TLRs and the airway epithelium in the cystic fibrosis lung.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. These mutations disrupt CFTR function within epithelial cells. Although the defect affects ion transport in many organs, the major cause of morbidity and mortality in individuals with CF is the progressive lung disease characterized by inflammation and unremitting bacterial infection with Pseudomonas, Staphylococcus, Haemophilus, Aspergillus and Burkholderia species. Although CF airways exhibit high numbers of immune cells and elevated levels of proinflammatory cytokines, the lung's innate immune defenses fail to clear bacterial infections. These observations suggest a modified immune response in the CF lung (due to the CFTR mutation and associated secondary alterations of the airway epithelium). Airway epithelial cells not only function as a physical barrier against inhaled pathogens, but also play an important role in innate immune responses of the host. Microorganisms are recognized through a variety of pattern recognition receptors (PRR), mainly Toll-like receptors (TLRs) that are abundantly expressed in airway epithelial cells. TLR activation in a milieu potentially rich in microbial (and endogenous) TLR agonists probably adds to the chronic inflammatory phenotype in CF airway epithelia. Therefore, the expression, function and activation of TLRs in CF airway epithelia have become the focus of intensive research. In this chapter, we give an overview of the current understanding of TLR signaling in CF and its potential role in the pathogenesis of CF lung disease

Reduced surface toll-like receptor-4 expression and absent interferon-γ-inducible protein-10 induction in cystic fibrosis airway cells.

ABSTRACT As part of the innate and adaptive immune system, airway epithelial cells secrete proinflammatory cytokines after activation of Toll-like receptors (TLRs) by pathogens. Nevertheless, cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. The authors have shown that in CF bronchial epithelial cells, a reduced surface expression of TLR-4 causes a diminished interleukin (IL)-8 and IL-6 response upon lipopolysaccharide (LPS) stimulation. However, there is no information regarding activation of the MyD88 (myeloid differentiation primary response gene 88)-independent TLR-4 signaling pathway by LPS, which results in the activation of adaptive immune responses by secretion of the T cell-recruiting chemokine interferon-γ-inducible protein (IP)-10. Therefore, the authors investigated the induction of IP-10 in CF bronchial epithelial cell line CFBE41o- and its CFTR-corrected isotype under well-differentiating conditions. TLR-4 surface expression was significantly reduced in CFBE41o- by a factor of 2, compared to the CFTR-corrected cells. In CFTR-corrected cells, stimulation with LPS increased IP-10 secretion. Incubating cells with siRNA directed against TLR-4 inhibited the LPS stimulated increase of IP-10 in CFTR-corrected cells. The reduced TLR-4 surface expression in CF cells causes the loss of induction of IP-10 by LPS. This could compromise adaptive immune responses in CF due to a reduced T-cell recruitment

A small molecule neutrophil elastase inhibitor, KRP-109, inhibits cystic fibrosis mucin degradation.

BACKGROUND: Neutrophil elastase (NE) rapidly degrades gel-forming airway mucins in cystic fibrosis (CF) sputum. We hypothesized that KRP-109, a small molecule NE inhibitor, would inhibit CF mucin degradation in vitro. METHODS: Sputa were collected from CF patients (n=5) chronically or intermittently infected with Pseudomonas aeruginosa (P.a.). Mucin degradation was analyzed using western blot. Protease inhibitor studies were performed using alpha1-proteinase inhibitor (A1-PI Prolastin®) and KRP-109. Elastase activity assays were performed using spectrophotometry. RESULTS: There were significant differences in the amount of active NE in different CF sputum samples. KRP-109 decreased the NE driven mucin degradation in vitro. Pseudomonas elastases appeared to blunt elastase inhibition by A1-PI or KRP-109. CONCLUSION: Inhibitors of neutrophil and Pseudomonas-derived elastases might rescue mucus clearance and reverse airway obstruction in CF

Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction.

Proteases have been shown to degrade airway mucin proteins and to damage the epithelium impairing mucociliary clearance. There are increased proteases in the COPD airway but changes in protease-antiprotease balance and mucin degradation have not been investigated during the course of a COPD exacerbation. We hypothesized that increased protease levels would lead to mucin degradation in acute COPD exacerbations. Methods: We measured neutrophil elastase (NE) and alpha 1 protease inhibitor (A1-PI) levels using immunoblotting, and conducted protease inhibitor studies, zymograms, elastin substrate assays and cigarette smoke condensate experiments to evaluate the stability of the gel-forming mucins, MUC5AC and MUC5B, before and 5-6 weeks after an acute pulmonary exacerbation of COPD (n=9 subjects). Results: Unexpectedly, mucin concentration and mucin stability were highest at the start of the exacerbation and restored to baseline after 6 weeks. Consistent with these data, immunoblots and zymograms confirmed decreased NE concentration and activity and increased A1-PI at the start of the exacerbation. After recovery there was an increase in NE activity and a decrease in A1-PI levels. In vitro, protease inhibitor studies demonstrated that serine proteases played a key role in mucin degradation. Mucin stability was further enhanced upon treating with cigarette smoke condensate (CSC). Conclusion: There appears to be rapid consumption of secreted proteases due to an increase in antiproteases, at the start of a COPD exacerbation. This leads to increased mucin gel stability which may be important in trapping and clearing infectious and inflammatory mediators, but this may also contribute acutely to mucus retention

Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis<em> in vitro</em>.

Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1&alpha; (HIF-1&alpha;), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1&alpha; were performed on lung sections of CFTR-/- and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1&alpha;, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1&alpha; stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1&alpha;, downregulate HO-1 expression in CFBE41o- cells