Epithelial Reparative Capacity Regulates Extracellular Matrix Dynamics and Innate Immunity

The mammalian lung supports the transport and diffusion of inspired and expired gasses that are critical for aerobic life. With every inspiration the lung is exposed to environmental agents including microbes, virus, and environmental pollutants. In the event that injury occurs the epithelium is repaired by an abundant facultative progenitor pool and a sequestered population of adult tissue stem cells. Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease, and bronchopulmonary dysplasia, are characterized by extensive epithelial remodeling resulting in a reduction to the number of non-ciliated bronchiolar Clara cells. Given the established role for Clara cells as abundant facultative progenitors, these data suggest that epithelial repair has been compromised. In addition to affects on the epithelium, these diseases are also accompanied by extensive subepithelial fibroproliferation, mesenchymal remodeling, and elevated extracellular matrix deposition as well as a profound increase to lung inflammation. It has been postulated, but never tested in vivo that mesenchymal remodeling and uncontrolled deposition of extracellular matrix may be a result of impaired airway epithelial reparative capacity. Moreover, the finding that airway epithelial cells are essential for modulation of innate immunity suggests that the enhanced inflammatory response described in chronic lung disease may be a result of attenuated airway epithelial cell function. Therefore, this dissertation tests the hypothesis that airway epithelial reparative capacity moderates extracellular matrix deposition and innate immunity. Through the use of in vivo models of injury, inflammation, and attenuated Clara cell function, this dissertation research work identifies a previously uncharacterized process in which extracellular matrix is dynamically and reversibly regulated during productive epithelial repair and severely disrupted by blocking stem cell mediated repair. In addition, the use of mouse models of decreased Clara cell abundance and secretion demonstrate airway epithelium modulates pulmonary innate immunity through regulation of macrophage behavior and inhibition of pulmonary inflammation. This work defines two phenotypes that are the result of attenuated epithelial repair and supports the paradigm that epithelial reparative capacity may be a principal determinant of lung disease

Removal of Ozone by the Extrathoracic and Intrathoracic Airways in Man

Extrathoracic and intrathoracic ozone removal were studied in eighteen healthy, nonsmoking volunteers using a controlled environmental chamber. Ozone-containing air was drawn through a polyethylene sampling tube placed through one nostril into the posterior pharynx, and analyzed with a rapidly responding ozone analyzer. Measurements were made using a randomized split-split plot design for each subject at ozone concentrations of 0.1 ppm, 0.2 ppm and 0.4 ppm, using three breathing modes (nasal, oronasal and mouth), and at two different breathing frequencies (12 and 24 BPM). For extrathoracic ozone removal, significant effects were demonstrated for mode of breathing and breathing frequency (both with p< 0.001), but not for ambient ozone concentration. For intrathoracic ozone removal, significant effects were demonstrated for ambient ozone concentration (p=0.032) and for breathing frequency (p<0.001), but not for mode of breathing. Mechanisms are postulated to explain the results obtained.Master of Public Healt

Isolation and functional analysis of cellular components of the bronchiolar stem cell hierarchy

Mouse bronchiolar stem cells have been identified in vivo based on functional characteristics including naphthalene resistance, long-term retention of labeled DNA precursors, and dual expression of markers for airway (CCSP) and alveolar (pro-SPC) epithelium. Further characterization would benefit from establishment of rigorous enrichment strategies allowing analysis of their behavior in vitro and following transplantation, and the establishment of a defining gene expression signature. We have determined that Epithelial Cell Adhesion Molecule (EpCAM) and Integrin α6 are expressed on the cell surface of both alveolar and bronchiolar epithelial cells and that low levels of Sca-1 expression characterize the bronchiolar epithelium. Within the Sca-1low EpCAMpos Integrinα6pos population of bronchiolar epithelial cells, autofluorescence (AF) levels distinguish the facultative transit-amplifying population which is AFhi from bronchiolar stem cells which are AFlow. Use of transgenic animal models allowing expansion or depletion of the stem cell compartment and use of lineage tracing strategies have allowed us to determine the identity of cells isolated based on their cell surface phenotype and autofluorescence characteristics. Injury models associated with depletion of terminally differentiated ciliated cells (ozone) or facultative transit amplifying population (naphthalene) were used to validate the functional characteristics of the two fractions of bronchiolar progenitors. In conclusion, we have developed and validated a fractionation approach for the generation of highly purified preparations of bronchiolar stem and Clara cells from the mouse lung. These data enable establishment of robust in vitro and transplantation assays to further validate the functional behavior of stem and facultative TA (Clara) cells and allows analysis of gene expression profile of the two populations towards a better understanding of unique characteristics of the bronchiolar stem cell compartment

Early events of Jaagsiekte sheep retrovirus infection in the ovine lung

Ovine pulmonary adenocarcinoma (OPA) is a chronic respiratory disease of adult sheep caused by Jaagsiekte sheep retrovirus (JSRV). The primary route of disease transmission is by inhalation of the virus, which then infects respiratory epithelial cells initiating oncogenesis and tumour growth. Clinical signs are seen towards end stage disease and include laboured breathing and weight loss. In these animals, the production of copious amounts of virus rich fluid which pours from the nose when the hind legs are lifted is a common finding and pathognomic for OPA. Once these signs are apparent, the disease is invariably fatal. The worldwide prevalence of OPA has both economic and welfare implications. However, there is currently no effective preclinical test or vaccine to control spread of disease. This is primarily due to the lack of detectable immunological response at any stage of disease pathogenesis. Theories of central and peripheral tolerance have been proposed as explanations for this. OPA is also regarded as a potential model for human lung cancer. As in sheep, clinical presentation in humans is not until tumour growth is extensive by which time treatment is unsuccessful. This makes it difficult to study the initial stages of disease, and identify potential markers for early detection or targets for therapeutics. The aim of this study was to investigate these previously unexplored early stages of disease pathogenesis for OPA. The primary areas of interest were identification of the target cells for JSRV infection in the lung and analysis of the innate response following this infection. Samples for analysis were provided by the intratracheal inoculation of specific pathogen free lambs with an infectious molecular clone JSRV[21]. Lambs were euthanased 3, 10 and 72-91 days following inoculation and lung samples representing different stages of disease pathogenesis were collected from each lamb. Mock infected and non infected lambs were included as negative controls at each time point. Immunohistochemistry was used to localise virus expression to specific epithelial cell types within the ovine respiratory tract. The same techniques also offered a means of studying the postnatal development of the ovine respiratory tract. The innate response to infection and subsequent tumour growth was measured in terms of cytokine production. RNA was extracted from adjacent samples to those used for IHC, and qRTPCR measured mRNA levels of a number of inflammatory cytokines and chemokines at each time point. Immunohistochemical analysis of the ovine respiratory tract found evidence of cytodifferentiation during postnatal development. This has implications for the susceptibility of lambs to infectious and noxious insults during this period. JSRV was found to target multiple cell types in the ovine lung. These included Clara cells, type II pneumocytes and cells which did not express either of these mature markers. Analysis of cytokine expression in lung tissue both before and during JSRV expression and tumour growth found little evidence of a host response to virus expression. Changes in the levels of cytokine mRNA were detected for those primarily involved in tumour growth and survival. These included IL-8 and IDO, both of which have been found to be increased in some cases of human of lung tumour. These findings increase the understanding of the pathogenesis of OPA, and improve its validity as an animal model for human lung cancer

The INDEX Project - Critical Appraisal of the Setting and Implementation of Indoor Exposure Limits in the EU

The INDEX project (Critical Appraisal of the Setting and Implementation of Indoor Exposure Limits in the EU), coordinated by the EU/JRC and funded by DG SANCO, has been finished in December 2004. The project was carried out in collaboration with a Steering Committee of leading European experts in the area of indoor air pollution. Scope of INDEX was to identify priorities and to assess the needs for a Community strategy and action plan in the area of indoor air pollution. The key issues that have been addressed within the project are: - the setting up of a list of compounds to be measured and regulated in indoor environments with priority, on the basis of health impact criteria - to provide suggestions and recommendations on potential exposure limits for these compounds and - to provide information on links with existing knowledge, ongoing studies, legislation etc. at world scale. Suggestions and recommendations on potential exposure limits or other exposure control actions were defined for five prioritised compounds; formaldehyde, nitrogen dioxide, carbon monoxide, benzene, and naphthalene.JRC.I.5-Physical and chemical exposure

Idiopathic pulmonary fibrosis: exploration of aberrant epithelial wound repair and stem cell-mediated regenerative approaches

Idiopathic pulmonary fibrosis (IPF) is a fatal form of fibrotic lung disease. The pathogenesis of IPF is unclear. An aberrant alveolar epithelial wound repair is likely to be involved in the disease process. Alveolar bronchiolisation, a process where bronchiolar Clara cells migrate into the affected alveoli, is a manifestation of abnormal alveolar wound repair. The role of Clara cells during alveolar injury repair in IPF is controversial. This study was undertaken to investigate the role of Clara cells in alveolar epithelial wound repair and pulmonary fibrosis. Currently, there is no curative treatment for IPF; therefore, stem-cell mediated regenerative therapy has been suggested. In this study, the paracrine role hMSC and hESC on pulmonary epithelial wound repair has also been evaluated. A direct-contact co-culture in vitro model was utilised to evaluate the role of Clara cells on alveolar epithelial cell wound repair. Immunohistochemistry was conducted on IPF lung tissue samples to replicate the in vitro findings ex vivo. The paracrine role of hMSC and hESC on pulmonary epithelial cells was evaluated by utilising the in vitro wound repair system.This study demonstrates that Clara cells induce apoptosis in AEC through a TRAILdependent mechanism, resulting in significant inhibition of wound repair. Furthermore in the IPF lungs, TRAIL-expressing Clara cells were detected within the fibrotic alveoli, together with widespread AEC apoptosis. This study also demonstrates that hMSC enhance AEC and SAEC wound repair via a paracrine mechanism through stimulation of cell migration; whereas, secretory factors of differentiated hESC promote AEC wound repair through stimulation of both cell proliferation and migration.Through this study I propose a novel hypothesis which implies that the extensive profibrotic remodelling associated with IPF could be driven by TRAIL-expressing Clara cells inducing AEC apoptosis through a TRAIL-dependent mechanism. My study also supports the notion of clinical application of hMSC and hESC or their secretory products as regenerative therapeutic modality for IPF