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.

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.

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.

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.

Middermal Elastolysis: Dermal Fibroblasts Cooperate with Inflammatory Cells to the Elastolytic Disorder.

Little is known about the cause and pathophysiology of middermal elastolysis (MDE). In this condition, variable inflammatory infiltrate may be present or not together with loss of elastic fibres in the middermis that spares both papillary and lower reticular dermis. MDE may be a consequence of abnormal extracellular matrix degradation related to an imbalance between elastolytic enzymes released from inflammatory and resident cells and their naturally occurring inhibitors. However, the cause of this imbalance is still an object of investigation. In order to shed light on the role of fibroblasts in MDE, we used fibroblast cultures from MDE and control subjects to evaluate matrix metalloproteinases (MMPs) and their major inhibitor TIMP-1, which in combination with neutrophil or macrophage proteases released in inflamed areas may influence the elastolytic burden. We demonstrate that fibroblasts derived from MDE produce in vitro low levels of TIMP-1, the major inhibitor of MMPs. Elevated levels of MMP-2, MMP-14, and TIMP-2 capable to activate in a cooperative manner pro-MMP-2 are present in MDE tissue samples. Additionally, significant reaction for MMP-1 is present in the same MDE areas. These data all together suggest that ECM changes in MDE are due to cooperation of different cell populations (i.e., inflammatory cells and fibroblasts).

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.

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.

Genetic Deletion and Pharmacological Inhibition of PI3K γ Reduces Neutrophilic Airway Inflammation and Lung Damage in Mice with Cystic Fibrosis-Like Lung Disease.

PURPOSE: Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF). Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kγ plays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kγ on airway inflammation and structural lung damage in a mouse model of CF lung disease. METHODS: ßENaC overexpressing mice (ßENaC-Tg) were backcrossed with PI3Kγ-deficient (PI3Kγ (KO)) mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF). Furthermore, we assessed the effect of a specific PI3Kγ inhibitor (AS-605240) on inflammatory cell number in BALF. RESULTS: Genetic deletion of PI3Kγ decreased neutrophil numbers in BALF of PI3Kγ (KO)/ßENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kγ inhibitor AS-605240 decreased the number of neutrophils in BALF of ßENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme. CONCLUSIONS: These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kγ in reducing chronic neutrophilic inflammation in CF-like lung disease in vivo.

Skeletal muscle oxidative metabolism in an animal model of pulmonary emphysema: formoterol and skeletal muscle dysfunction.

Skeletal muscle dysfunction is a significant contributor to exercise limitation in pulmonary emphysema. This study investigated skeletal muscle oxidative metabolism before and after aerosol exposure to a long-acting ß-agonist (LABA), such as formoterol, in the pallid mouse (B6.Cg-Pldnpa/J), which has a deficiency in serum α(1)-antitrypsin (α(1)-PI) and develops spontaneous pulmonary emphysema. C57 BL/6J and its congener pallid mice of 8-12 and 16 months of age were treated with vehicle or formoterol aerosol challenge for 120 seconds. Morphological and morphometric studies and evaluations of mitochondrial adenosine diphosphate-stimulated respiration and of cytochrome oxidase activity on skeletal muscle were performed. Moreover, the mtDNA content in skeletal muscle and the mediators linked to muscle mitochondrial function and biogenesis, as well as TNF-α and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), were also evaluated. The lungs of pallid mice at 12 and 16 months of age showed patchy areas of airspace enlargements, with the destruction of alveolar septa. No significant differences were observed in basal values of mitochondrial skeletal muscle oxidative processes between C57 BL/6J and pallid mice. Exposure to LABA significantly improved mitochondrial skeletal muscle oxidative processes in emphysematous mice, where the mtDNA content was significantly higher with respect to 8-month-old pallid mice. This effect was compared with a significant increase of PGC-1α in skeletal muscles of 16-month-old pallid mice, with no significant changes in TNF-α concentrations. In conclusion, in emphysematous mice that showed an increased mtDNA content, exposure to inhaled LABA can improve mitochondrial skeletal muscle oxidative processes. PGC-1α may serve as a possible mediator of this effect.

Receptor for advanced glycation end products contributes to postnatal pulmonary development and adult lung maintenance program in mice.

The role of the receptor for advanced glycation end products (RAGE) in promoting the inflammatory response through activation of NF-κB pathway is well established. Recent findings indicate that RAGE may also have a regulative function in apoptosis, as well as in cellular proliferation, differentiation, and adhesion. Unlike other organs, lung tissue in adulthood and during organ development shows relatively high levels of RAGE expression. Thus a role for the receptor in lung organogenesis and homeostasis may be proposed. To evaluate the role of RAGE in lung development and adult lung homeostasis, we generated hemizygous and homozygous transgenic mice overexpressing human RAGE, and analyzed their lungs from the fourth postnatal day to adulthood. Moderate RAGE hyperexpression during lung development influenced secondary septation, resulting in an impairment of alveolar morphogenesis and leading to significant changes in morphometric parameters such as airspace number and the size of alveolar ducts. An increase in alveolar cell apoptosis and a decrease in cell proliferation were demonstrated by the terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling reaction, active caspase-3, and Ki-67 immunohistochemistry. Alterations in elastin organization and deposition and in TGF-ß expression were observed. In homozygous mice, the hyperexpression of RAGE resulted in histological changes resembling those changes characterizing human bronchopulmonary dysplasia (BPD). RAGE hyperexpression in the adult lung is associated with an increase of the alveolar destructive index and persistent inflammatory status leading to "destructive" emphysema. These results suggest an important role for RAGE in both alveolar development and lung homeostasis, and open new doors to working hypotheses on the pathogenesis of BPD and chronic obstructive pulmonary disease.

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.

Genetic ablation of the fpr1 gene confers protection from smoking-induced lung emphysema in mice.

Cigarette smoke (CS) is the main causative factor of chronic obstructive pulmonary disease (COPD). Current research supports the concept that airway inflammation is central to the development and progression of the disease. Studies have demonstrated that neutrophils are increased in COPD lungs and that neutrophil-associated products correlate with the development and severity of COPD. The peptide FMLP is an active component of CS. FMLP interacts on the neutrophil and macrophage membranes with a high-affinity receptor subtype (FPR1) and with a low-affinity subtype FPRL1, promoting a chemotactic response, superoxide anion production, and degranulation. Bacterial colonization of the lower respiratory tract and lung cell damage may represent further sources of formyl peptides in patients with COPD. We investigated the role of FPR in a mouse model on lung inflammation and emphysema induced by CS. Here, we report the novel observation that genetic ablation of the FPR1 gene (Fpr1) confers protection from smoking-induced lung emphysema in mice. Compared with wild-type mice, Fpr1 knockout mice displayed marked decreases in the lung migration of neutrophils and macrophages after CS exposure. Upon transgenic approach, the changes in cell numbers were accompanied by marked modulation of the expression of genes implicated in the inflammatory response. Administration of the FPR1 antagonist cyclosporine H to wild-type mice attenuated the acute inflammatory response evoked by CS. These findings may have clinical significance because current smokers and subjects with emphysema showed increased FPR expression in bronchoalveolar fluids and on peripheral neutrophils. Modulating the FPR1 signal should be explored as a potential new therapy.

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 P2Y(2)R deficient ((-/-)) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y(2)R. Moreover, P2Y(2)R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y(2)R(-/-) and wild type chimera animals revealed that P2Y(2)R 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 P2Y(2)R.

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.

Extracellular adenosine triphosphate and chronic obstructive pulmonary disease.

RATIONALE: Extracellular ATP promotes inflammation, but its role in chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES: To analyze the expression of ATP and its functional consequences in never-smokers, asymptomatic smokers, and patients with COPD. METHODS: ATP was quantified in bronchoalveolar lavage fluid (BALF) of never-smokers, asymptomatic smokers, and patients with COPD of different severity. The expression of specific ATP (purinergic) receptors was measured in airway macrophages and blood neutrophils from control subjects and patients with COPD. The release of mediators by macrophages and neutrophils and neutrophil chemotaxis was assessed after ATP stimulation. MEASUREMENTS AND MAIN RESULTS: Chronic smokers had elevated ATP concentrations in BALF compared with never-smokers. Acute smoke exposure led to a further increase in endobronchial ATP concentrations. Highest ATP concentrations in BALF were present in smokers and ex-smokers with COPD. In patients with COPD, BALF ATP concentrations correlated negatively with lung function and positively with BALF neutrophil counts. ATP induced a stronger chemotaxis and a stronger elastase release in blood neutrophils from patients with COPD, as compared with control subjects. In addition, airway macrophages from patients with COPD responded with an increased secretion of proinflammatory and tissue-degrading mediators after ATP stimulation. These findings were accompanied by an up-regulation of specific purinergic receptors in blood neutrophils and airway macrophages of patients with COPD. CONCLUSIONS: COPD is characterized by a strong and persistent up-regulation of extracellular ATP in the airways. Extracellular ATP appears to contribute to the pathogenesis of COPD by promoting inflammation and tissue degradation.

Sarcopenia in Chronic Kidney Disease: Focus on Advanced Glycation End Products as Mediators and Markers of Oxidative Stress.

Sarcopenia is common in chronic kidney disease (CKD), and it is independently associated with morbidity and mortality. Advanced glycation end products (AGE) are mainly known as aging products. In CKD, AGE accumulate due to increased production and reduced kidney excretion. The imbalance between oxidant/antioxidant capacities in CKD patients is one of the main factors leading to AGE synthesis. AGE can, in turn, promote CKD progression and CKD-related complications by increasing reactive oxygen species generation, inducing inflammation, and promoting fibrosis. All these derangements can further increase AGE and uremic toxin accumulation and promote loss of muscle mass and function. Since the link between AGE and sarcopenia in CKD is far from being fully understood, we revised hereby the data supporting the potential contribution of AGE as mediators of oxidative stress in the pathogenesis of sarcopenia. Understanding how AGE and oxidative stress impact the onset of sarcopenia in CKD may help to identify new potential markers of disease progression and/or therapeutic targets.

The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease.

After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.

SARS-CoV-2 COVID-19 susceptibility and lung inflammatory storm by smoking and vaping.

The current pandemic of COVID-19 has caused severe morbidity and mortality across the globe. People with a smoking history have severe disease outcomes by COVID-19 infection. Epidemiological studies show that old age and pre-existing disease conditions (hypertension and diabetes) result in severe disease outcome and mortality amongst COVID-19 patients. Evidences suggest that the S1 domain of the SARS-CoV-2 (causative agent of COVID-19) membrane spike has a high affinity towards the angiotensin-converting enzyme 2 (ACE2) receptor found on the host's lung epithelium. Likewise, TMPRSS2 protease has been shown to be crucial for viral activation thus facilitating the viral engulfment. The viral entry has been shown to cause 'cytokine storm' involving excessive production of pro-inflammatory cytokines/chemokines including IL-6, TNF-α, IFN-γ, IL-2, IL-7, IP-10, MCP-3 or GM-CSF, which is augmented by smoking. Future research could target these inflammatory-immunological responses to develop effective therapy for COVID-19. This mini-review provides a consolidated account on the role of inflammation and immune responses, proteases, and epithelial permeability by smoking and vaping during SARS-CoV2 infection with future directions of research, and provides a list of the potential targets for therapies particularly controlling cytokine storms in the lung.