Dataset Information

Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice.

ABSTRACT: Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for lipopolysaccharide (LPS). Transient receptor potential ankyrin 1 (TRPA1), a Ca²⁺-permeant channel, has been suggested as a non-TLR receptor membrane-bound sensor of LPS. We recently reported that TRPA1 is expressed in lung epithelial cells (LECs) and mediates lung inflammation induced by cigarette smoke. However, the role of TRPA1 in LPS-induced lung inflammation has not been conclusively defined, and its underlying cellular mechanisms remain unclear. In this study, our in vitro results showed that LPS sequentially produced a cascade of events, including the elevation of intracellular Ca²⁺, the activation of NADPH oxidase, increase in intracellular reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kB (NF-?B) signaling, and the induction of IL-8. The increase in intracellular Ca²⁺ was inhibited by HC030031 (a TRPA1 antagonist) but was unaffected by TAK-242 (a TLR-4 inhibitor). The activation of NADPH oxidase was prevented by its inhibitor apocynin, EGTA (an extracellular Ca²⁺ chelator), and HC030031. The increase in intracellular ROS was attenuated by apocynin, N-acetyl-cysteine (NAC, a ROS scavenger), EGTA, and HC030031. The activation of the MAPK/NF-?B signaling was halted by NAC, EGTA, and HC030031. IL-8 induction was suppressed by HC030031 and TRPA1 siRNA, and further reduced by the combination of HC030031 and TAK-242. Our in vivo studies showed that trpa1^-/- mice exhibited a reduced level of LPS-induced lung inflammation compared with wild-type mice as evidenced by the alleviations of increases in vascular permeability, inflammatory cell infiltration, inflammatory cytokine levels, oxidative stress, and MAPK signaling activation. Thus, in LECs, LPS may activate TRPA1 resulting in an increase in Ca²⁺ influx. The increased intracellular Ca²⁺ leads to NADPH oxidase activation, which causes an increase in intracellular ROS. The intracellular ROS activates the MAPK/NF-?B signaling resulting in IL-8 induction. This mechanism may possibly be at work to induce lung inflammation in mice.

SUBMITTER: Ko HK

PROVIDER: S-EPMC7705107 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice.

Ko Hsin-Kuo HK Lin An-Hsuan AH Perng Diahn-Warng DW Lee Tzong-Shyuan TS Kou Yu Ru YR

Frontiers in physiology 20201117

Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for lipopolysaccharide (LPS). Transient receptor potential ankyrin 1 (TRPA1), a Ca<sup>2+</sup>-permeant channel, has been suggested as a non-TLR receptor membrane-bound sensor of LPS. We recently reported that TRPA1 is expressed in lung epithelial cells (LECs) and mediates lung inflammation induced by cigarette smoke. However, the role of TRPA1 in LPS-induced lung inflammation has not been conclusively ...[more]

PMID: 33281629

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Project description:BackgroundAcute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.MethodsWild-type (WT) mice and Spred-2(-/-) mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2(-/-) mice and stimulated with LPS. In in vitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.ResultsLPS-induced acute lung inflammation was significantly exacerbated in Spred-2(-/-) mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-?, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2(-/-) mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.ConclusionsThe ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.

Project description:Acute lung injury (ALI) is mainly caused by inflammation and is associated with high mortality rates. Emerging evidence has suggested that microRNAs (miRNAs or miRs) serve a significant function in ALI. However, the fundamental mechanism underlying ALI remain to be fully elucidated. Although miR-16 has been reported to be involved in the occurrence and development of a number of diseases its association with ALI has not been previously investigated. Therefore, the present study aimed to explore the role of miR-16 in the lipopolysaccharide (LPS)-induced ALI model. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were measured by ELISA in the blood samples of rats with ALI and in the normal human bronchial epithelial (NHBE) cell line. The role of miR-16 in inflammation was evaluated using gene overexpression and silencing experiments in NHBE cells by reverse transcription-quantitative PCR. In addition, the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 were also determined using ELISA. The potential interaction between miR-16 and TLR4 was assessed using bioinformatics analysis by the TargetScan database and then verified in 293T cells using luciferase reporter assay. The expression of miR-16 was notably decreased in the lung tissues of rats with LPS-induced ALI compared with the PBS treated-group. Additionally, the levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 were reduced following transfection of NHBE cells with miR-16 mimics compared with those in the miR-negative control group. Western blot analysis revealed that miR-16 overexpression could downregulate TLR4 expression in NHBE cells compared with that in the miR-NC group. Luciferase reporter assay confirmed that TLR4 may be directly targeted by miR-16. The effect of miR-16 on TLR4 was rescued in NHBE cells following treatment with LPS. Overall, these aforementioned findings suggest that miR-16 may serve a protective role against LPS-mediated inflammatory responses in NHBE cells by regulating TLR4, where this mechanism may be considered to be a novel approach for treating ALI in the future.

Project description:The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.

Dataset Information

Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice.

Publications

Lung Epithelial TRPA1 Mediates Lipopolysaccharide-Induced Lung Inflammation in Bronchial Epithelial Cells and Mice.

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