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages

Bitter taste receptor-stimulated nitric oxide innate immune responses are reduced by loss of CFTR function in nasal epithelial cells and macrophages

MEX3B inhibits collagen production in eosinophilic nasal polyps by downregulating epithelial cell TGFBR3 mRNA stability

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