Gaetano Caramori

A molecular mechanism of action of theophylline: Induction of histone deacetylase activity to decrease inflammatory gene expression

The molecular mechanism for the anti-inflammatory action of theophylline is currently unknown, but low-dose theophylline is an effective add-on therapy to corticosteroids in controlling asthma. Corticosteroids act, at least in part, by recruitment of histone deacetylases (HDACs) to the site of active inflammatory gene transcription. They thereby inhibit the acetylation of core histones that is necessary for inflammatory gene transcription. We show both in vitro and in vivo that low-dose theophylline enhances HDAC activity in epithelial cells and macrophages. This increased HDAC activity is then available for corticosteroid recruitment and predicts a cooperative interaction between corticosteroids and theophylline. This mechanism occurs at therapeutic concentrations of theophylline and is dissociated from phosphodiesterase inhibition (the mechanism of bronchodilation) or the blockade of adenosine receptors, which are partially responsible for its side effects. Thus we have shown that low-dose theophylline exerts an anti-asthma effect through increasing activation of HDAC which is subsequently recruited by corticosteroids to suppress inflammatory genes.

Increased expression of nuclear factor-κB in bronchial biopsies from smokers and patients with COPD

The expression of nuclear factor (NF)-κB is an indicator of cellular activation and of inflammatory mediator production. The aim of the present study was to characterise the expression and localisation of p65, the major subunit of NF-κB, in the bronchial mucosa of patients with chronic obstructive pulmonary disease (COPD), and to examine the relationship between p65 expression and disease status. Bronchial biopsies were obtained from 14 smokers with COPD, 17 smokers with normal lung function and 12 nonsmokers with normal lung function. The number of p65 positive (+) cells was quantified by immunohistochemistry and the expression of p65 in bronchial biopsies from the three groups was examined by Western blotting (WB). Smokers with normal lung function and patients with COPD had increased numbers of p65+ cells in the epithelium and increased p65 nuclear expression. In COPD patients the number of epithelial p65+ cells correlated with the degree of airflow limitation. WB analysis showed an increase in p65 in smokers with normal lung function and COPD patients (p<0.05). Bronchial biopsies in smokers with normal lung function and chronic obstructive pulmonary disease patients show increased expression of p65 protein, predominantly in the bronchial epithelium. Disease severity is associated with an increased epithelial expression of nuclear factor-κB.

Cross-talk between pro-inflammatory transcription factors and glucocorticoids

Asthma is a chronic inflammatory disease of the airway that is characterized by cellular infiltration and activation. These processes are induced by overexpression of chemokines and cytokines, such as eotaxin, IL‐1β and GM‐CSF. These mediators are downstream targets for the transcription factors activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB), which control the expression of most immunomodulatory genes and whose activity and expression are elevated in asthma. Glucocorticoids are the most effective anti‐inflammatory drugs used in the treatment of chronic inflammatory diseases such as asthma. They act by binding to a specific glucocorticoid receptor (GR) that on activation translocates to the nucleus and either increases (transactivates) or decreases (transrepresses) the expression of responsive genes. Transrepression is the major mechanism of glucocorticoid action in inhibiting inflammatory gene expression. Thus, the ability of the transciption factors AP‐1 and NF‐κB to induce gene transcription is attenuated by GR. Although only 5–10% of asthmatic subjects are glucocorticoid‐insensitive, these subjects account for over 50% of the health‐care costs for asthma (>

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

6 billion per annum). Examining these patients also gives an insight into important aspects of glucocorticoid action in controlling inflammation and into the development of potential new drugs. Biochemical and genomic studies have indicated abnormal induction of the c‐Jun N‐terminal kinase (JNK) pathway in some of these patients. The ability of most patients to respond to dexamethasone with induction of histone acetylation correlated with nuclear translocation of GR. However, a subgroup of these patients had an inability to correctly interact with the basal transcription complex in spite of high levels of nuclear GR. This suggests that cross‐talk between pro‐ and anti‐inflammatory transcription factors may modulate activation of the transcriptional complex and thereby reduce steroid actions.

T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

New targets for drug development in asthma

There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)‐17 as their effector cytokine under the control of IL‐22 and IL‐23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and IL‐23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age‐matched control subjects. The expression of IL‐17A, IL‐17F, IL‐21, IL‐22, IL‐23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL‐22+ and IL‐23+ immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL‐17A+ and IL‐22+ immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non‐smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL‐22+ cells correlated significantly with the number of both CD4+ and CD8+ cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL‐17A and IL‐22. Increased expression of the Th17‐related cytokines IL‐17A, IL‐22 and IL‐23 in COPD patients may reflect their involvement, and that of specific IL‐17‐producing cells, in driving the chronic inflammation seen in COPD.

Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice.

Asthma is a chronic inflammatory disease that affects about 300 million people worldwide, a total that is expected to rise to about 400 million over the next 15-20 years. Most asthmatic individuals respond well to the currently available treatments of inhaled corticosteroids and beta-adrenergic agonists; however, 5-10% have severe disease that responds poorly. Improved knowledge of asthma mechanisms has led to the recognition of different asthma phenotypes that might reflect distinct types of inflammation, explaining the effectiveness of anti-leucotrienes and the anti-IgE monoclonal antibody omalizumab in some patients. However, more knowledge of the inflammatory mechanisms within the airways is required. Improvements in available therapies-such as the development of fast-onset, once-a-day combination drugs with better safety profiles-will occur. Other drugs, such as inhaled p38 MAPK inhibitors and anti-oxidants, that target specific pathways or mediators could prove useful as monotherapies, but could also, in combination with corticosteroids, reduce the corticosteroid insensitivity often seen in severe asthma. Biological agents directed against the interleukin-13 pathway and new immunoregulatory agents that modulate functions of T-regulatory and T-helper-17 cells are likely to be successful. Patient-specific treatments will depend on the development of discriminatory handprints of distinct asthma subtypes and are probably over the horizon. Although a cure is unlikely to be developed in the near future, a greater understanding of disease mechanisms could bring such a situation nearer to reality.

Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview

RATIONALE There is an increasing prevalence of reduced responsiveness to glucocorticoid therapy in severe asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanism of this remains unknown. Recent studies have shown that histone deacetylase activity, which is critical to glucocorticoid function, is altered by oxidant stress and may be involved in the development of glucocorticoid insensitivity. OBJECTIVES To determine the role of phosphoinositol-3-kinase (PI3K) in the development of cigarette smoke-induced glucocorticoid insensitivity. METHODS Wild-type, PI3Kgamma knock-out and PI3Kdelta kinase dead knock-in transgenic mice were used in a model of cigarette smoke-induced glucocorticoid insensitivity. Peripheral lung tissue was obtained from six healthy nonsmokers, nine smokers with normal lung function, and eight patients with COPD. MEASUREMENTS AND MAIN RESULTS In vitro oxidative stress activates PI3K and induced a relative glucocorticoid resistance, which is restored by PI3K inhibition. In vivo, cigarette smoke exposure in mice increased tyrosine nitration of histone deacetylase 2 in the lung, correlating with reduced histone deacetylase 2 activity and reduced glucocorticoid function. Histone deacetylase 2 activity and the antiinflammatory effects of glucocorticoids were restored in PI3Kdelta kinase dead knock-in but not PI3Kgamma knock-out smoke-exposed mice compared with wild type mice, correlating with reduced histone deacetylase 2 tyrosine nitration. Glucocorticoid receptor expression was significantly reduced in smoke-exposed mice, in smokers with normal lung function, and in patients with COPD. CONCLUSIONS These data show that therapeutic inhibition of PI3Kdelta may restore glucocorticoid function in oxidative stress-induced glucocorticoid insensitivity.

Chronic Obstructive Pulmonary Disease and Lung Cancer: New Molecular Insights

In the last decade, the analysis of bronchial biopsies and lung parenchyma obtained from chronic obstructive pulmonary disease (COPD) patients compared with those from smokers with normal lung function and non‐smokers has provided new insights on the role of the different inflammatory and structural cells, their signalling pathways and mediators, contributing to a better knowledge of the pathogenesis of COPD. This review summarizes and discusses the lung pathology of COPD patients with emphasis on inflammatory cell phenotypes that predominate in different clinical conditions. In bronchial biopsies, a cascade of events takes place during progression from mild‐to‐severe disease. T lymphocytes, particularly CD8+ cells and macrophages are the prevalent inflammatory cells in the lung of healthy smokers and patients with mild COPD, while total and activated neutrophils predominate in severe COPD. The number of CD4+, CD8+ cells and macrophages expressing nuclear factor‐kappa B (NF‐κB), STAT‐4 and IFN‐γ proteins as well as endothelial adhesion molecule‐1 in endothelium is increased in mild/moderate disease. In contrast, activated neutrophils (MPO+ cells) and increased nitrotyrosine immunoreactivity develops in severe COPD. In bronchial biopsies obtained during COPD exacerbations, some studies have shown an increased T cell and granulocyte infiltration. Regular treatment with high doses of inhaled glucocorticoids does not significantly change the number of inflammatory cells in bronchial biopsies from patients with moderate COPD. The profile in lung parenchyma is similar to bronchial biopsies. ‘Healthy’ smokers and mild/moderate diseased patients show increased T lymphocyte infiltration in the peripheral airways. Pulmonary emphysema is associated with a general increase of inflammatory cells in the alveolar septa. The molecular mechanisms driving the lymphocyte and neutrophilic prevalence in mild and severe disease, respectively, needs to be extensively studied. Up‐regulation of pro‐inflammatory transcription factors NF‐κB and STAT‐4 in mild, activated epithelial and endothelial cells in the more severe disease may contribute to this differential prevalence of infiltrating cells.

COPD increases the risk of squamous histological subtype in smokers who develop non-small cell lung carcinoma

Both chronic obstructive pulmonary disease (COPD) and lung cancer are major causes of death worldwide. In most cases this reflects cigarette smoke exposure which is able to induce an inflammatory response in the airways of smokers. Indeed, COPD is characterized by lower airway inflammation, and importantly, the presence of COPD is by far the greatest risk factor for lung cancer amongst smokers. Cigarette smoke induces the release of many inflammatory mediators and growth factors including TGF-β, EGFR, IL-1, IL-8 and G-CSF through oxidative stress pathways and this inflammation may persist for decades after smoking cessation. Mucus production is also increased by these inflammatory mediators, further linking airway inflammation to an important mechanism of lung cancer. A greater understanding of the molecular and cellular pathobiology that distinguishes smokers with lung cancer from smokers with and without COPD is needed to unravel the complex molecular interactions between COPD and lung cancer. By understanding the common signalling pathways involved in COPD and lung cancer the hope is that treatments will be developed that not only treat the underlying disease process in COPD, but also reduce the currently high risk of developing lung cancer in these patients.