Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke

8openThe protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.openDe Cunto, Giovanna; Lunghi, Benedetta; Bartalesi, Barbara; Cavarra, Eleonora; Fineschi, Silvia; Ulivieri, Cristina; Lungarella, Giuseppe; Lucattelli, MonicaDE CUNTO, Giovanna; Lunghi, Benedetta; Bartalesi, Barbara; Cavarra, Eleonora; Fineschi, Silvia; Ulivieri, Cristina; Lungarella, Giuseppe; Lucattelli, Monic

Effect of roflumilast on inflammatory cells in the lungs of cigarette smoke-exposed mice

<p>Abstract</p> <p>Background</p> <p>We reported that roflumilast, a phosphodiesterase 4 inhibitor, given orally at 5 mg/kg to mice prevented the development of emphysema in a chronic model of cigarette smoke exposure, while at 1 mg/kg was ineffective. Here we investigated the effects of roflumilast on the volume density (V_V) of the inflammatory cells present in the lungs after chronic cigarette smoke exposure.</p> <p>Methods</p> <p>Slides were obtained from blocks of the previous study and V_Vwas assessed immunohistochemically and by point counting using a grid with 48 points, a 20× objective and a computer screen for a final magnification of 580×. Neutrophils were marked with myeloperoxidase antibody, macrophages with Mac-3, dendritic cells with fascin, B-lymphocytes with B220, CD4+ T-cells with CD4+ antibody, and CD8+T-cells with CD8-α. The significance of the differences was calculated using one-way analysis of variance.</p> <p>Results</p> <p>Chronic smoke exposure increased neutrophil V_Vby 97%, macrophage by 107%, dendritic cell by 217%, B-lymphocyte by 436%, CD4+ by 524%, and CD8+ by 417%. The higher dose of roflumilast prevented the increase in neutrophil V_Vby 78%, macrophage by 82%, dendritic cell by 48%, B-lymphocyte by 100%, CD4+ by 98% and CD8+ V_Vby 88%. The lower dose of roflumilast did not prevent the increase in neutrophil, macrophage and B-cell V_Vbut prevented dendritic cells by 42%, CD4+ by 55%, and CD8+ by 91%.</p> <p>Conclusion</p> <p>These results indicate (<it>i</it>) chronic exposure to cigarette smoke in mice results in a significant recruitment into the lung of inflammatory cells of both the innate and adaptive immune system; (<it>ii</it>) roflumilast at the higher dose exerts a protective effect against the recruitment of all these cells and at the lower dose against the recruitment of dendritic cells and T-lymphocytes; (<it>iii</it>) these findings underline the role of innate immunity in the development of pulmonary emphysema and (<it>iiii</it>) support previous results indicating that the inflammatory cells of the adaptive immune system do not play a central role in the development of cigarette smoke induced emphysema in mice.</p

Th2 immune response and respiratory bronchiolitis-associated interstitial lung disease in p66Shc KO smoking mice

Some interstitial lung diseases, namely Desquamative Interstitial Pneumonia, Respiratory Bronchiolitis-associated Interstitial Lung Disease (RB-ILD), and Pul- monary Langerhans cell Histiocytosis, are etiologically linked to cigarette smoke (CS). However, very little is known about the pathogenetic mechanisms and the reason why only a minority of tobacco smokers develop these diseases. The targeted deletion of p66Shc gene, involved in the modulation of antioxidant genes, confers to mice resistance to oxidative stress, extends life span and corre- lates with reduced levels of p53 dependent apoptosis. These mice exposed to CS develop changes similar to those characterizing human RB-ILD. In particular, p66Shc KO (p66Shc–/–) mice develop patchy lung changes with bronchiolocentric distribution characterized by an accumulation of pigmented macrophages within bronchiolar lumina and the adjacent alveoli, peribronchiolar infiltrates of histiocytes and lymphocytes (prevalently CD4+ T and B cells), and patchy areas of peribronchiolar and septal fibrosis. The alveolar macrophages in p66Shc–/– mice are often multinucleated and contain cytoplasmic granules which stain with Prussian blue. At electron microscopy, they show an activated pattern and large amount of siderosomes and residual bodies (aging pigments) in the cytoplasm. About 85-90% of p66Shc–/– macrophages show a Th2 pattern of activation with induction of arginase and chitinase, and suppression of iNOS. A prevailing Th2 dominated immune response in p66Shc–/– mice is confirmed by the presence of inflammatory cell positive for IL-4 and IL-13. These factors may be involved in the pathogenic mechanism(s) leading to RB-ILD

Two mouse models for studying smoke-related interstitial lung diseases

Rationale. Some subgroups of interstitial lung diseases, namely desquamative interstitial pneumonia (DIP), respiratory bronchiolitis-associated interstitial pneumonia (RB-ILD), and pulmonary Langerhans cell histiocytosis, are caused by cigarette smoke (CS) in susceptible individuals. Despite smoke-related interstitial lung diseases (SRILDs) have been subjected to extensive investigation during the last years, very little is know about the pathogenetic mechanisms of these diseases and why only a susceptible minority of tobacco smokers develop a clinically significant disease. Methods & Results. We observed that the combination of chronic CS exposure with targeted mutation of genes involved in the expression of factors important for adaptive immune responses (namely p56LcK), or for the modulation of antioxidant genes (such as p66Shc), results in the development of SRILDs. These mice mimic the histopathological traits, which characterise either DIP or RB—ILD in man. As in human RB-ILD, p66Shc KO mice develop after CS exposure patchy lung changes with a bronchiolocentric distribution characterized by pigmented macrophages within bronchiolar lumina, peribronchiolar inflammation and fibrosis. In p56LcK KO mice, chronic macrophage pneumonia is the main feature. Unlike the previous model, the alveolar septal thickening and inflammation in this DIP model tends to be diffuse and uniform from field to field. Species differences notwithstanding, the data obtained in our mice suggest that DIP cannot be regarded as a late stage of RB—ILD but may represent an earlier stage of cryptogenic fibrosing alveolitis. Conclusions. Further studies on these unique animal models may shed light on the pathogenetic mechanisms and the missing links existing among the various SRILDs and can provide valuable information on both the reversibility and the evolution of the lung changes characterising DIP and/or RB—ILD

Effect of roflumilast on inflammatory cell volume density in lungs of mice chronically exposed to cigarette smoke

Rationale: The phosphodiesterase 4 (PDE4) inhibitor roflumilast was previously shown to fully prevent emphysematous-like changes in lungs of mice that were chronically exposed to cigarette smoke. This study assessed the effect of roflu- milast on inflammatory cell volume density (VV) changes in lung tissue of mice chronically exposed to cigarette smoke. Methods: C57BL/6J mice were either exposed to room air (N=5) or cigarette smoke for 7 months (N = 5). Another group of animals (N = 5) exposed to cigarette smoke received roflumilast 5 mg/kg per os on 5 days/week for 7 months. VV was assessed immunohistochemically and by point counting in lung sections. Cells were labeled with specific antibodies: neutrophils with myeloperoxidase, macrophages with Mac-3, dendritic cells with fascin, B-lymphocytes with B220, CD4+ T-cells with CD4+ antibody. Results: In mice chronically exposed to cigarette smoke, VV of neutrophils, macrophages, dendritic cells, B-lymphocytes, and CD4+ T-cells increased 1.8- to more than 6-fold as compared to air exposure (all p < 0.01). Roflumilast did not affect baseline VV but prevented VV increases of neutrophils by 78% (p < 0.05), macrophages by 70%, dendritic cells by 48%, B-lymphocytes by 100%, and CD4+ T-cells by 98% (all p < 0.01). Conclusions: In this mouse model of chronic cigarette smoke exposure, increases in broad inflammatory cell populations involved in both the innate and adaptive immune responses were demonstrated. This study suggests that roflumilast may prevent cigarette smoke-induced emphysema development through inhibition of the recruitment of neutrophils, macrophages, and cells of the adaptive immune system

Preliminary conservation status assessment of cave-dwelling planarians (Platyhelminthes, Tricladida) of Italian Alps and Apennines.

Despite being a fundamental source of biodiversity, several highly diverse taxa of aquatic invertebrates, including Platyhelminthes, remain still poorly known and poorly considered in protection programs. This is the case especially of several invertebrate species that inhabit groundwater. In this environment, invertebrates play significant roles that are strictly connected to the usefulness of these systems for human survival. The groundwater biodiversity of continental Italy is largely unknown and its importance neglected in national and regional legislation. One of the most poorly studied group of the Italian groundwater fauna are planarians. Most of the known species are endemic of small single karst areas or a single cave, with their distribution being never investigated after the original description. The aims of this study are to provide an update conservation status assessment of cave-dwelling planarians known from Italian Alps and Apennines. In particular, we want to analyze the major threats for their survival and the factors determining the possibility to contact and observe these important invertebrates for the subterranean systems. From October 2016 to September 2017, we explored the known localities of nine taxa and a relevant number of surrounding caves. Our results suggest that most of the cave-dwelling planarians species of continental Italy are threatened by water pollution and habitat destruction; moreover, our research underlines that there is a large conservation issue dealing with stenoendemic planarians that may involve other cave-dwelling invertebrates with narrow ranges. Generally, the underground habitat of most species appears to be deeply compromised and changed since species description

Roflumilast prevents the increase in inflammatory cells in the lungs of mice chronically exposed to cigarette smoke: an immunohistochemical study

RATIONALE We have previously reported that roflumilast, an investigational phosphodiesterase 4 (PDE4) inhibitor, fully prevented the development of emphysema in C57BL/6J mice chronically exposed to cigarette smoke (AJRCCM 2005; 172: 848-53). In the present study, we investigated the effect of roflumilast on changes in inflammatory cell volume density (VV) in mice exposed to cigarette smoke for 7 months. METHODS C57BL/6J mice were either exposed to room air (N=5) or cigarette smoke for 7 months (N=5). Additionally, a group of cigarette smoke exposed animals received roflumilast 5 mg/kg per os (N=5). VV was assessed immunohistochemically and by point counting. Neutrophils were marked with myeloperoxidase antibody, macrophages with Mac-3 antibody, dendritic cells with fascin antibody, B-lymphocytes with B220 antibody, and CD4+ T-cells with CD4+ antibody. RESULTS Chronic cigarette smoke exposure of mice, as compared to air exposure, increased neutrophil VV (2.3±0.3 x 10-4 mL/g vs 1.2±0.2 x 10-4 mL/g, p<0.01), macrophage VV (14.4±0.4 x 10-4 mL/g vs 8.0±0.5 x 10-4 mL/g, p<0.01), dendritic cell VV (6.1±0.4 x 10-4 mL/g vs 1.9±0.3 x 10-4 mL/g, p<0.01), B-lymphocyte VV (3.4±0.4 x 10-4 mL/g vs 0.6±0.3 x 10-4 mL/g, p<0.01), and CD4+ T-cell VV (6.7±1.0 x 10-4 mL/g vs 1.1±0.3 x 10-4 mL/g, p<0.01). Roflumilast prevented the increase in neutrophil VV by 78% (p<0.05), macrophage VV by 70% (p<0.01), dendritic cell VV by 48% (p<0.01), B-lymphocyte VV by 100% (p<0.01), and CD4+ T-cell VV by 98% (p<0.01). CONCLUSIONS These results indicate that chronic cigarette smoke exposure in mice elicits an increase of cells involved in both the innate and adaptive immune responses. Roflumilast administration significantly prevented the recruitment of these cells

Effects of cigarette smoke on lung antioxidant defences, induction of goblet cell metaplasia and micropapillomatosis in DBA/2 and ICR mice

The role of strain differences in the response to cigarette smoke was investigated in mice. Basal total antioxidant capacity of bronchoalveolar lavage (BAL) fluids assessed as “Trolox equivalent antioxidant capacity” in DBA/2 mice was 61±12 nmolTrolox/ml BAL. After acute exposure to cigarette smoke (5 cigarettes within 20 min) the total BAL fluids antioxidant capacity of the DBA/2 mice decreased by 32% (p<0.01). Basal total antioxidant capacity of BAL fluids in ICR mice was similar to that of the DBA/2 mice (63±22 nmolTrolox/ml BAL). However, after acute cigarette smoke ICR mice increased their BAL antioxidant capacity by 57 % (p<0.01). Mice of these two strains were then exposed either to room air or to chronic cigarette smoke (3 cigarettes/day, 5 days/week) for 7 months. The animals were then sacrificed, the lungs stained with periodic acid-Schiff and the peripheral airways examined. The airways of both strains exposed to room air were normal. The peripheral airways of ICR mice exposed to cigarette smoke (N=5) showed no abnormalities and their epithelium was devoid of goblet cells. All DBA/2 mice exposed to cigarette smoke (N=8) exhibited a large number of goblet cells (goblet cell metaplasia) in their peripheral airways. Additionally, 7/8 mice had a diffuse micropapillomatosis in some cases with infiltrating character. All these results indicate that the response and sensitivity to the effects of cigarette smoke is strain-dependent