Marco Failla

TGF-β1 targets the GSK-3β/β-catenin pathway via ERK activation in the transition of human lung fibroblasts into myofibroblasts

Transforming growth factor-beta1 (TGF-beta1) is known to induce the transition of human lung fibroblasts to myofibroblasts, a primary event in the pathogenesis of idiopathic pulmonary fibrosis. The molecular pathways involved in myofibroblast transformation are only partially identified. We found that a 24-h treatment with TGF-beta1 (10 ng/ml) induced alpha-smooth actin (SMA) expression and collagen production in human lung fibroblasts. These effects were abrogated by PD98059, a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway. TGF-beta1 treatment activated the MAPK pathway, as shown by an increased phosphorylation of extracellular-regulated kinases (ERK)1/2 after 30 min of exposure. TGF-beta1 also increased the expression of the Ser-9-phosphorylated inactive form of glycogen synthase kinase-3beta (GSK-3beta), an effect that was largely attenuated by PD98059. A nuclear translocation of beta-catenin in human lung fibroblasts was observed 2h after TGF-beta1 addition both by confocal microscopy and nuclear protein analysis. At this time, TGF-beta1 also increased the total levels of beta-catenin, an effect that was prevented by PD98059. Similarly to TGF-beta1, the GSK-3beta inhibitor lithium chloride (10mM), increased the total levels of beta-catenin and promoted alpha-SMA expression and collagen production. This study demonstrates that TGF-beta1 induces alpha-SMA expression and collagen production in human lung fibroblasts via ERK1/2 activation, GSK-3beta inhibition and nuclear beta-catenin translocation. The evidence that the silencing of beta-catenin by siRNAs was able to prevent the induction of alpha-SMA expression in TGF-beta1-treated fibroblasts further supports the hypothesis of a contribution of the GSK-3beta/beta-catenin pathway in the pathogenesis of idiopathic pulmonary fibrosis.

Inhibition or knock out of Inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury

BackgroundIn the present study, by comparing the responses in wild-type mice (WT) and mice lacking (KO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the development of on the lung injury caused by bleomycin administration. When compared to bleomycin-treated iNOSWT mice, iNOSKO mice, which had received bleomycin, exhibited a reduced degree of the (i) lost of body weight, (ii) mortality rate, (iii) infiltration of the lung with polymorphonuclear neutrophils (MPO activity), (iv) edema formation, (v) histological evidence of lung injury, (vi) lung collagen deposition and (vii) lung Transforming Growth Factor beta1 (TGF-β1) expression.MethodsMice subjected to intratracheal administration of bleomycin developed a significant lung injury. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from bleomycin-treated iNOSWT mice.ResultsThe intensity and degree of nitrotyrosine staining was markedly reduced in tissue section from bleomycin-iNOSKO mice. Treatment of iNOSWT mice with of GW274150, a novel, potent and selective inhibitor of iNOS activity (5 mg/kg i.p.) also significantly attenuated all of the above indicators of lung damage and inflammation.ConclusionTaken together, our results clearly demonstrate that iNOS plays an important role in the lung injury induced by bleomycin in the mice.

Pharmacological inhibition of leukotrienes in an animal model of bleomycin-induced acute lung injury

Leukotrienes are increased locally in idiopathic pulmonary fibrosis. Furthermore, a role for these arachidonic acid metabolites has been thoroughly characterized in the animal bleomycin model of lung fibrosis by using different gene knock-out settings.We investigated the efficacy of pharmacological inhibition of leukotrienes activity in the development of bleomycin-induced lung injury by comparing the responses in wild-type mice with mice treated with zileuton, a 5-lipoxygenase inhibitor and MK-571, a cys-leukotrienes receptor antagonist.Mice were subjected to intra-tracheal administration of bleomycin or saline and were assigned to receive either MK-571 at 1 mg/Kg or zileuton at 50 mg/Kg daily. One week after bleomycin administration, BAL cell counts, lung histology with van Gieson for collagen staining and immunohistochemical analysis for myeloperoxidase, IL-1 and TNF-α were performed.Following bleomycin administration both MK-571 and zileuton treated mice exhibited a reduced degree of lung damage and inflammation when compared to WT mice as shown by the reduction of:(i) loss of body weight, (ii) mortality rate, (iii) lung infiltration by neutrophils (myeloperoxidase activity, BAL total and differential cell counts), (iv) lung edema, (v) histological evidence of lung injury and collagen deposition, (vi) lung myeloperoxidase, IL-1 and TNF-α staining.This is the first study showing that the pharmacological inhibition of leukotrienes activity attenuates bleomycin-induced lung injury in mice. Given our results as well as those coming from genetic studies, it might be considered meaningful to trial this drug class in the treatment of pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.

Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

RationaleGap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition.MethodsWe investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF).ResultsHere we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF.ConclusionThese results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

16,16-Dimethyl Prostaglandin E2 Efficacy on Prevention and Protection from Bleomycin-Induced Lung Injury and Fibrosis

In this study, we evaluated the protective effect and therapeutic potential of the prostaglandin E(2) (PGE(2)) synthetic analog 16,16-dimethyl-PGE(2) (dmPGE(2)) in the animal model of pulmonary fibrosis induced by bleomycin. Mice subjected to intratracheal administration of bleomycin (1 mg/kg) received a dmPGE(2) dose of 30 microg/kg/day by continuous subcutaneous infusion. Bronchoalveolar lavage (BAL); immunohistochemical analysis for IL-1, TNF-alpha, and nitrotyrosine; measurement of fluid content in lung; myeloperoxidase activity assay; and lung histology were performed 1 week later. Lung histology and Sircol assay for collagen deposition were performed 3 weeks after treatments. Changes of body weight and survival rate were also evaluated at 1 and 3 weeks. Compared with bleomycin-treated mice, dmPGE(2) co-treated mice exhibited a reduced degree of body weight loss and mortality rate as well as of lung damage and inflammation, as shown by the significant reduction of: (1) lung infiltration by leukocytes; (2) myeloperoxidase activity; (3) IL-1, TNF-alpha, and nitrotyrosine immunostaining; (4) lung edema; and (5) histologic evidence of lung injury and collagen deposition. In a separate set of experiments, dmPGE(2) treatment was started 3 days after bleomycin administration, and the evaluation of lung damage and inflammation was assessed 4 days later. Importantly, delayed administration of dmPGE(2) also was able to protect from inflammation and lung injury induced by bleomycin. These results, indicating that dmPGE(2) is able to prevent and to reduce bleomycin-induced lung injury through its regulatory and anti-inflammatory properties, encourage further research to find new options for the treatment of pulmonary fibrosis.

Intranasal steroid reduces exhaled bronchial cysteinyl leukotrienes in allergic patients

Background Allergic rhinitis (AR) precedes and is often associated with bronchial asthma. Indeed, local and systemic inflammations in both conditions are very similar. Cysteinyl‐leukotrienes (cys‐LTs) are generated during early‐ and late‐phase allergic reactions and induce smooth‐muscle contraction, microvascular leakage, and mucous hypersecretion. Cys‐LTs are detected in exhaled breath condensate (EBC) of asthmatics and regardless of bronchial symptoms, they are also found in EBC of rhinitic patients.

Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells

BackgroundT lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts.MethodsHuman lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay.ResultsIn lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 ± 0.9 and 0.7 ± 0.15 to 9.1 ± 1.5 and 38.6 ± 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 ± 0.7, 18.9 ± 1.9 and 6.6 ± 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 ± 0.7, 9.4 ± 1.4 and 3.5 ± 1.0. CD3 expression in resting lymphocytes was 11.9 ± 1.4 and was significantly reduced by fibroblasts to 6.4 ± 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54,4% ± 6.12 to 30.8 ± 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 ± 0.02 nm to 0.16 ± 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation of LFA-1, CD28 and CD69.ConclusionFindings from this study suggest that fibroblasts play a role in the local regulation of the immune response, being able to modulate effector functions of cells recruited into sites of inflammation.

Exhaled bronchial cysteinyl leukotrienes in allergic patients.

Purpose of review To review the current knowledge of noninvasive monitoring of allergic airway inflammation by analysis of leukotrienes in the exhaled breath condensate. Recent findings Treatment of respiratory allergies involves chronic treatment based on clinical symptoms and pulmonary function tests. Evaluation of local inflammation would be desirable but is currently not feasible because of the difficulty in sampling the airways. Recently, exhaled breath condensate collection and analysis has polarized much interest in the respiratory field. Although some methodological issues are still under scrutiny, airways inflammatory markers can be assayed with this technique. In particular, exhaled breath condensate leukotrienes have been thoroughly investigated in the setting of bronchial asthma and allergic rhinitis in adults and children. Exhaled leukotrienes are increased in patients with asthma and rhinitis during the pollen season, correlate with exacerbations and asthma severity, and are reduced by specific anti-inflammatory treatment and allergen avoidance. Summary Some issues still prevent the use of exhaled breath condensate in clinical practice but in the research setting it has been proved to be useful for noninvasive monitoring of allergic inflammation in the lung. In particular, exhaled leukotrienes may represent valuable biomarkers for diagnostic and therapeutic purposes in allergic patients.