Altered histone deacetylase activity and iNOS expression in cells isolated from induced sputum of COPD patients treated with tiotropium.

Chronic obstructive pulmonary disease (COPD) is the only major disease with increasing death rate. In COPD, progressive reduction in quality of life is closely related to the increasing limitation of airflow due to chronic bronchitis, cell hyperplasia, fibrosis, and irreversible lung damage. Signaling pathways involved in inflammatory processes in COPD and inflammatory response to therapy are unknown. Our aim was to isolate cells from induced sputum of COPD patients treated with formoterol or formoterol + tiotropium and assess enzymatic activity of histone deacetylases (HDACs) acetylated histone 4 (AcH4) and expression of inducible nitric oxide synthase (iNOS). HDACs are important in signal transduction and inflammation. iNOS is generating nitric oxide (NO) relevant to blood pressure regulation, inflammation and infections. Thirty stable COPD patients (21 males and 9 females, mean age 67 years) receiving 12 µg b.i.d. formoterol were assayed before and after 3 months add-on therapy consisting of 18 µg q.i.d. tiotropium. In all patients, spirometry, lung volumes, and DLCO were performed before and after tiotropium therapy and all patients were subjected to sputum induction. Sputum cells were isolated and processed to obtain cytosolic and nuclear fractions. HDAC activity was measured in nuclear fraction using colorimetric assay. Expression AcH4 and iNOS was quantified using Western blot. In patients receiving both drugs, FEV1 and lung volumes significantly improved compared with formoterol-only treated patients. Mean HDAC activity was slightly decreased (P < 0.05), while AcH4 levels and iNOS expression were significantly elevated in tiotropium-treated patients (increase by about 65 %; P < 0.01 and 77 %; P < 0.01 respectively). Our data show that beneficial effects of tiotropium in add-on therapy to formoterol may be related to altered histone signaling and increased iNOS expression.

Siglec-8 in induced sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is related to infiltration and activation of inflammatory cells in airways and pulmonary tissue. In COPD, neutrophils are prominent, while eosinophilic influx is typical to asthma. Inflammatory cells express sialic acid-binding immunoglobulin like lectins called Siglecs, a family of innate immune receptors that are transmembrane I-type lectins binding sialic acid. One member of the Siglec family, Siglec-8, is expressed mostly in eosinophils and may be an important therapeutic target in asthma or COPD. The aim of our project was to quantify Siglec-8 expression in induced sputum cells of COPD patients treated with long-acting beta2-agonists (LABA) or combined with long-acting antimuscarinic agents (LAMA) - tiotropium bromide. Thirty stable COPD patients (21 males and 9 females, mean age 67 years) receiving 12 µg BID formoterol therapy were assessed before and after 3 months' add-on therapy consisting of 18 µg QID tiotropium. In all patients, spirometry, lung volumes, and DLCO were performed before and after therapy. The patients were subjected to sputum induction before and after therapy. Sputum cells were isolated and processed to obtain cell membranes. Siglec-8 protein expression was assessed using Western blot. In patients receiving tiotropium and formoterol, improved FEV1 and lung volumes were observed compared with formoterol-only treated patients. The mean Siglec-8 level was significantly higher in eosinophilic subgroup of COPD patients compared with non-eosinophilic patients before therapy 40,000 vs. 15,000 Adj. Vol. INT/mm(2). Our data show that Siglec-8 may be involved in COPD pathogenesis and may influence COPD phenotyping.

Tiotropium increases PPARγ and decreases CREB in cells isolated from induced sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and chronic inflammation of airways and lung parenchyma. Our aim was to assess two important elements of intracellular signaling involved in regulation of inflammation in COPD in patients subjected to long-acting beta2-agonist or long-acting beta2-agonist plus long-acting antimuscarinic: peroxisome proliferator-activated receptor gamma (PPARγ) protein, which has antiinflammatory and immunomodulatory properties and cAMP response element binding protein (CREB) and activated (CREB-P) protein which has histone acetyltransferase activity and increases histone acetylation and transcriptional activation of chromatin. Twenty one stable COPD patients (18 males and 3 females, mean age 65 years) receiving 12 µg B.I.D formoterol were assayed before and after 3 month add-on therapy, consisting of 18 µg Q.D. tiotropium. In all patients, sputum induction, spirometry, lung volumes, and DLCO were performed before and after therapy. Sputum cells were isolated and processed to isolate cytosolic and nuclear fractions. PPARγ, CREB, or CREB-P proteins were quantified in subcellular fractions using Western blot. Tiotropium add-on therapy improved respiratory parameters: FEV1 and lung volumes. After therapy mean expression of PPARγ in cell nuclei was significantly increased by about 180%, while CREB and phosphorylated CREB levels in cytosol and nuclei were decreased by about 30%. Our data show that the mechanism whereby tiotropium reduces exacerbations may be associated not only with persistent increase in airway functions and reduced hyperinflation mediated by muscarinic receptors, but also with possible anti-inflammatory effects of the drug, involving increased PPARγ and decreased CREB signaling.

Tiotropium increases cytosolic muscarinic M3 receptors and acetylated H3 histone proteins in induced sputum cells of COPD patients.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible progressive airflow limitation related to tobacco smoking. This limitation is caused by chronic inflammation of the airways and lung parenchyma and is associated with increased activity of parasympathetic system. The most effective bronchodilators in COPD are muscarinic receptor antagonists (MRA), which reverse, at least in part, compromised respiratory function. MRA also contribute to control inflammatory processes via interactions with inflammatory signaling molecules. The use of the long-acting cholinolytic bronchodilatator - tiotropium, with high affinity to M3 receptors, is suggested as a first line maintenance treatment in COPD patients. MATERIAL AND METHODS: In this study we assessed M3 receptor protein expression in induced sputum of 27 stable COPD patients before and after therapy consisting of 18 µg once daily tiotropium for 12 weeks. Lung function tests including spirometry, lung volumes, and DLCO were performed before and after therapy in all COPD patients. The patients were subjected to the sputum induction procedures before and after therapy. Sputum cells were isolated, sample-specific cell profiles were characterized, and the cells were processed to isolate pure cytosolic fractions. Cytosolic M3 protein and HDAC2 levels and nuclear acetylated histone H3 (AcH3) expression was quantified using specific antibodies against human proteins and Western blot with enhanced luminescence detection. RESULTS: Therapy significantly increased the mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) volume (P<0.05). The mean expression of M3 protein was higher by 37% after therapy (P<0.05), HDAC2 expression was not altered, while AcH3 level was increased by about 90% (P<0.01), compared with the corresponding data before therapy. HDAC2 expression before therapy was positively correlated with AcH3 expression (r = 0.74), while after therapy no correlation was detected. FEV1, FCV, and cytosolic M3 protein expression did not correlate with other biochemical parameters tested. CONCLUSIONS: Twelve weeks of tiotropium therapy in COPD patients improves clinical indices of lung function and involves alterations in sputum cell chromatin acetylation and also increased cholinergic M3 receptor internalization.