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Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation
Published on: July 6, 2019
TMEM16A protein attenuates lipopolysaccharide-mediated inflammatory response of human lung epithelial cell line A549
Aili Zhang1, Xixin Yan, Honglin Li
11Department of Respirology, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.
Objective:
To observe the expression of endogenous TMEM16A in rat alveolar type II epithelial cells (AT-II) and A549, and study the effect of TMEM16A on lipopolysaccharide (LPS)-induced proinflammatory cytokine secretion.
Methods:
Rat AT-II cells were isolated and TMEM16A protein expression in rat AT-II cells was measured by Western blot. TMEM16A mRNA and protein expressions in A549 were measured by real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. TMEM16A gene was transfected into A549 using Lipofectamine 2000. Transfected cells were selected in the presence of G418 to create a stable TMEM16A overexpression A549 cell line. The expression of TMEM16A in A549 was knocked down by lentiviral vector-mediated RNA interference. TNF-α and IL-8 levels were determined by enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter assay system was used to measure the transcriptional activity of NF-κB.
Results:
(1) Endogenous TMEM16A was expressed in rat AT-II and A549. (2) TMEM16A expression in A549 significantly increased at 24 hours and 36 hours, and then decreased at 48 hours after LPS treatment. (3) TMEM16A mRNA and protein expressions were increased in the stable TMEM16A overexpression A549 cell line. (4) TMEM16A overexpression decreased the LPS-induced TNF-α and IL-8 secretions. (5) TMEM16A mRNA and protein expressions were knocked down in TMEM16A-siRNA lentivirus transfected A549. (6) TMEM16A knockdown increased the LPS-induced TNF-α and IL-8 secretions. (7) TMEM16A overexpression inhibited LPS-induced NF-κB activation.
Conclusions:
TMEM16A is expressed in AT-II. TMEM16A in A549 inhibits LPS-induced NF-κB activation and decreases proinflammatory cytokines release, protecting A549 from acute LPS-mediated damage.
Insights
The anoctamin-1 (TMEM16A) protein is present in alveolar cells and influences inflammatory responses. TMEM16A overexpression reduces lipopolysaccharide-induced cytokine release, while knockdown increases it, suggesting a protective role.
Area of Science:
- Cell Biology
- Immunology
- Respiratory Medicine
Background:
- Anoctamin-1 (TMEM16A) is an ion channel protein with diverse physiological roles.
- Its involvement in inflammatory responses within lung epithelial cells requires further elucidation.
- Lipopolysaccharide (LPS) is a potent inducer of inflammation in the respiratory system.
Purpose of the Study:
- To investigate the expression of endogenous TMEM16A in rat alveolar type II (AT-II) epithelial cells and A549 cells.
- To determine the effect of TMEM16A on lipopolysaccharide (LPS)-induced secretion of proinflammatory cytokines.
- To explore the role of TMEM16A in regulating NF-κB activation.
Main Methods:
- TMEM16A expression was assessed in rat AT-II and A549 cells using Western blot and real-time quantitative PCR.
- Stable TMEM16A-overexpressing and knockdown A549 cell lines were generated via transfection and lentiviral RNA interference.
- Proinflammatory cytokine levels (TNF-α, IL-8) and NF-κB transcriptional activity were measured using ELISA and dual-luciferase reporter assays, respectively.
Main Results:
- Endogenous TMEM16A expression was confirmed in both rat AT-II and A549 cells.
- LPS treatment transiently modulated TMEM16A expression in A549 cells.
- TMEM16A overexpression attenuated LPS-induced TNF-α and IL-8 release, whereas TMEM16A knockdown exacerbated it.
- TMEM16A overexpression inhibited LPS-induced NF-κB activation.
Conclusions:
- TMEM16A is expressed in alveolar type II cells.
- TMEM16A plays an inhibitory role in LPS-induced NF-κB activation and proinflammatory cytokine release in A549 cells.
- TMEM16A may confer protection against acute LPS-mediated lung injury.
