Sekretomodulatorische Effekte des proinflammatorischen Zytokins GM-CSF auf die Muzinsekretion an der isolierten Rattentrachea nativer und NO2-exponierter Tiere

Eine Facette des klinischen Erscheinungsbildes der „chronic obstructive pulmonary disease“ (COPD) ist die chronische Bronchitis mit einem überwiegend inflammatorischen Korrelat. Der Pathogenese der chronischen Bronchitis liegen komplexe pathophysiologische Vorgänge zugrunde, welche bislang nur unvollstädig bekannt und im Einzelnen nicht vollständig geklärt sind. Die Definition der „global initiative on obstructive lung disease“ (GOLD) erwähnt eine abnormale inflammatorische Reaktion der Lunge auf schädliche Partikel und Gase und rückt somit die Entzündung ins Zentrum der Pathogenese. Zigarettenrauch und wichtige Umweltnoxen wie Stickstoffdioxid (NO2) führen über die hervorgerufene Entzündung bei Menschen und anderen Spezies zu Veränderungen des Atemwegsepithels und zur Stimulation der Produktion von proinflammatorischen Zytokinen wie dem Granulozyten-Makrophagen-Kolonie-stimulierenden Faktor (GM-CSF). Die strukturellen Veränderungen schließen auch die Muzin-produzierenden epithelialen Komponenten ein. Ein relevantes klinisches Symptom der chronischen Bronchitis stellt die Hypersekretion dar. Die Regulation der Muzinsynthese und -sekretion unterliegt komplexen neuralen, neuroendokrinen und parakrinen Mechanismen. Mit dem Ziel, zugrunde liegende Regulationsmechanismen der Mukussekretion bei der chronisch inflammatorischen Erkrankung COPD näher zu charakterisieren, liegt der Schwerpunkt dieser Arbeit in der Bedeutung des proinflammatorischen Zytokins GM-CSF. Folgenden Fragen, welchen eine Bedeutung bei der Regulation der Mukussekretion zukommt, wird in der vorliegenden Arbeit nachgegangen: Welchen Einfluss hat die Lang- und Kurzzeitexposition von NO2 auf die tracheobronchiale Muzinsekretion? Welchen Einfluss zeigt die peptiderge Substanz Substanz P (SP) auf die Muzinsekretion exponierter Tiere auch im Speziesvergleich? Wirkt das proinflammatorische Zytokin GM-CSF sekretagog? Gibt es interaktive Prozesse zwischen dem Zytokin GM-CSF und dem Tachykinin SP im Hinblick auf die Sekretomodulation? Zur experimentellen Klärung dieser Fragestellungen wurde ein etabliertes Tiermodell gewählt. Für das Studium der Reaktionsweise entzündlich alterierter Atemwege erfolgte die inhalative Exposition gegenüber NO2 im Rattenmodell. Die Muzinsekretion wurde an Trachealexplantaten von Fischerratten (F344-Ratten) und Sprague-Dawley-Ratten (SD-Ratten) durch die Anwendung der etablierten Ussing-Kammer-Methode untersucht. Zur Quantifizierung der Sekretion trachealer submuköser Drüsen erfolgte die radioaktive Markierung der Muzine mit 35S. Zum Studium der trachealen Muzinsekretion erfolgte die inhalative Exposition mit Induktion einer Schädigung und Herbeiführung einer akuten und subakuten bis chronischen Tracheobronchitis. Mit standardisierter Methodik wurden bei männlichen Ratten der jeweiligen Spezies Expositionszeiten von 1, 3 und 20 Tagen sowie darüber hinaus teilweise von 2 und 28 Tagen gegenüber NO2 bei einer Konzentration von 10 ppm gewählt. Kontrolltiere wurden einer Atmosphäre ohne NO2 ausgesetzt. Neben der Basalsekretion wurde zunächst an den Trachealexplantaten die maximal stimulierte Sekretion durch Acetylcholin (ACh) in Stadien der artifiziellen Entzündung und im Nativzustand erfasst. Mit diesen Grundlagen kann ein sekretomodulatorischer Effekt des Peptides SP und eine Dosis-Wirkungs-Beziehung des proinflammatorischen Zytokins GM-CSF im zeitlichen Verlauf unter NO2-Exposition untersucht werden. Zudem wurde hinsichtlich des Sekretionsergebnisses die Frage nach einem additiven Effekt von GM-CSF und SP an langzeit-exponierten Tracheen untersucht. Die Sekretionsanalyse ergab, dass eine NO2-Exposition von 10 ppm die Muzinsekretion der Rattentrachea beider Spezies zeitabhängig nicht signifikant beeinflusst. Es zeigten sich allerdings gleichgerichtete Tendenzen ohne Signifikanz im Sekretionsverlauf. Im Speziesvergleich zeigten dabei SD-Ratten eine größere sekretorische Potenz im Vergleich zu F344-Ratten im Sinne eines nahezu parallel verschobenen Sekretionsniveaus auf. Darüber hinaus wurde die Sekretionsrate nativ und bis zu einer Expositionsdauer von 20 Tagen bei beiden Spezies durch Acetylcholin im Vergleich zur Basalsekretion signifikant gesteigert. Hierbei fanden sich im direkten Vergleich zu den nativen Fischerratten wiederum signifikante Differenzen nach 3 sowie 20 Tagen. Signifikante sekretagoge Effekte von Substanz P ließen sich bei Fischerratten und SD-Ratten sowohl bei den Kontrolltieren als auch bei nahezu allen untersuchten Expositionsgruppen aufzeigen. GM-CSF zeigte in unserem Rattenmodell der COPD dosis- und expositionszeitabhängig eine Stimulation der trachealen Muzinsekretion. Eine signifikante Sekretionsantwort in der 3-Tages-Expositionsgruppe konnte für 0,1 ng/ml GM-CSF und für 0,4 ng/ml GM-CSF demonstriert werden. Zudem fand sich eine Signifikanz in der 20-Tages-Expositionsgruppe für 0,1 und 0,15 sowie 0,2 ng/ml GM-CSF. Für die Dosis von 0,2 ng/ml GM-CSF ergab sich ein hochsignifikantes Ergebnis. Die gleichzeitige Gabe von GM-CSF und SP zeigte in der Langzeitexposition keinen messbaren additiven sekretagogen Effekt und ergab das Sekretionsniveau von SP. Dies wurde nach 20 Tagen Expositionsdauer untersucht, da für diese Gruppe für beide Einzelsubstanzen jeweils eine signifikante Sekretionsantwort gesehen wurde. Die mutmaßlich unterschiedlichen sekretagogen Mechanismen von SP und GM-CSF sind vereinbar mit der Beobachtung, dass SP auch unabhängig vom Vorhandensein einer Atemwegsinflammation wirksam ist. Diese Ergebnisse liefern einen Beitrag zum Verständnis der Komplexität der bei der Regulation der Muzinsekretion beteiligten Pathomechanismen des Tracheobronchialepithels in der chronischen Bronchitis. Der sekretagoge Effekt von GM-CSF in der chronischen Inflammation wurde demonstriert. Mit dem Ziel einer Beeinflussung der Hypersekretion bei der COPD ist hier ein zielgerichteter therapeutischer Ansatz denkbar. Der Einsatz von neutralisierenden Antikörpern gegen GM-CSF oder dessen Rezeptor ist als spezifisches Therapiekonzept vorstellbar. Dieses ist in Ergänzung zu den etablierten antiinflammatorischen therapeutischen Möglichkeiten bei der COPD zu sehen und ermöglicht im Gegensatz dazu ein zielgerichtetes Eingreifen in die inflammatorische Kaskade. Es bedarf weiterer Untersuchungen, um die Rolle einer solchen Herangehensweise zu konkretisieren

The Mechanism Of Tobacco-Induced Decrements In Mucociliary Clearance

Chronic obstructive pulmonary disease (COPD), characterized by progressive loss of lung function including mucus obstruction and airway remodeling, is primarily caused by cigarette smoking. This pulmonary phenotype resembles that of cystic fibrosis (CF), which results from genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel important in maintaining airway surface hydration and mucus clearance. Cigarette smoke exposure has been found to induce acquired CFTR dysfunction, leading to airway dehydration, abnormal mucin production, and impaired mucus transport. Smoke exposure also induces mucus hypersecretion and ciliary dysfunction; together, these changes result in defective mucus clearance and subsequent muco-obstruction. E-cigarettes, a popular alternative to traditional cigarettes, are commonly perceived to be safer than smoking. However, evidence has shown that e-cigarette components and vapor can cause defects in the airways similar to tobacco smoke. Thus, we hypothesized that e-cigarette use can induce acquired CFTR dysfunction in a similar manner to cigarette smoking, which may contribute to COPD pathogenesis. We demonstrate that e-cigarette vapor not only induces acquired CFTR dysfunction, but it also contains respiratory toxicants known to impair CFTR activity, suggesting that e-cigarettes may still lead to pulmonary decrements with chronic use. Additionally, muco-obstruction paired with observations of mucus abnormalities in CF and cigarette smoke-induced acquired CFTR dysfunction, led to the hypothesis that cigarette smoke exposure alters the airway functional microanatomy and mucus viscoelasticity, contributing to mucus transport decrements. Using real-time imaging that allows co-localized measures of the mucociliary transport (MCT) apparatus in conjunction with a novel COPD animal model, we establish that chronic smoke exposure impairs MCT in a manner that is dependent on the functional microanatomy as well as mucus properties. Here, we provide evidence that cigarette alternatives can induce airway epithelial ion transport defects that may contribute to COPD pathogenesis, and that mucociliary dysfunction and mucus abnormalities are important in COPD. Based on our data, augmenting secretion by way of CFTR channel activation or by cholinergic stimulation may improve ion transport and overcome airway surface dehydration and MCT decrements, respectively; these, along with muco-active agents to reduce mucus viscoelasticity, are promising therapeutic avenues for slowing COPD progression

The EGFR-ADAM17 Axis in Chronic Obstructive Pulmonary Disease and Cystic Fibrosis Lung Pathology

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) share molecular mechanisms that cause the pathological symptoms they have in common. Here, we review evidence suggesting that hyperactivity of the EGFR/ADAM17 axis plays a role in the development of chronic lung disease in both CF and COPD. The ubiquitous transmembrane protease A disintegrin and metalloprotease 17 (ADAM17) forms a functional unit with the EGF receptor (EGFR), in a feedback loop interaction labeled the ADAM17/EGFR axis. In airway epithelial cells, ADAM17 sheds multiple soluble signaling proteins by proteolysis, including EGFR ligands such as amphiregulin (AREG), and proinflammatory mediators such as the interleukin 6 coreceptor (IL-6R). This activity can be enhanced by injury, toxins, and receptor-mediated external triggers. In addition to intracellular kinases, the extracellular glutathione-dependent redox potential controls ADAM17 shedding. Thus, the epithelial ADAM17/EGFR axis serves as a receptor of incoming luminal stress signals, relaying these to neighboring and underlying cells, which plays an important role in the resolution of lung injury and inflammation. We review evidence that congenital CFTR deficiency in CF and reduced CFTR activity in chronic COPD may cause enhanced ADAM17/EGFR signaling through a defect in glutathione secretion. In future studies, these complex interactions and the options for pharmaceutical interventions will be further investigated

Studies on Nontypeable Haemophilus Influenzae Biofilm Persistence & Redox Homeostasis in Chronic Bacterial Diseases

Bacterial biofilms, or surface-adherent bacterial communities surrounded by a self-produced extracellular polymeric matrix, are ubiquitous. They are found throughout Earth’s ecosystems, in the soil and waterways, and more pressingly for the medical community, they are found within humans. Bacterial biofilms are associated with a multitude of diseases and their involvement significantly complicates treatment given that bacteria growing within a biofilm are extremely more resistant to antibiotics and immune effectors. Thus, the need to better understand biofilm biology and find ways to disrupt and/or sensitize biofilms to medical treatment is significant. The goal of the research presented herein was to investigate the role of nontypeable Haemophilus influenzae (NTHi) biofilms in chronic obstructive disease and study the viability of targeting bacterial redox homeostasis as an anti-biofilm strategy in multiple disease contexts. In the first portion of the research presented herein, we established a NTHi infection model utilizing the smoke exposed ferret COPD animal model. We characterized the infection dynamics of NTHi, particularly we were able to establish that NTHi forms biofilms within the smoke exposed ferret airways, and the host responses to NTHi, and the airway functional and structural changes induced by NTHi infection. Establishing this physiologically relevant model of COPD using the most common bacterial pathogen of COPD sets the stage for significant advancements in our understandings of the complex interactions occurring within the smoke exposed airways. iv Building upon this work, we turned our attention to investigating anti-biofilm strategies by targeting bacterial redox homoeostasis pathways. We went on to generate five thiol redox pathway mutants and characterize their ability to respond to oxidative stress using both in vitro techniques and both a smoke exposed murine infection model and a chinchilla otitis media model. We demonstrated disruption of these genes in the thiol redox pathways sensitized NTHi biofilms to oxidative stress and compromised the ability of NTHi to establish a successful infection in both animal models. Together, highlighting the potentiality of thiol redox homeostasis as a druggable anti-biofilm target that has clinical relevance in multiple NTHi disease contexts

Animal Models of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the USA and currently there are minimal therapies specific for the treatment of COPD. To advance our knowledge on COPD pathogenesis and develop new therapeutics, animal models are needed that represent key clinical and pathologic features of the human disease. The primary animal models utilized to study COPD rely on several factors associated with disease progression, i.e. genetic and epigenetic changes, environmental exposures and the microbial flora of the lungs. Here, a systematic approach was taken to summarize and evaluate the current animal models employed to study COPD pathogenesis, comorbidities and exacerbations. The strengths and limitations of these disease models are also delineated. The rodent COPD models have been extensively utilized but several studies have highlighted the potential of larger animals as an additional approach. Due to the inherent heterogeneity of COPD, the usefulness of certain animal models may be limiting but still represent helpful means to explore gene functional studies, testing new therapeutics and the exploring the significance of microbial floral changes. Therefore, interpreting the findings from animal models for the study of COPD represents a critical approach in deciding possible future human therapeutics

Animal models and mechanisms of tobacco smoke-induced chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide, and its global health burden is increasing. COPD is characterized by emphysema, mucus hypersecretion, and persistent lung inflammation, and clinically by chronic airflow obstruction and symptoms of dyspnea, cough, and fatigue in patients. A cluster of pathologies including chronic bronchitis, emphysema, asthma, and cardiovascular disease in the form of hypertension and atherosclerosis variably coexist in COPD patients. Underlying causes for COPD include primarily tobacco use but may also be driven by exposure to air pollutants, biomass burning, and workplace related fumes and chemicals. While no single animal model might mimic all features of human COPD, a wide variety of published models have collectively helped to improve our understanding of disease processes involved in the genesis and persistence of COPD. In this review, the pathogenesis and associated risk factors of COPD are examined in different mammalian models of the disease. Each animal model included in this review is exclusively created by tobacco smoke (TS) exposure. As animal models continue to aid in defining the pathobiological mechanisms of and possible novel therapeutic interventions for COPD, the advantages and disadvantages of each animal model are discussed

Mucus Matters: Mucociliary Physiology In Chronic Lung Diseases

The mucociliary escalator is an innate defense mechanism in the lung comprised of the ciliated, pseudostratified epithelium that lines the conducting airways and the mucus that sits atop the cilia. Mucus traps inhaled particulate matter/pathogens, and the cilia brush works in concert to propel the mucin gel proximally. Therefore, proper mucociliary physiology is critical to maintaining lung health. A metachronal wave propagated across the epithelium can occur when the phase of the ciliary stroke is shifted in time relative to its neighbors. Metachrony has been hypothesized to help overcome the viscoelastic forces required for the propulsion of mucus. Still, its effect on mucociliary transport rate has not been characterized due to previous limitations in imaging technology. The first portion of my thesis was to describe the influence of metachronal beating on mucociliary transport (MCT) rates in human bronchi and ferret tracheae and investigate the role of calcium signaling on metachronal wave generation using μOCT. We found that mucociliary transport was significantly faster when metachrony was present. Treatment of ferret tracheae with either cell-permeant chelator of intracellular Ca2+, a nonpermeant Ca2+ channel competitive antagonist, or an inhibitor of calaxin was sufficient to abolish metachrony and had a deleterious effect on MCT. A gain-of-function promoter variant for MUC5B (rs35705950) is a significant risk factor for developing idiopathic pulmonary fibrosis (IPF); yet, the role of MUC5B mucin or abnormal mucociliary physiology in IPF pathogenesis is unknown. Bleomycin (BLM)-exposed rodent models do not develop sustained fibrosis or exhibit IPF-like airway remodeling. Ferrets, unlike mice, have a human-like distribution of Muc5b in the lung and natively express the riskconferring variant that induces high MUC5B expression in humans. Therefore, I established a BLM-ferret model for the second portion of my thesis. Our BLMinduced pulmonary fibrosis ferret demonstrated sustained fibrosis by μCT, hallmarks of restrictive physiology by flexivent, and pulse oxygenation decrement through 12-weeks following a single dose of BLM. Additionally, BLM ferrets recapitulate features of human IPF not observed in lower species, such as aberrant bronchiolization of the distal lung associated with fibrotic remodeling and pathologic lesions including fibroblastic foci and honeycomb cysts. These studies are significant for 1) adding fundamental knowledge about how metachronal waves influence mucociliary physiology and 2) developing the ferret model of pulmonary fibrosis, which will be a valuable tool to elucidate the impact of a human-like mucin microenvironment on fibrosis pathogenesis

Models of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a major global health problem and is predicted to become the third most common cause of death by 2020. Apart from the important preventive steps of smoking cessation, there are no other specific treatments for COPD that are as effective in reversing the condition, and therefore there is a need to understand the pathophysiological mechanisms that could lead to new therapeutic strategies. The development of experimental models will help to dissect these mechanisms at the cellular and molecular level. COPD is a disease characterized by progressive airflow obstruction of the peripheral airways, associated with lung inflammation, emphysema and mucus hypersecretion. Different approaches to mimic COPD have been developed but are limited in comparison to models of allergic asthma. COPD models usually do not mimic the major features of human COPD and are commonly based on the induction of COPD-like lesions in the lungs and airways using noxious inhalants such as tobacco smoke, nitrogen dioxide, or sulfur dioxide. Depending on the duration and intensity of exposure, these noxious stimuli induce signs of chronic inflammation and airway remodelling. Emphysema can be achieved by combining such exposure with instillation of tissue-degrading enzymes. Other approaches are based on genetically-targeted mice which develop COPD-like lesions with emphysema, and such mice provide deep insights into pathophysiological mechanisms. Future approaches should aim to mimic irreversible airflow obstruction, associated with cough and sputum production, with the possibility of inducing exacerbations

Toxicol Pathol

Smoking is a major risk factor for heart attack, stroke, and lung cancer. Tobacco smoke (TS) causes bronchitis, emphysema, persistent cough, and dyspnea. Smoking cessation minimizes risks of TS-related disease. To determine whether smoking cessation could reverse TS-induced pulmonary changes, 10-week-old male spontaneously hypertensive rats were exposed to TS or filtered air (FA) for 39 weeks and allowed to live out their normal lifespan. Significantly (| 64 .05) decreased survival was noted by 21 months in TS versus FA rats. In TS rats, persistent peribronchiolar, perivascular, alveolar, and subpleural inflammation were observed with pervasive infiltration of pigmented foamy macrophages and plausible intra-alveolar fibrosis and osseous metaplasia. Alveolar airspace was significantly (| 64 .05) increased in TS versus FA rats as was the volume of stored epithelial mucosubstances in the left central axial airway. Increased mucin contributes to airflow obstruction and increased lung infection risks. Findings suggest TS-induced changes do not attenuate with smoking cessation but result in irreversible damage similar to chronic obstructive pulmonary disease. The observed persistent pulmonary changes mirror common TS effects such as chest congestion, sputum production, and shortness of breath long after smoking cessation and represent important targets for treatment of former smokers.P30 ES023513/ES/NIEHS NIH HHS/United StatesU54 OH007550/OH/NIOSH CDC HHS/United States2021-04-01T00:00:00Z31870229PMC71131187487vault:3520