Bronchial epithelial cells release inflammatory markers linked to airway inflammation and remodeling in response to TLR5 ligand flagellin

Abstract

Background/aims: Flagellin, which is abundant in gram-negative bacteria, including Pseudomonas, is reported to influence on inflammatory responses in various lung diseases. However, its effect on airway epithelial cells in contribution to asthma pathogenesis is not elucidated yet. We aimed to investigate the effect of TLR5 ligand flagellin on the transcriptomic profile of primary human epithelial cells and to determine the markers of airway inflammation.

Methods: Normal human bronchial epithelial (NHBE) cells were grown and differentiated in air-liquid interface (ALI) culture for 14-16 days. The cells were treated with flagellin in vitro at 10 and 100 ng/ml for 3 and 24 h. The conditioned media and cells were harvested to validate inflammatory markers involved in airway inflammation using ELISA, Western blot, and quantitative PCR methods. RNA-sequencing was performed to investigate the transcriptional response to flagellin in ALI-NHBE cells.

Results: Altered transcriptional responses to flagellin in differentiated bronchial epithelial cells were determined, including genes encoding chemokines, matrix metalloproteinases, and antimicrobial biomolecules. Pathway analysis of the transcriptionally responsive genes revealed enrichment of signaling pathways. Flagellin induced the mRNA expressions of proinflammatory cytokines and chemokines, and secretion of GM-CSF, CXCL5, CCL5 and CXCL10. Flagellin enhanced the protein expression of MMP-13 in TGF-β1 and TGF-β2 pretreated cell lysates and Wnt/β-catenin signaling.

Conclusions: These findings suggest that flagellin could be a potent inducer of inflammatory markers that may contribute to airway inflammation and remodeling.

Keywords: Airway; Epithelial cell; Flagellin; Inflammation; TLR5.

Fig. 1

The expression patterns of gene expression and protein production of cytokines in differentiated NHBE cells stimulated with flagellin. Relative mRNA expression levels of CSF2 (A), CXCL8 (B) and IL33 (C). D-E. Protein levels of GM-CSF and IL-8. Normalization for quantitative PCR was performed using GAPDH gene as an internal control. The sampling time points were 3 h (open bar) and 24 h (closed bar) after stimulation. Data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ∗p < 0.05 indicates significant differences from control group.

Fig. 2

The expression patterns of gene expression and protein production of chemokines in differentiated NHBE cells stimulated with flagellin. Relative mRNA expression levels of CXCL5 (A), CCL5 (B), CXCL10 (C) and CXCL11 (D). E-H. Protein levels of chemokines. Normalization for qPCR was performed using GAPDH gene as an internal control. The sampling time points were 3 h (open bar) and 24 h (closed bar) after stimulation. Data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ∗p < 0.05 indicates significant differences from control group.

Fig. 3

Expression patterns of 2 representative genes involved in airway remodeling and Wnt/β-catenin signaling pathway. NHBE cells were pretreated with TGF-β1 and TGF-β2 (10 ng/ml for 1 h) and stimulated with flagellin (100 ng/ml for 24 h). Data are presented as mean ± standard error of the mean (SEM) of 2 independent experiments. A-B. The mRNA expression of genes from flagellin unstimulated (open bar) and stimulated (closed bar) samples were evaluated. ∗p < 0.05 indicates significant differences from unstimulated group. C. Representative Western blot analysis of MMP-9 and MMP-13 with pretreated and stimulated sample. D. Effect of flagellin on Wnt/β-catenin signaling pathway.