Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease. Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells. S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients. Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity. Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

Current methods for the risk assessment of environmental exposures commonly involve questionnaires, stationary monitoring, and personal air sampling. However, as these approaches do not capture the body's internal response, they lend minimal understanding to the biologic consequence of exposure. In order to address the unmet need of connecting external exposure measurements with signatures of internal exposure, this thesis examines the overarching hypothesis that transcriptomic changes in the human airway epithelium can serve as indicators of physiologic responses to inhaled pollutants. This is an extension of previous work that has demonstrated an airway ''field of injury'' effect where cigarette smoke exposure alters gene-expression in epithelial cells lining the respiratory tract. Specifically, I examine transcriptomic changes and the biologic responses associated with exposure to the following pollutants: environmental tobacco smoke (Aim 1), household air pollution from smoky coal combustion (Aim 2), and electronic cigarette vapor (Aim 3). First, I performed whole-genome transcriptional profiling of the nasal epithelium in children and adults and detected gene-expression changes associated with exposure to environmental tobacco smoke. Next, I employed similar approaches to detect a signature of coal smoke exposure in the buccal epithelium of healthy, non-smoking females exposed to household air pollution Xuanwei, China. The findings from these studies suggest that upper airway gene-expression can reflect the host response to prolific sources of environmental exposures that are major risk factors for chronic lung disease. Lastly, I examine the cellular and physiologic consequences of electronic cigarette (ECIG) aerosol exposure by analyzing transcriptomic profiles of human bronchial epithelial cells that have either been (1) differentiated and exposed in vitro or (2) acquired via bronchoscopy from the airway epithelium of ECIG users. The studies detailed in this dissertation offer valuable insight that will accelerate the efforts to evaluate the health effects of both well-established and emerging types of inhaled exposures in large-scale population studies. Furthermore, the transcriptomic strategies woven throughout the following chapters push for a novel assessment paradigm that may enable the public health community to rapidly characterize the physiologic host response to inhalation exposures of different sources, and to evaluate the biologic consequences of exposure-reduction initiatives. / 2017-05-01T00:00:00Z

The human body encounters a number of chemical exposures on a daily basis, which may have short- or long-term health implications. Previously it has been demonstrated that the entire respiratory tract of an individual reacts to exposures like tobacco smoke in a similar manner, and that common molecular changes can be measured in airway epithelium. I propose that cataloguing the exposure of airway epithelial cells to tobacco cigarette (TCIG) smoke and its constituents, electronic cigarette (ECIG) aerosol and other drugs and small molecules can significantly increase the understanding of chemical exposure and identify common gene expression alterations. First, I determined the molecular impact of ECIG aerosol exposure on human airway epithelium in vitro, including alterations in genes related to xenobiotic metabolism, oxidative stress, and ciliated cells. These changes were generally less pronounced than the effects of TCIG exposure, and were more pronounced in ECIG products containing nicotine than those without nicotine. Furthermore, gene expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users. Second, I examined the impact of TCIG exposure and TCIG constituents on premalignant airway cells, to better understand the progression or regression of precancerous lesions. These data could also identify the constituents of TCIGs and the precancerous mutations that increase the risk for malignancy. Third, in an effort to build a high-throughput methodology for chemical exposures, I exposed primary lung cell lines to small molecule therapeutics and identified lung-specific and lung cell-type-specific effects of exposure, suggesting that profiling additional cell lines would further inform airway gene expression in response to exposure and that organ-specific exposure profiling may provide valuable insight into drug discovery for common diseases. Overall, transcriptomic profiles from the airway epithelium reflect exposure to various inhaled and chemical perturbations. These gene expression profiles indicate common changes across a multitude of airway exposures as well as unique alterations specific to a given perturbation. Gene expression profiling can therefore be used to detail the potential response to a compendium of chemical exposures including those that are either well-established or potential risk factors for chronic lung diseases. / 2019-07-09T00:00:00Z

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O objetivo do presente trabalho foi propiciar o acesso in vivo ao epitélio respiratório e estudar a frequência de batimento ciliar (FBC) e a diferença de potencial transepitelial (DP) em um modelo murino de doença alérgica das vias aéreas induzida por ovoalbumina. Camundongos Swiss foram sensibilizados com ovoalbumina (OVA) através de duas injeções intraperitoneais de alérgico com o adjuvante hidróxido de alumínio (dias 0 e 14) e quatro inalações de OA 1% (dias 22, 24, 26 e 28). O grupo controle (S) foi tratado com salina 0,9 % seguindo o mesmo protocolo. Após 48h da última inalação, os camundongos foram anestesiados, a traquéia foi exposta longitudinalmente (1x4 mm) e o epitélio pode ser visualizado. A FBC foi mensurada pela técnica estroboscópica antes (basal) e logo após a administração inalatória das drogas (salbutamol e brometo de ipratrópio). A DP foi mensurada nos grupos S e OVA. Foram avaliados o lavado broncoalveolar e o remodelamento do epitélio da cavidade nasal, traquéia e vias aéreas distais. Nenhuma diferença foi encontrada na FBC basal entre os grupos (OVA e S), no entanto o grupo OVA mostrou uma DP basal significativamente menor. A inalação de salbutamol (3.5.10-3M ou 3.5.10-4M) elevou a FBC nos grupos estudados (p<0,05). O brometo de ipratrópio (10- 4M e 6.10-4M) não influenciou a FBC basal. Nossos resultados mostraram que é possível avaliar a FBC e a DP in vivo em um modelo murino de doença pulmonar alérgica crônica, e indicam que o processo inflamatório não afeta a FBC, mas contribui para o aumento de muco nas vias aéreas com conseqüências deletérias ao transporte mucociliar facilitando a retenção / The aim of the present work was to propitiate the in vivo assessment of the respiratory epithelium. The effects of salbutamol and ipratropium bromide on ciliary beat frequency (CBF) in a murine model of allergic airway disease were addressed. Transepithelial electric potential difference (PD) was also measured in order to verify the integrity of the epithelial barrier. Mice were sensitized with ovalbumin (OVA) by two intraperitoneal injections of allergen (days 0 and 14) and four inhalations of OVA 1% (days 22, 24, 26 and 28). The control group was treated with saline following the same procedures. After 48 hs of the last inhalation, mice were anesthetized, trachea was opened longitudinally (1 x 4 mm) and the ciliated epithelium could be visualized. CBF was measured by a modification on the videoscopic technique. We measured the CBF before and just after the administration of aerosolized substances. The PD was also measured on groups OVA and S. Additionally, the eosinophil cell count was measured on broncoalveolar lavage (BAL) in order to access the magnitude of airway inflammation. No difference on baseline CBF was noticed between groups (OVA and S), however the OVA group had a significantly lower PD. The administration of aerosolized capsaicin (3.10-9M) and salbutamol (3.5.10-3M or 3.5.10-4M) increased CBF in all groups studied. Ipratropium bromide (10-4M and 6.10- 4M) did not influence the CBF. The eosinophil cell count in broncoalveolar lavage was higher in OVA group compared to S group. CBF and PD results indicate that the inflammatory process does not affect the ciliary beat frequency but augments the amount of mucus in the airway, with deleterious consequences to the mucociliary transport facilitating mucus retention. Our results demonstrated for the first time the possibiliy of studying airway epithelium in an in vivo murine model of allergic airway disease

<p>S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.</p><p>Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.</p><p>Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate <i>in vitro</i> evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.</p><p>Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.</p>

<p>Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. </p><p>A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest.</p><p>Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. </p><p>A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies.</p><p>Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients.</p><p>Successful <i>in vitro </i>transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.</p>

Throughout many parts of the world house dust mites (HDM) are considered as a major source of indoor aeroallergens and they are powerful inducers of allergic diseases. Proteolytic HDM allergens are recognised as being able to directly activate respiratory epithelial cells and thereby actively participate in innate immune responses. Although several major HDM allergens lack proteolytic activity, their possible ability to similarly interact with epithelial cells is not known. The overall aim of this thesis was therefore to elucidate if and how major non-proteolytic group 2 allergens from different mite species interact with respiratory epithelial cells. The effects of the structurally related Der p 2, Der f 2 and Eur m 2 from different HDM species as well as the storage mite allergen Lep d 2 were studied in vitro using human respiratory epithelial cells. Also the non-proteolytic, but structurally dissimilar, Fel d 1 from cat, Can f 2 from dog, Bet v 1 from birch and Phl p 5a from timothy were studied. In this thesis evidence that major group 2 mite allergens activate bronchial epithelial cells is presented. Following allergen exposure the secreted amount of the inflammatory mediators G-CSF, GM-CSF, IL-6, IL-8, MCP-1, MIP-3α and sICAM-1 was increased. Surface expression of ICAM-1 was also increased following allergen exposure. Moreover, Fel d 1 and Can f 2 induced secretion of the same mediators from bronchial epithelial cells, representing two additional protein structures being able to directly induce cell activation. In experiments using specific inhibitors and siRNA transfection, it was shown that the mite allergens engage TLR4 and activation through MyD88, MAPK and NF-κB signal transduction pathways. In conclusion, the novel findings in this thesis provide knowledge on how major aeroallergens, in addition to their ability to provoke specific adaptive immune responses, may aggravate a respiratory airway disease by adjuvant-like activation of inflammatory responses in bronchial epithelial cells. This differs from previously reported allergen-induction of epithelial cells by the clear independency of proteolytic activation.