Francesca Polverino

A novel insight into adaptive immunity in chronic obstructive pulmonary disease: B cell activating factor belonging to the tumor necrosis factor family.

RATIONALE Chronic obstructive pulmonary disease (COPD) is a disorder characterized by an abnormal inflammatory response that persists even after smoking cessation, yet the underlying mechanisms are not fully understood. OBJECTIVES To investigate the expression of B-cell activating factor of tumor necrosis factor family (BAFF), a crucial mediator in the crosstalk between innate and adaptive immune responses, in patients with COPD and to explore its correlation with disease severity. METHODS Using immunohistochemistry, expression of BAFF was examined in lung specimens from 21 smokers with COPD (FEV(1) = 57 ± 5% predicted), 14 control smokers (FEV(1) = 99 ± 2% predicted) and 8 nonsmokers (FEV(1) = 104 ± 4% predicted). BAFF was quantified in alveolar macrophages and alveolar walls, in bronchiolar and parenchymal lymphoid follicles, and in peripheral airways and pulmonary arterioles. MEASUREMENTS AND MAIN RESULTS In alveolar macrophages and parenchymal lymphoid follicles, BAFF expression was increased in smokers with COPD compared with control smokers and nonsmokers (P < 0.05 for all comparisons). In both compartments, BAFF was also up-regulated in control smokers as compared with nonsmokers (P = 0.03 and P = 0.01). Moreover, BAFF was overexpressed in bronchiolar lymphoid follicles, alveolar walls, peripheral airways, and pulmonary arterioles from smokers with COPD compared with nonsmokers (P < 0.05 for all). Among patients with COPD, BAFF(+) macrophages were inversely related to FEV(1) (P = 0.03, Spearmans rho [r(S)] = -0.48), FEV(1)/FVC (P = 0.02, r(S) = -0.50), and Pa(O(2)) values (P = 0.01, r(S) = -0.55). CONCLUSIONS This study demonstrated overexpression of BAFF in peripheral lung of patients with COPD, mainly in alveolar macrophages and lymphoid follicles. Moreover, BAFF expression was correlated to the degree of lung function impairment and hypoxia, suggesting that it may have a possible impact on disease severity.

Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element–binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

A Chronic Obstructive Pulmonary Disease Susceptibility Gene, FAM13A, Regulates Protein Stability of β-Catenin

RATIONALE A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown. OBJECTIVES To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility. METHODS Fam13a null mice (Fam13a(-/-)) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a(-/-) and Fam13a(+/+) littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase-polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13As function. MEASUREMENTS AND MAIN RESULTS In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a(-/-)) were resistant to chronic cigarette smoke-induced emphysema compared with Fam13a(+/+) mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3β and β-catenin, inducing β-catenin degradation. Fam13a(-/-) mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a β-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting β-catenin signaling. Moreover, human COPD lungs had decreased protein levels of β-catenin and increased protein levels of FAM13A. CONCLUSIONS We show that FAM13A may influence COPD susceptibility by promoting β-catenin degradation.

Protective role for club cell secretory protein-16 (CC16) in the development of COPD.

Club cell secretory protein-16 (CC16) is the major secreted product of airway club cells, but its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unclear. We measured CC16 airway expression in humans with and without COPD and CC16 function in a cigarette smoke (CS)-induced COPD murine model. Airway CC16 expression was measured in COPD patients, smokers without COPD and non-smokers. We exposed wildtype (WT) and CC16−/−mice to CS or air for up to 6 months, and measured airway CC16 expression, pulmonary inflammation, alveolar septal cell apoptosis, airspace enlargement, airway mucin 5AC (MUC5AC) expression, small airway remodelling and pulmonary function. Smokers and COPD patients had reduced airway CC16 immunostaining that decreased with increasing COPD severity. Exposing mice to CS reduced airway CC16 expression. CC16−/− mice had greater CS-induced emphysema, airway remodelling, pulmonary inflammation, alveolar cell apoptosis, airway MUC5AC expression, and more compliant lungs than WT mice. These changes were associated with increased nuclear factor-κB (NF-κB) activation in CC16−/− lungs. CS-induced acute pulmonary changes were reversed by adenoviral-mediated over-expression of CC16. CC16 protects lungs from CS-induced injury by reducing lung NF-κB activation. CS-induced airway CC16 deficiency increases CS-induced pulmonary inflammation and injury and likely contributes to the pathogenesis of COPD. Cigarette smoke exposure reduces airway levels of anti-inflammatory CC16 to thereby contribute to the genesis of COPD http://ow.ly/GOMiZ

B Cell–Activating Factor. An Orchestrator of Lymphoid Follicles in Severe Chronic Obstructive Pulmonary Disease

RATIONALE Patients with chronic obstructive pulmonary disease (COPD) have increased pulmonary lymphoid follicle (LF) counts. B cell-activating factor of tumor necrosis factor family (BAFF) regulates B cells in health, but its role in COPD pathogenesis is unclear. OBJECTIVES To determine whether BAFF expression in pulmonary LFs correlates with COPD severity, LF size or number, and/or readouts of B-cell function in LFs. METHODS We correlated BAFF immunostaining in LFs in lung explants or biopsies from nonsmoking control subjects (NSC), smokers without COPD (SC), and patients with COPD with the number and size of LFs, and LF B-cell apoptosis, activation, and proliferation. We analyzed serum BAFF levels and BAFF expression in B cells in blood and bronchoalveolar lavage samples from the same subject groups. We assessed whether: (1) cigarette smoke extract (CSE) increases B-cell BAFF expression and (2) recombinant BAFF (rBAFF) rescues B cells from CSE-induced apoptosis by inhibiting activation of nuclear factor-κB (NF-κB). MEASUREMENTS AND MAIN RESULTS Patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD had increased numbers and larger pulmonary LFs than patients with GOLD stages I-II COPD and SC. We identified two main types of pulmonary LFs: (1) type A, the predominant type in GOLD stages I-II COPD and SC, characterized by abundant apoptotic but few BAFF-positive cells (mostly B cells); and (2) type B, the main type in GOLD stage IV COPD, characterized by abundant BAFF-positive cells but few apoptotic cells (mostly B cells). BAFF levels were also higher in blood and bronchoalveolar lavage B cells in patients with COPD versus NSC and SC. Surprisingly, rBAFF blocked CSE-induced B-cell apoptosis by inhibiting CSE-induced NF-κB activation. CONCLUSIONS Our data support the hypothesis that B-cell BAFF expression creates a self-perpetuating loop contributing to COPD progression by promoting pulmonary B-cell survival and LF expansion.

A Pilot Study Linking Endothelial Injury in Lungs and Kidneys in Chronic Obstructive Pulmonary Disease

Rationale: Patients with chronic obstructive pulmonary disease (COPD) frequently have albuminuria (indicative of renal endothelial cell injury) associated with hypoxemia. Objectives: To determine whether (1) cigarette smoke (CS)‐induced pulmonary and renal endothelial cell injury explains the association between albuminuria and COPD, (2) CS‐induced albuminuria is linked to increases in the oxidative stress‐advanced glycation end products (AGEs) receptor for AGEs (RAGE) pathway, and (3) enalapril (which has antioxidant properties) limits the progression of pulmonary and renal injury by reducing activation of the AGEs‐RAGE pathway in endothelial cells in both organs. Methods: In 26 patients with COPD, 24 ever‐smokers without COPD, 32 nonsmokers who underwent a renal biopsy or nephrectomy, and in CS‐exposed mice, we assessed pathologic and ultrastructural renal lesions, and measured urinary albumin/creatinine ratios, tissue oxidative stress levels, and AGEs and RAGE levels in pulmonary and renal endothelial cells. The efficacy of enalapril on pulmonary and renal lesions was assessed in CS‐exposed mice. Measurements and Main Results: Patients with COPD and/or CS‐exposed mice had chronic renal injury, increased urinary albumin/creatinine ratios, and increased tissue oxidative stress and AGEs‐RAGE levels in pulmonary and renal endothelial cells. Treating mice with enalapril attenuated CS‐induced increases in urinary albumin/creatinine ratios, tissue oxidative stress levels, endothelial cell AGEs and RAGE levels, pulmonary and renal cell apoptosis, and the progression of chronic renal and pulmonary lesions. Conclusions: Patients with COPD and/or CS‐exposed mice have pulmonary and renal endothelial cell injury linked to increased endothelial cell AGEs and RAGE levels. Albuminuria could identify patients with COPD in whom angiotensin‐converting enzyme inhibitor therapy improves renal and lung function by reducing endothelial injury.

ADAM9 Is a Novel Product of Polymorphonuclear Neutrophils: Regulation of Expression and Contributions to Extracellular Matrix Protein Degradation during Acute Lung Injury

A disintegrin and a metalloproteinase domain (ADAM) 9 is known to be expressed by monocytes and macrophages. In this study, we report that ADAM9 is also a product of human and murine polymorphonuclear neutrophils (PMNs). ADAM9 is not synthesized de novo by circulating PMNs. Rather, ADAM9 protein is stored in the gelatinase and specific granules and the secretory vesicles of human PMNs. Unstimulated PMNs express minimal quantities of surface ADAM9, but activation of PMNs with degranulating agonists rapidly (within 15 min) increases PMN surface ADAM9 levels. Human PMNs produce small quantities of soluble forms of ADAM9. Surprisingly, ADAM9 degrades several extracellular matrix (ECM) proteins, including fibronectin, entactin, laminin, and insoluble elastin, as potently as matrix metalloproteinase-9. However, ADAM9 does not degrade types I, III, or IV collagen or denatured collagens in vitro. To determine whether Adam9 regulates PMN recruitment or ECM protein turnover during inflammatory responses, we compared wild-type and Adam9−/− mice in bacterial LPS- and bleomycin-mediated acute lung injury (ALI). Adam9 lung levels increase 10-fold during LPS-mediated ALI in wild-type mice (due to increases in leukocyte-derived Adam9), but Adam9 does not regulate lung PMN (or macrophage) counts during ALI. Adam9 increases mortality, promotes lung injury, reduces lung compliance, and increases degradation of lung elastin during LPS- and/or bleomycin-mediated ALI. Adam9 does not regulate collagen accumulation in the bleomycin-treated lung. Thus, ADAM9 is expressed in an inducible fashion on PMN surfaces where it degrades some ECM proteins, and it promotes alveolar–capillary barrier injury during ALI in mice.

Adam8 Limits the Development of Allergic Airway Inflammation in Mice

To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite–treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.

Anatomy and neuro-pathophysiology of the cough reflex arc.

Coughing is an important defensive reflex that occurs through the stimulation of a complex reflex arc. It accounts for a significant number of consultations both at the level of general practitioner and of respiratory specialists. In this review we first analyze the cough reflex under normal conditions; then we analyze the anatomy and the neuro-pathophysiology of the cough reflex arc. The aim of this review is to provide the anatomic and pathophysiologic elements of evaluation of the complex and multiple etiologies of cough.

A novel nonhuman primate model of cigarette smoke-induced airway disease.

Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)-exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD.