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

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-β1), 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-β1, 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

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 CX3CR1(gfp/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

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

Chronic Exposure to Cigarette Smoke Induces Pulmonary Hypertension and Vascular Remodelling in Mice Over−Expressing Protease−Activated Receptor−2 (PAR−2).

INTRODUCTION: The mechanism(s) involved in the development of pulmonary hypertension (PH) in chronic obstructive pulmonary disease is still object of research. Recent studies indicate that cigarette smoke (CS) may have, at least in some individuals, a direct effect on the intrapulmonary vessels with up−regulation of mediators that lead to vascular structural remodelling and dynamic changes in vascular function. A role for proteases in PH has been recently put forward. In the present study, we examined the role of PAR−2 in the pathogenesis of lung vascular remodelling induced in mice by chronic exposure to CS. METHODS: Mice were exposed to the smoke of 3 cigarettes / day, 5 days / week. At various times, lungs were analysed by morphology, morphometry, immunohistochemistry and molecular biology techniques. Ventricular pressure was measured by using a miniature catheter. RESULTS: After 7 months of CS exposure, FVB mice over−expressing PAR−2 developed emphysema associated with PH (~ 45% increase in mean right ventricular pressure). A marked vascular remodelling of the middle and small intrapulmonary vessels preceded the onset of PH. No vascular changes were seen in WT mice, which develop only emphysema after chronic exposure to CS. The effect of tobacco smoke in transgenic PAR−2 mice resulted in an imbalance between vasoconstrictors (especially ET−1) and vasodilators (i.e. VEGF, eNOS and iNOS) and enhanced production of growth factors involved in fibroblast−SMC transaction (PDGF and TGFb) and vascular cell proliferation (PDGF). CONCLUSIONS: PAR−2 signalling can influence the production and the release of many of these factors, which ultimately lead to vascular remodelling and aberrant vascular physiology. The level of expression of PAR−2 may play an important role in the development of PH in human smoker