Isabel Vega-Carrascal

Dysregulation of TIM-3–Galectin-9 Pathway in the Cystic Fibrosis Airways

The T-cell Ig and mucin domain-containing molecules (TIMs) have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. It has been reported that proinflammatory cytokine dysregulation and neutrophil-dominated inflammation are the main causes of morbidity in cystic fibrosis (CF). However, the role of TIM receptors in CF has not been investigated. In this study, we demonstrated that TIM-3 is constitutively overexpressed in the human CF airway, suggesting a link between CF transmembrane conductance regulator (CFTR) function and TIM-3 expression. Blockade of CFTR function with the CFTR inhibitor-172 induced an upregulation of TIM-3 and its ligand galectin-9 in normal bronchial epithelial cells. We also established that TIM-3 serves as a functional receptor in bronchial epithelial cells, and physiologically relevant concentrations of galectin-9 induced TIM-3 phosphorylation, resulting in increased IL-8 production. In addition, we have demonstrated that both TIM-3 and galectin-9 undergo rapid proteolytic degradation in the CF lung, primarily because of neutrophil elastase and proteinase-3 activity. Our results suggest a novel intrinsic defect that may contribute to the neutrophil-dominated immune response in the CF airways.

Galectin-9 Signaling through TIM-3 Is Involved in Neutrophil-Mediated Gram-Negative Bacterial Killing: An Effect Abrogated within the Cystic Fibrosis Lung

The T cell Ig and mucin domain–containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.

A novel neutrophil derived inflammatory biomarker of pulmonary exacerbation in cystic fibrosis

BACKGROUND The focus of this study was to characterize a novel biomarker for cystic fibrosis (CF) that could reflect exacerbations of the disease and could be useful for therapeutic stratification of patients, or for testing of potential drug treatments. This study focused exclusively on a protein complex containing alpha-1 antitrypsin and CD16b (AAT:CD16b) which is released into the bloodstream from membranes of pro-inflammatory primed neutrophils. METHODS Neutrophil membrane expression and extracellular levels of AAT and CD16b were quantified by flow cytometry, Western blot analysis and by 2D-PAGE. Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and AAT:CD16b complex were quantified in CF plasma (n=38), samples post antibiotic treatment for 14 days (n=10), chronic obstructive pulmonary disease (n=10), AAT deficient (n=10) and healthy control (n=14) plasma samples by ELISA. RESULTS Cell priming with IL-8 and TNF-alpha caused release of the AAT:CD16b complex from the neutrophil cell membrane. Circulating plasma levels of IL-8, TNF-alpha and AAT:CD16b complex were significantly higher in patients with CF than in the other patient groups or healthy controls (P<0.05). Antibiotic treatment of pulmonary exacerbation in patients with CF led to decreased plasma protein concentrations of AAT:CD16b complex with a significant correlation with improved FEV1 (r=0.81, P=0.003). CONCLUSION The results of this study have shown that levels of AAT:CD16b complex present in plasma correlate to the inflammatory status of patients. The AAT:CD16b biomarker may become a useful addition to the clinical diagnosis of exacerbations in CF.

The role of TIM-containing molecules in airway disease and their potential as therapeutic targets

T cell immunoglobulin and mucin-domain (TIM)-containing molecules have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. Despite the initial discovery linking TIM-containing molecules and the airway hyperreactivity regulatory locus in mice, there is a paucity of studies on the function of TIM-containing molecules in lung inflammatory disease. Initially, studies were limited to mice models of asthma. More recently however, TIM-containing molecules have been implicated in an ever-expanding list of airway conditions that includes pneumonia, tuberculosis, influenza, sarcoidosis, lung cancer, and cystic fibrosis. This present review discusses the role of TIM-containing molecules and their ligands in the lung, as well as their potential as therapeutic targets in airway disease.

141 TIM-3 is required for neutrophil mediated Gram-negative bacterial killing: an effect abrogated within the cystic fibrosis lung

Introduction: Sputum and blood inflammatory markers, including MMP-9, TNFalpha and IL-8, are elevated in CF and these same biomarkers increase during pulmonary exacerbations. In this prospective study, we examine whether urinary inflammatory biomarkers are detectable and reflect clinical status in CF adults. Methods: Subjects were recruited at our regional centre, data included: demographics, spirometry and clinical status (‘stable’ or ‘exacerbation’). Subjects were chronically infected with P. aeruginosa. Pulmonary exacerbations were defined according to the Fuchs criteria. Urinary MMP-9, TIMP-1, TIMP-2, neutrophil elastase, alpha-1-antitrypsin and neutrophil gelatinase-associated lipocalin (NGAL) were measured. Results: 14 subjects (8 male) had provided 24 urine samples at the time of abstract submission, FEV1% predicted (mean±SD) 49±14.2%. A1AT was significantly higher in ‘stable’ compared to ‘exacerbation’ samples [535.0 (210.1–653.1) ng/ml vs. 86.0 (32.5–185.3) ng/ml, p< 0.006]. TIMP-2/A1AT ratio [0.0095 (0.0055– 0.019) vs. 0.046 (0.022–0.115), p = 0.013], TIMP-1/A1AT ratio [0.002 (0.002– 0.003) vs. 0.01 (0.002–0.04), p = 0.03] and NGAL/A1AT ratio [0.129 (0.01−0.3) vs. 0.535 (0.23–1.07), p = 0.04] were significantly lower in ‘stable’ compared to ‘exacerbation’ samples. All other inflammatory markers tested were detected, but no significant differences were seen in this preliminary analysis. Conclusions: We demonstrate for the first time that relevant inflammatory biomarkers are present in the urine of CF adults. The assessment of urinary inflammatory markers may prove to be a useful non-invasive method to diagnose pulmonary exacerbations, as well as to assess treatment response.