Giovanna De Cunto

P2X7 receptor signaling in the pathogenesis of smoke-induced lung inflammation and emphysema.

Extracellular ATP is up-regulated in the airways of patients with chronic obstructive pulmonary disease, and may contribute to the pathogenesis of the disease. However, the precise mechanisms are poorly understood. Our objective was to investigate the functional role of the ATP receptor P2X(7) in the pathogenesis of cigarette smoke (CS)-induced lung inflammation and emphysema in vivo. Expression of the P2X(7) receptor (P2X(7)R) was measured in lung tissue und immune cells of mice with CS-induced lung inflammation. In a series of experiments using P2X(7) antagonists and genetically engineered mice, the functional role of the P2X(7)R in CS-induced lung inflammation was explored. CS-induced inflammation was associated with an up-regulation of the P2X(7)R on blood and airway neutrophils, alveolar macrophages, and in whole lung tissue. Selective intrapulmonary inhibition of the P2X(7)R reduced CS-induced lung inflammation and prevented the development of emphysema. Accordingly, P2X(7)R knockout mice showed a reduced pulmonary inflammation after acute CS exposure. Experiments with P2X(7)R chimera animals revealed that immune cell P2X(7)R expression plays an important role in CS-induced lung inflammation and emphysema. Extracellular ATP contributes to the development of CS-induced lung inflammation and emphysema via activation of the P2X(7)R. Inhibition of this receptor may be a new therapeutic target for the treatment of chronic obstructive pulmonary disease.

Purinergic Receptor Inhibition Prevents the Development of Smoke-Induced Lung Injury and Emphysema

Extracellular ATP acts as a “danger signal” and can induce inflammation by binding to purinergic receptors. Chronic obstructive pulmonary disease is one of the most common inflammatory diseases associated with cigarette smoke inhalation, but the underlying mechanisms are incompletely understood. In this study, we show that endogenous pulmonary ATP levels are increased in a mouse model of smoke-induced acute lung inflammation and emphysema. ATP neutralization or nonspecific P2R-blockade markedly reduced smoke-induced lung inflammation and emphysema. We detected an upregulation the purinergic receptors subtypes on neutrophils (e.g., P2Y2R), macrophages, and lung tissue from animals with smoke-induced lung inflammation. By using P2Y2R deficient (−/−) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y2R. Moreover, P2Y2R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y2R−/− and wild type chimera animals revealed that P2Y2R expression on hematopoietic cell plays the pivotal role in the observed effect. We demonstrate, for the first time, that endogenous ATP contributes to smoke-induced lung inflammation and then development of emphysema via activation of the purinergic receptor subtypes, such as P2Y2R.

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

BackgroundWe 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 (VV) of the inflammatory cells present in the lungs after chronic cigarette smoke exposure.MethodsSlides were obtained from blocks of the previous study and VV was 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.ResultsChronic smoke exposure increased neutrophil VV by 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 VV by 78%, macrophage by 82%, dendritic cell by 48%, B-lymphocyte by 100%, CD4+ by 98% and CD8+ VV by 88%. The lower dose of roflumilast did not prevent the increase in neutrophil, macrophage and B-cell VV but prevented dendritic cells by 42%, CD4+ by 55%, and CD8+ by 91%.ConclusionThese results indicate (i) 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; (ii) 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; (iii) these findings underline the role of innate immunity in the development of pulmonary emphysema and (iiii) 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.

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.

Early response of gene clusters is associated with mouse lung resistance or sensitivity to cigarette smoke.

We have investigated the effects of cigarette smoke exposure in three different strains of mice. DBA/2 and C57BL/6J are susceptible to smoke and develop different lung changes in response to chronic exposure, whereas ICR mice are resistant to smoke and do not develop emphysema. The present study was carried out to determine early changes in the gene expression profile of mice exposed to cigarette smoke with either a susceptible or resistant phenotype. The three strains of mice were exposed to smoke from three cigarettes per day, 5 days/wk, for 4 wk. Microarray analysis was carried out on total RNA extracted from the lung using the Affymetrix platform. Cigarette smoke modulates several clusters of genes (i.e., proemphysematous, acute phase response, and cell adhesion) in smoke-sensitive DBA/2 or C57BL/6J strains, but the same genes are not altered by smoke in ICR resistant mice. Only a few genes were commonly modulated by smoke in the three strains of mice. This pattern of gene expression suggests that the response to smoke is strain-dependent and may involve different molecular signaling pathways. Real-time quantitative PCR was used to verify the pattern of modulation of selected genes and their potential biological relevance. We conclude that gene expression response to smoke is highly dependent on the mouse genetic background. We speculate that the definition of gene clusters associated, to various degrees, with mouse susceptibility or resistance to smoke may be instrumental in defining the molecular basis of the individual response to smoke-induced lung injury in humans.

Differential thiol status in blood of different mouse strains exposed to cigarette smoke

C57Bl/6J, DBA/2 and ICR mouse strains are known to possess different susceptibilities to developing emphysema after exposure to cigarette smoke with DBA/2 and C57Bl/6J strains being significantly more susceptible to pulmonary damage than the ICR strain. This study was aimed at analysing the occurrence of systemic oxidative stress in the blood of these different mouse strains after exposure to cigarette smoke. This study did not observe a significant decrease in glutathione in erythrocytes or in plasma cysteine, cysteinylglycine, homocysteine and glutathione in C57Bl/6J or DBA/2 mice, whereas a significant increase in the corresponding oxidized forms was observed in plasma. However, the ICR strain showed a significant increase in glutathione in erythrocytes and a significant decrease in most of the oxidized forms of cysteine, cysteinylglycine, homocysteine and glutathione in plasma after the same exposition. These experiments demonstrate that exposure to cigarette smoking induces systemic oxidative stress only in some mouse strains which are susceptible to developing emphysema.

CX3CR1+ Cell–Mediated Salmonella Exclusion Protects the Intestinal Mucosa during the Initial Stage of Infection

During Salmonella Typhimurium infection, intestinal CX3CR1+ cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection, migrate into the intestinal lumen to capture bacteria. However, until now, the biological relevance of the intraluminal migration of CX3CR1+ cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent BALB/c mice. In contrast, in sampling-deficient/migration-deficient CX3CR1−/− mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1+ cells directly into the intestinal lumen, consistent with intraluminal CX3CR1+ cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial fecal load in CX3CR1+/gfp compared with CX3CR1gfp/gfp mice following oral infection. Furthermore, by using real-time in vivo imaging we observed that CX3CR1+ cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect Ab responses to a noninvasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1+ cell–based mechanism of immune exclusion is a defense mechanism against pathogens that complements the mucous and secretory IgA Ab–mediated system in the protection of intestinal mucosal surface.

Vulnerability and Genetic Susceptibility to Cigarette Smoke–Induced Emphysema in Mice

Cigarette smoke (CS) has been identified as the most important risk factor for the development of chronic obstructive pulmonary disease (COPD). Studies of cigarette/tobacco smoke–induced COPD have primarily utilized small laboratory animals, particularly inbred laboratory mice, as the model of choice. The first comprehensive review of animal models of emphysema relating to the pathogenesis of COPD was published in 2002 (1), and a more recent review provided updates on this topic (2). The use of this species has several advantages, including a relatively low cost, a rapid reproductive cycle, large litter sizes, and the availability of antibodies and molecular probes developed specifically for mouse research. In addition, many inbred strains and their mutants (transgenic and knockout) are available and a high overall genetic homology exists between the mouse and human genomes. Finally, mouse genes can be manipulated to create global as well as conditional mutants (using CRISPR/Cas and tissue-specific Cre transgenes). However, the mouse has limitations as an animal model of COPD in that it does not fully recapitulate the human disease. One should keep this limitation in mind when planning experiments and interpreting results obtained in this species (3). Importantly, mice are obligatory nose breathers, so they exhibit a different pattern of particle filtration in the nares and upper respiratory tract compared with humans. However, this does not invalidate the methods commonly used to expose mice to cigarette/tobacco smoke, i.e., by nose only and by whole body, because both approaches model second-hand smoke exposure. Moreover, mouse submucosal glands are restricted to the trachea, and mice lack a significant population of goblet cells in their bronchi and bronchioles. The appearance of clusters of goblet cells in their bronchi/bronchioles after cigarette/tobacco smoke exposure is likely due to metaplasia and not hyperplasia. Additionally, mice do not have respiratory bronchioles and therefore lack the anatomical basis for the development of centrilobular emphysema, a lesion of respiratory bronchioles. Instead, they develop patchy emphysema. Mice can be exposed to CS delivered by a smoking machine via nose-only (4) or whole-body (5) exposure. The types of cigarettes, chemical emissions, and particulates used, the number of cigarettes smoked per day, and the duration of exposure (in months) have not been standardized, so these aspects differ among laboratories. Generally, either reference (R series) cigarettes from the Kentucky Tobacco Research and Development Center at the University of Kentucky (Lexington, KY) or the commercially available Marlboro Red cigarettes are used. The latter have the advantage of being widely available and commonly used by smokers. CS studies have provided a fundamental understanding of the complex pathogenic cellular and molecular processes, including genetic/epigenetic mechanisms, that are involved in the development of COPD and the testing of potential therapeutics. Not all strains of mice develop emphysema after chronic CS exposure. In susceptible strains, the severity of emphysema is limited and does not reach the extent seen in human smokers or in other murine models of emphysema. The different susceptibilities to emphysema observed in three strains of mice have been linked to strain-dependent differences in antioxidant defenses in response to CS (6). ICR mice were shown to have increased lung antioxidant defenses (possibly Nrf2, glutathione, glutaredoxin-1, and peroxiredoxin-6) when acutely exposed to CS, whereas C57BL/6J and DBA2 mice did not (6). Of interest, C57BL/6J and DBA2 mice developed significant emphysema when exposed to CS for 6–7 months, whereas ICR mice did not (6). Chronic CS exposure causes other cellular alterations; for example, impaired ciliated epithelial cell function was observed in C57BL/6J mice exposed to whole-body CS for up to 1 year (7). Another study of several mouse strains (i.e., NZWLac/J, A/J, SJ/L, and AKR/J) confirmed the strain dependency response to CS and identified other emphysema-resistant or susceptible strains (8). However, the genetic basis for that variability has not been studied. Recently, a recombinant inbred strain of mice, referred to as the Collaborative Cross (CC), was derived from random mixing of eight different founder strains (9). This CC strain has a high phenotypic diversity that closely mimics human disparity/diversity, thus enhancing investigators’ ability to map the causative loci underlying complex disease-related traits (9). Future studies using the CC strain will provide a better understanding of tobacco/CS-induced COPD/emphysema models at the level of systems genetics. In this issue of the Journal, Radder and colleagues (pp. 367– 375) show that susceptibility to CS-induced emphysema as measured by alveolar chord length is a variable and continuous trait in 34 inbred strains of mice (10). They demonstrate various degrees of susceptibility, ranging from no response (resistant CBA/J) to extremely susceptible (A/J). The C57BL/6J mice were the second most susceptible to CS. By testing the association of this quantitative trait across the genome and then integrating mouse and human genome-wide scans, Radder and colleagues identified Abi3bp as a novel candidate gene contributing to emphysema susceptibility. This gene encodes for ABI family member 3 binding protein (also known as TARSH or eratin), a putative tumor suppressor that is predominantly expressed in the lung. Intriguingly, this protein controls the growth and differentiation of stem and tumor cells while it also promotes senescence in some cells via a p21-dependent pathway (11). Cellular senescence is one of several molecular mechanisms that have been shown to mediate COPD pathogenesis (12). The potential role of the Abi3bp gene in pulmonary emphysema requires more investigation in humans and laboratory animals (i.e., knockout mice for Abi3bp) to establish the mechanistic contribution of this gene to emphysema susceptibility. In addition to the study limitations identified by the authors, only female mice were used, and sex-specific differences may be important (13). Also, the authors were unable to detect any regions that strictly met multiple testing correction thresholds. Importantly, alveolar chord length is not a correlate/measure for emphysema, and emphysema is only one pathological entity of COPD. The moderate functional and anatomical responses to

NTPDase1/CD39 and aberrant purinergic signalling in the pathogenesis of COPD.

Purinergic receptor activation via extracellular ATP is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Nucleoside triphosphate diphosphohydrolase-1/CD39 hydrolyses extracellular ATP and modulates P2 receptor signalling. We aimed to investigate the expression and function of CD39 in the pathogenesis of cigarette smoke-induced lung inflammation in patients and preclinical mouse models. CD39 expression and soluble ATPase activity were quantified in sputum and bronchoalveolar lavage fluid (BALF) cells in nonsmokers, smokers and COPD patients or mice with cigarette smoke-induced lung inflammation. In mice, pulmonary ATP and cytokine concentrations, inflammation and emphysema were analysed in the presence or absence of CD39. Following acute cigarette smoke exposure CD39 was upregulated in BALF cells in smokers with further increases in COPD patients. Acute cigarette smoke exposure induced CD39 upregulation in murine lungs and BALF cells, and ATP degradation was accelerated in airway fluids. CD39 inhibition and deficiency led to augmented lung inflammation; treatment with ATPase during cigarette smoke exposure prevented emphysema. Pulmonary CD39 expression and activity are increased in COPD. CD39 deficiency leads to enhanced emphysema in mice, while external administration of a functional CD39 analogue partially rescues the phenotype. The compensatory upregulation of pulmonary CD39 might serve as a protective mechanism in cigarette smoke-induced lung damage. The upregulation of pulmonary ATPase is a protective mechanism in cigarette smoke-induced lung inflammation http://ow.ly/S5YcC

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.