Immunohistochemical Study of Airways Fibrous Remodeling in Smoking Mice.

The fibrotic remodeling in chronic obstructive pulmonary disease (COPD) is held responsible for narrowing of small airways and thus for disease progression. Oxidant damage and cell senescence factors are recently involved in airways fibrotic remodeling. Unfortunately, we have no indications on their sequential expression at anatomical sites in which fibrotic remodeling develops in smoking subjects. Using immunohistochemical techniques, we investigated in two strains of mice after cigarette smoke (CS) exposure what happens at various times in airway areas where fibrotic remodeling occurs, and if there also exists correspondence among DNA damage induced by oxidants, cellular senescence, the presence of senescence-secreted factors involved in processes that affect transcription, metabolism as well as apoptosis, and the onset of fibrous remodeling that appears at later times in mice exposed to CS. A clear positivity for fibrogenic cytokines TGF-ß, PDGF-B, and CTGF, and for proliferation marker PCNA around airways that will be remodeled is observed in both strains. Increased expression of p16ink4A senescence marker and MyoD is also seen in the same areas. p16ink4A and MyoD can promote cell cycle arrest, terminal differentiation of myofibroblasts, and can oppose their dedifferentiation. Of interest, an early progressive attenuation of SIRT-1 is observed after CS exposure. This intracellular regulatory protein can reduce premature cell senescence. These findings suggest that novel agents, which promote myofibroblast dedifferentiation and/or the apoptosis of senescent cells, may dampen progression of airway changes in smoking COPD subjects.

Smoking Cessation in Mice Does Not Switch off Persistent Lung Inflammation and Does Not Restore the Expression of HDAC2 and SIRT1.

Once COPD is established, pulmonary lesions can only progress and smoking cessation by itself is not sufficient to switch off persistent lung inflammation. Similarly, in former-smoker mice, neutrophil inflammation persists and lung lesions undergo progressive deterioration. The molecular mechanisms underlying disease progression and the inefficiency of smoking cessation in quenching neutrophilic inflammation were studied in male C57 Bl/6 mice after 6 months of rest from smoking cessation. As compared with the mice that continued to smoke, the former-smoker mice showed reduced expression of histone deacetylases HDAC2 and SIRT1 and marked expression of p-p38 MAPK and p-Ser10. All these factors are involved in corticosteroid insensitivity and in perpetuating inflammation. Former-smoker mice do show persistent lung neutrophilic influx and a high number of macrophages which account for the intense staining in the alveolar structures of neutrophil elastase and MMP-9 (capable of destroying lung scaffolding) and 8-OHdG (marker of oxidative stress). "Alarmins" released from necrotic cells together with these factors can sustain and perpetuate inflammation after smoking cessation. Several factors and mechanisms all together are involved in sustaining and perpetuating inflammation in former-smoker mice. This study suggests that a better control of COPD in humans may be achieved by precise targeting of the various molecular mechanisms associated with different phenotypes of disease by using a cocktail of drug active toward specific molecules.

Lung ultrasound features and relationships with respiratory mechanics of evolving BPD in preterm rabbits and human neonates.

Evolving bronchopulmonary dysplasia (BPD) is characterized by impaired alveolarization leading to lung aeration inhomogeneities. Hyperoxia-exposed preterm rabbits have been proposed to mimic evolving BPD; therefore, we aimed to verify if this model has the same lung ultrasound and mechanical features of evolving BPD in human neonates. Semiquantitative lung ultrasound and lung mechanics measurement was performed in 25 preterm rabbits (28 days of gestation) and 25 neonates (mean gestational age ≈ 26 wk) with evolving BPD. A modified rabbit lung ultrasound score (rLUS) and a validated neonatal lung ultrasound score (LUS) were used. Lung ultrasound images were recorded and evaluated by two independent observers blinded to each other's evaluation. Lung ultrasound findings were equally heterogeneous both in rabbits as in human neonates and encompassed all the classical lung ultrasound semiology. Lung ultrasound and histology examination were also performed in 13 term rabbits kept under normoxia as further control and showed the absence of ultrasound and histology abnormalities compared with hyperoxia-exposed preterm rabbits. The interrater absolute agreement for the evaluation of lung ultrasound images in rabbits was very high [ICC: 0.989 (95%CI: 0.975-0.995); P < 0.0001], and there was no difference between the two observers. Lung mechanics parameters were similarly altered in both rabbits and human neonates. There were moderately significant correlations between airway resistances and lung ultrasound scores in rabbits (ρ = 0.519; P = 0.008) and in neonates (ρ = 0.409; P = 0.042). In conclusion, the preterm rabbit model fairly reproduces the lung ultrasound and mechanical characteristics of preterm neonates with evolving BPD.NEW & NOTEWORTHY We have reported that hyperoxia-exposed preterm rabbits and human preterm neonates with evolving BPD have the same lung ultrasound appearance, and that lung ultrasound can be fruitfully applied on this model with a brief training. The animal model and human neonates also presented the same relationship between semiquantitative ultrasound-assessed lung aeration and airway resistances. In conclusion, this animal model fairly reproduce evolving BPD as it is seen in clinical practice.

Alveolar Macrophage Phenotype and Compartmentalization Drive Different Pulmonary Changes in Mouse Strains Exposed to Cigarette Smoke.

COPD can manifest itself with different clinical phenotypes characterized by different disease progression and response to therapy. Although a remarkable number of studies have been carried out, little is known about the mechanisms underlying phenotypes that could guide the development of viable future therapies. Several murine strains mirror some human phenotypes after smoke exposure. It was of interest to investigate in these strains whether different pattern of activation of macrophages, and their distribution in lungs, is associated to changes characterizing different phenotypes. We chose C57Bl/6, and Lck deficient mice, which show significant emphysema, DBA/2 mice that develop changes similar to those of "pulmonary fibrosis/emphysema syndrome", p66Shc ko mice that develop bronchiolitis with fibrosis but not emphysema, and finally ICR mice that do not develop changes at 7 months after smoke exposure. Unlike other strains, ICR mice show very few activated macrophages (Mac-3 positive) mostly negative to M1 or M2 markers. On the other hand, a large population of M1 macrophages predominates in the lung periphery of DBA/2, C57Bl/6 and in Lck deficient mice, where emphysema is more evident. M2 macrophages are mainly observed in subpleural and intraparenchymal areas of DBA/2 mice and around bronchioles of p66Shc ko mice where fibrotic changes are present. We observed slight but significant differences in mRNA expression of iNOS, ECF-L, arginase 1, IL-4, IL-13 and TGF-ß between air- and smoke-exposed mice. These differences together with the different compartmentalization of macrophages may offer an explanation for the diversity of lesions and their distribution that we observed among the strains.

Innate Immunity and Cell Surface Receptors in the Pathogenesis of COPD: Insights from Mouse Smoking Models.

Chronic obstructive pulmonary disease (COPD) is mainly associated with smoking habit. Inflammation is the major initiating process whereby neutrophils and monocytes are attracted into the lung microenvironment by external stimuli present in tobacco leaves and in cigarette smoke, which promote chemotaxis, adhesion, phagocytosis, release of superoxide anions and enzyme granule contents. A minority of smokers develops COPD and different molecular factors, which contribute to the onset of the disease, have been put forward. After many years of research, the pathogenesis of COPD is still an object of debate. In vivo models of cigarette smoke-induced COPD may help to unravel cellular and molecular mechanisms underlying the pathogenesis of COPD. The mouse represents the most favored animal choice with regard to the study of immune mechanisms due to its genetic and physiological similarities to humans, the availability of a large variability of inbred strains, the presence in the species of several genetic disorders analogous to those in man, and finally on the possibility to create models "made-to-measure" by genetic manipulation. The review outlines the different response of mouse strains to cigarette smoke used in COPD studies while retaining a strong focus on their relatability to human patients. These studies reveal the importance of innate immunity and cell surface receptors in the pathogenesis of pulmonary injury induced by cigarette smoking. They further advance the way in which we use wild type or genetically manipulated strains to improve our overall understanding of a multifaceted disease such as COPD. The structural and functional features, which have been found in the different strains of mice after chronic exposure to cigarette smoke, can be used in preclinical studies to develop effective new therapeutic agents for the different phenotypes in human COPD.

Functional contribution of sphingosine-1-phosphate to airway pathology in cigarette smoke-exposed mice.

BACKGROUND AND PURPOSE: A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH: C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS: Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS: S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.

Ongoing Lung Inflammation and Disease Progression in Mice after Smoking Cessation: Beneficial Effects of Formyl-Peptide Receptor Blockade.

The most important risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoking. Until now, smoking cessation (SC) is the only treatment effective in slowing down the progression of the disease. However, in many cases SC may only relieve the airflow obstruction and inflammatory response. Consequently, a persistent lung inflammation in ex-smokers is associated with progressive deterioration of respiratory functions. This is an increasingly important clinical problem whose mechanistic basis remains poorly understood. Available therapies do not adequately suppress inflammation and are not able to stop the vicious cycle that is at the basis of persistent inflammation. In addition, in mice after SC an ongoing inflammation and progressive lung deterioration is observed. After 4 months of smoke exposure mice show mild emphysematous changes. Lung inflammation is still present after SC, and emphysema progresses during the next 6-month period of observation. Destruction of alveolar walls is associated with airways remodeling (goblet cell metaplasia and peribronchiolar fibrosis). Modulation of formyl-peptide receptor signaling with antagonists mitigates inflammation and prevents deterioration of lung structures. This study suggests an important role for N-formylated peptides in the progression and exacerbation of COPD. Modulating formyl-peptide receptor signal should be explored as a potential new therapy for COPD.

Ajulemic acid exerts potent anti-fibrotic effect during the fibrogenic phase of bleomycin lung.

BACKGROUND: Ajulemic acid (AjA) is a synthetic analogue of tetrahydrocannabinol that can prevent and limit progression of skin fibrosis in experimental systemic sclerosis. In this study we investigated whether AjA also prevents and modulates lung fibrosis induced by bleomycin (BLM) when administered in mice during the inflammatory or the fibrogenic phase of the model. METHODS: The anti-inflammatory and antifibrotic efficacy of AjA was evaluated in DBA/2 mice treated orally once a day starting either at day 0 (preventive treatment) or at day 8 (therapeutic treatment) after a single intratracheal instillation of BLM. AjA was given at a dose of 1 mg/kg or 5 mg/kg. Mice were sacrificed at day 8, 14 and 21 after BLM and lungs were processed for histology and morphometry, and examined for HO-proline content and for the expression of transforming growth factor beta 1 (TGF-ß1), phosphorylated Smad2/3 (pSMAD2/3), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and peroxisome proliferator-activated receptor-gamma (PPAR-γ). RESULTS: In the 1st week after BLM challenge, an acute inflammation characterized by neutrophil and macrophage accumulation was the main change present in lung parenchyma. The "switch" between inflammation and fibrosis occurs between day 8 and 14 after BLM instillation and involves the bronchi and vasculature. In the subsequent week (at day 21 after BLM instillation) bronchiolocentric fibrosis with significant increase of tissue collagen develops. The fibrotic response evaluated by morphometry and quantified as HO-proline in lung tissue at day 21 after BLM treatment was significantly reduced in mice receiving either AjA in the inflammatory or in early fibrogenic phase. AjA induces marked change in the expression pattern of products implicated in fibrogenesis, such as TGF-ß1, pSMAD2/3, CTGF and α-SMA. In addition, AjA increases significantly the number of PPAR-γ positive cells and its nuclear localization. CONCLUSIONS: AjA treatment, starting either at day 0 or at day 8 after BLM challenge, counteracts the progression of pulmonary fibrosis. The anti-fibrotic effectiveness of AjA is irrespective of timing of compound administration. Further clinical studies are necessary to establish whether AjA may represent a new therapeutic option for treating fibrotic lung diseases.

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.

The 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.

Histopathological data of iron and calcium in the mouse lung after asbestos exposure.

This data article contains data related to the research article entitled, "Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice" [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice.

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (µ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, µ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

Smoking p66Shc knocked out mice develop respiratory bronchiolitis with fibrosis but not emphysema.

The adaptor protein p66Shc regulates intracellular oxidant levels through the modulation of a forkhead-related transcription factor (FOXO3a). The genetic ablation of p66Shc (p66Shc-/-) renders mice resistant to oxidative stress and p53-dependent apoptosis. We investigated whether p66Shc ablation in mice modifies lung cellular and molecular responses to cigarette smoke (CS) exposure. No differences between wild type (WT) and p66Shc-/- mice were observed in terms of inflammation and oxidant burden after acute CS exposure; however,p66Shc ablation modifies specific features of chronic inflammation induced by repeated exposure to CS. Unlike WT mice, p66Shc-/- mice did not develop emphysema, showing protection toward oxidative damage to DNA and apoptosis as revealed by a trivial 8-hydroxyguanosine staining and faint TUNEL and caspase-3 positivity on alveolar epithelial cells. Unexpectedly, CS exposure in p66Shc-/- mice resulted in respiratory bronchiolitis with fibrosis in surrounded alveoli. Respiratory bronchiolitis was characterized by peribronchiolar infiltrates of lymphocytes and histiocytes, accumulation of ageing pigmented macrophages within and around bronchioles, and peribronchiolar fibrosis. The blockage of apoptosis interferes with the macrophage "clearance" from alveolar spaces, favouring the accumulation of aging macrophages into alveoli and the progressive accumulation of iron pigment in long-lived senescent cells. The presence of areas of interstitial and alveolar fibrosis in peripheral parenchyma often accompanied the bronchiolar changes. Macrophages from smoking p66Shc-/- mice elaborate M2 cytokines (i.e., IL-4 and IL-13) and enzymes (i.e., chitinase and arginase I), which can promote TGF-beta expression, collagen deposition, and fibrosis in the surrounding areas. We demonstrate here that resistance to oxidative stress and p53-dependent apoptosis can modify tissue responses to CS caused by chronic inflammation without influencing early inflammatory response to CS exposure.

Absence of proteinase-activated receptor-1 signaling in mice confers protection from fMLP-induced goblet cell metaplasia.

The morphological features of chronic obstructive pulmonary disease in man include emphysema and chronic bronchitis associated with mucus hypersecretion. These alterations can be induced in mice by a single intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a chemoattractant and degranulating agent for neutrophils. The mechanisms underlying excessive mucus production and, in particular, goblet cell hyperplasia/metaplasia in chronic obstructive pulmonary disease remain poorly understood. The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties during inflammation. In this study, we examined whether PAR-1 contributes to inflammation and lung damage induced by fMLP by comparing the response of PAR-1-deficient (PAR-1(-/-)) mice with that of wild-type (WT) mice. Mice were killed at various time points after fMLP instillation (200 microg/50 microl). WT mice developed emphysema and goblet cell metaplasia. The onset of pulmonary lesions was preceded by an increase in thrombin immunoreactivity in bronchial airways and alveolar tissue. This was followed by a decrease in PAR-1 immunoreactivity, and by an increase in IL-13 immunostaining on the luminal surface of airway epithelial cells. In PAR-1(-/-) mice, fMLP administration induced similar responses in terms of inflammation and emphysema, but these mice were protected from the development of goblet cell metaplasia. The involvement of PAR-1 in airway epithelial cell transdifferentiation was confirmed by demonstrating that intratracheal instillation of the selective PAR-1 agonist (TFLLR) induced goblet cell metaplasia in the airways of WT mice only. These data suggest that emphysema and goblet cell metaplasia occur independently, and that PAR-1 signaling through IL-13 stimulation may play an important role in inducing goblet cell metaplasia.

Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models.

BACKGROUND: The separation of emphysema from fibrosis is not as clear-cut as it was thought in early studies. These two pathologies may be present at the same time in human lungs and in mice either instilled with elastolytic enzymes or bleomycin or exposed to cigarette-smoke. According to a current view, emphysema originates from a protease/antiprotease imbalance, and a role for antiproteases has also been suggested in the modulation of the fibrotic process. In this study we investigate in experimental animal models of emphysema and fibrosis whether neutrophil elastase may constitute a pathogenic link between these two pathologies. METHODS: This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarette-smoke. In order to assess the protease-dependence of the BLM-induced lesion, a group mice was treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active toward neutrophil elastase. Lungs from each experimental group were used for the immunohistochemical assessment of transforming growth factor-beta (TGF-beta) and transforming growth factor-alpha (TGF-alpha) and for determination of the mean linear intercept as well as the percent volume densities of fibrosis and of emphysematous changes. Additionally, the lungs were also assessed for desmosine content and for the determination of elastase levels in the pulmonary interstitium by means of immunoelectron microscopy. RESULTS: We demonstrate that in BLM-treated mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) significant amount of elastase in alveolar interstitium is associated with an increased expression of TGF-beta and TGF-alpha; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase. Also, in a strain of mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is associated with a positive immunohistochemical reaction for reaction for both TGF-beta and TGF-alpha. CONCLUSION: The results of the present study strongly suggest that neutrophil elastase may represent a common pathogenic link between emphysema and fibrosis. Proteases and in particular neutrophil elastase could act as regulatory factors in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both.

Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs.

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.