TLR-Induced Inflammation in Cystic Fibrosis and Non-Cystic Fibrosis Airway Epithelial Cells

Abstract

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o−, which is homozygous for the ΔF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o−. Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o− cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-κB-linked reporter gene and increased IL-8 protein production in CFTE29o− cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.

Airway epithelial cell signaling in response to bacterial pathogens

“…5,6 Toll Like Receptors Among the eleven Toll like receptors (TLRs) that have been recognized to date, 7,8 TLRs 1-10 are expressed in airway epithelia. 9,10 However, not all of these TLRs and the associated adaptors are equally available on the surface of the airway epithelium where they can respond to luminal contaminants. Whereas TLR2 is apically expressed and TLR5 is readily mobilized in response to bacteria, 5,6 TLR4 is intracellular 11 and is not mobilized to the cell surface in response to the major lung pathogens P. aeruginosa or S. aureus.…”

“…The role of TLR2 in initiating pro-inflammatory signaling in professional immune cells, as well as airway epithelia in vitro has been established. 5,7,9,10 TLR2 mRNA expression is up-regulated in the lungs during both Gram-positive and Gram-negative infection. [15][16][17] In response to S. aureus systemic infection, TLR2 null mice have increased mortality.…”

IRAP+ endosomes restrict TLR9 activation and signaling

“…3a).% We wondered if the exacerbated proinflammatory TLR9 signaling might affect the innate immune response during a bacterial infection. Pseudomonas aeruginosa is a Gram-negative bacterium that activates several TLRs including TLR9 on alveolar macrophages (AM) and epithelial cells 24 . TLR9-deficient mice were shown to be resistant to P. aeruginosa infection, suggesting that TLR9 signaling can have deleterious effects in this model 25 .…”

miR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression

“…IL-1RI and TLRs are present on a variety of cell types, including both immune cells and epithelial cells within the lung. In the context of CF, airway epithelial cells have been shown to promote proinflammatory gene transcription following stimulation with their cognate agonists (1,2). For example, in airway epithelial cells of non-CF and CF origin triacylated lipopeptide, LPS or unmethylated CpG DNA can induce IL-6, IL-8, and TNF-a production via TLRs 2, 4, and 9 (1).…”