TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells

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 DeltaF508 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-kappaB-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.     

Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.     

Early bronchial inflammation in cystic fibrosis

Cystic fibrosis (CF) is the most common genetic autosomal recessive disease in caucasian north-american and european populations. The CF gene codes for a transmembrane glycoprotein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a chloride channel which regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells. Mutations of the CF gene lead to a dysregulation of chloride and sodium channel associated to airway mucus dehydration, neutrophil-dominated airway inflammation and chronic infection responsible for the morbidity and mortality of CF patients. Although a high number of studies has been devoted to the CFTR pleiotropic functions, the chronology of the physiopathological events leading to the airway inflammation linked to mutations of the CF gene is still an open question. The issue of whether airway inflammation takes place before infection or is a consequence of infection during CF pathogenesis is still controversial. It has been recently reported that in broncho-alveolar lavages collected in CF infants, there is an increased level of interleukin IL-8 and abnormal low level of IL-10. The decreased IL-10 production has been confirmed in peripheral blood monocytes as well as in airway cell lines. Under basal conditions, the increased expression of the pro-inflammatory IL-8 cytokine has also been recently observed in the airway liquid secreted by CF na?ve humanized airway xenografts and in the supernatant culture of CF human airway epithelial cells. These results suggest that CFTR dysfunction may result in a constitutive pro-inflammatory vs anti-inflammatory imbalance in CF disease. Recent data from the literature suggest that the failure of chloride transport, the maturation defect and mistraffricking of mutated CFTR, lead to its accumulation in the endoplasmic reticulum and activation of NF-kappa B, responsible for the imbalance in the CF airway cell cytokine production.     

Sequential analysis of surfactant,lung function and inflammation in cystic fibrosis patients

Background

In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.

Methods

As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV₁ 94% of predicted) at three times over a three year period.

Results

There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV₁, MEF_75/25%VC, and MEF_25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.

Conclusion

Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.     

Infection,inflammation and exercise in cystic fibrosis

Regular exercise is positively associated with health. It has also been suggested to exert anti-inflammatory effects. In healthy subjects, a single exercise session results in immune cell activation, which is characterized by production of immune modulatory peptides (e.g. IL-6, IL-8), a leukocytosis and enhanced immune cell functions. Upon cessation of exercise, immune activation is followed by a tolerizing phase, characterized by a reduced responsiveness of immune cells. Regular exercise of moderate intensity and duration has been shown to exert anti-inflammatory effects and is associated with a reduced disease incidence and viral infection susceptibility. Specific exercise programs may therefore be used to modify the course of chronic inflammatory and infectious diseases such as cystic fibrosis (CF).Patients with CF suffer from severe and chronic pulmonary infections and inflammation, leading to obstructive and restrictive pulmonary disease, exercise intolerance and muscle cachexia. Inflammation is characterized by a hyper-inflammatory phenotype. Patients are encouraged to engage in exercise programs to maintain physical fitness, quality of life, pulmonary function and health.In this review, we present an overview of available literature describing the association between regular exercise, inflammation and infection susceptibility and discuss the implications of these observations for prevention and treatment of inflammation and infection susceptibility in patients with CF.     

Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.     

Neutrophil caveolin-1 expression contributes to mechanism of lung inflammation and injury

Caveolin-1 present in immune cells may be involved in regulation of the inflammatory response. Here, using caveolin-1-null (Cav-1(-/-)) mice, we addressed the role of caveolin-1 in polymorphonuclear neutrophils (PMNs) in regulating PMN activation-mediated lung injury. In lungs of wild-type (Cav-1(+/+)) mice perfused at constant flow with Krebs-Henseleit solution, addition of Cav-1(+/+) PMNs (4 x 10(6) cells) into the perfusate followed by their activation with formyl-Met-Leu-Phe (fMLP, 1.0 muM) plus platelet-activating factor (1.0 nM) increased pulmonary microvessel filtration coefficient by 150% and wet-to-dry lung weight ratio by 50% as well as PMN accumulation in lungs. These responses were markedly reduced in lungs perfused with Cav-1(-/-) PMNs followed by addition of the same activating agents. fMLP-stimulated adhesion of Cav-1(-/-) PMNs to pulmonary microvascular endothelial cells and migration of Cav-1(-/-) PMNs across endothelial monolayers were also impaired compared with Cav-1(+/+) PMNs. Cav-1(-/-) PMNs showed 50-80% reduction in PMA- or fMLP-stimulated superoxide production compared with Cav-1(+/+) PMNs. In addition, Cav-1(-/-) PMNs had decreased migratory activity (50%) and adhesion to fibrinogen (40%) in response to fMLP. Rac1 and Rac2 were activated in Cav-1(+/+) PMNs after stimulation of fMLP but not in Cav-1(-/-) PMNs. Exogenous expression of caveolin-1 in COS-phox cells augmented the fMLP-induced Rac1 activation and superoxide production, indicating a direct role of caveolin-1 in the mechanism of superoxide production. Thus caveolin-1 expression in PMNs plays a key role in mediating PMN activation, adhesion, and transendothelial migration and in PMN activation-induced lung inflammation and vascular injury.     

Pathogenicity of microbes associated with cystic fibrosis

Cystic fibrosis patients are exceptionally prone to colonisation by a narrow spectrum of pathogenic bacteria. Since pulmonary infection presently, and for the foreseeable future, plays such a major role in CF lung disease, we review the microbes that are classically associated with CF and the virulence, inflammatory potential and resistance mechanisms which contribute to the reduction in life expectancy for colonised CF patients.     

Neutrophil restraint by green tea: inhibition of inflammation,associated angiogenesis,and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (-)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.     

Anaerobic bacteria infection in cystic fibrosis airway disease

Depletion of the periciliary liquid in "Cystic Fibrosis" airway disease results in reduced mucociliary transport, persistent mucus hypersecretion and consequently increased height of the luminal mucus layer, so hypoxic gradients in the mucus plugs are developed. Because of anaerobic lung zones, it is highly probable that anaerobic bacteria not detected by routine bacteriologic culture methods also reside within the mucus. Notwithstanding this evidence, microbiology laboratories working in the cystic fibrosis field do not generally use strict anaerobic bacteriologic cultures to determine the presence of anaerobic bacteria in the Cystic Fibrosis lung. The aim of this review is to focus on the published data regarding the finding of anaerobic bacteria in cystic fibrosis airway disease. Therefore, microbiology, diagnosis, antimicrobial susceptibility and possible impact on clinical management of anaerobic bacteria lung infection in cystic fibrosis are described.     

Glucocorticoid receptor gene polymorphisms associated with progression of lung disease in young patients with cystic fibrosis

Background

The variability in the inflammatory burden of the lung in cystic fibrosis (CF) patients together with the variable effect of glucocorticoid treatment led us to hypothesize that glucocorticoid receptor (GR) gene polymorphisms may affect glucocorticoid sensitivity in CF and, consequently, may contribute to variations in the inflammatory response.

Methods

We evaluated the association between four GR gene polymorphisms, TthIII, ER22/23EK, N363S and BclI, and disease progression in a cohort of 255 young patients with CF. Genotypes were tested for association with changes in lung function tests, infection with Pseudomonas aeruginosa and nutritional status by multivariable analysis.

Results

A significant non-corrected for multiple tests association was found between BclI genotypes and decline in lung function measured as the forced expiratory volume in one second (FEV₁) and the forced vital capacity (FVC). Deterioration in FEV₁ and FVC was more pronounced in patients with the BclI GG genotype compared to the group of patients with BclI CG and CC genotypes (p = 0.02 and p = 0.04 respectively for the entire cohort and p = 0.01 and p = 0.02 respectively for F508del homozygous patients).

Conclusion

The BclI polymorphism may modulate the inflammatory burden in the CF lung and in this way influence progression of lung function.     

Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.     

Primary inflammation in human cystic fibrosis small airways

Most cystic fibrosis (CF) patients die of lung failure, due to the combined effects of bacterial infection, neutrophil-mediated inflammation, and airway obstruction by hyperviscous mucus. To this day, it remains unclear where and how this pathological vicious circle is initiated in vivo. In particular, it has proven difficult to investigate whether inflammatory pathways are dysregulated in CF airways independently of infection. Also, the relative involvement of large (tracheobronchial) vs. small (bronchiolar) airways in CF pathophysiology is still unclear. To help address these issues, we used an in vivo model based on the maturation of human fetal CF and non-CF small airways in severe combined immunodeficiency mice. We show that uninfected mature CF small airway grafts, but not matched non-CF controls, undergo time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clear-cut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection.     

Extracellular pH and lung infections in cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na⁺/K⁺ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.