Doctor of Philosophy(PhD) / Over the last 20 years both asthma and obesity have increased in prevalence. What is the link? There are data to suggest that increasing obesity is a risk for the increase in prevalence of asthma. A number of mechanisms have been postulated including the effects of reduced lung volume on bronchial reactivity and mechanical changes with lower lung volumes. Other possibilities include other obesity-induced co-morbidities including gastro-oesophageal reflux. The aim of this thesis was to evaluate the link between asthma and obesity in both adult and childhood populations and to undertake experimental studies to examine the effects of changes in lung volume on bronchial reactivity. In chapter 1, the literature is reviewed. The current literature suggests that there is a link between diagnosis of asthma, new onset of asthma, symptoms of shortness of breath and wheeze. In chapter 2, data on 1997 adults in 3 population studies were analysed and the association between body mass index (BMI) and symptoms of shortness of breath and wheeze, diagnosis of asthma, medication usage for asthma, lung function and bronchial responsiveness were studied. This study showed that obesity was a risk for recent asthma (OR 2.04; 95%CI 1.02-3.76, p=0.048), symptoms of shortness of breath and wheeze (OR 2.6; 95%CI 1.46- 4.70, p=0.001), and medication usage for asthma (OR 2.53; 95%CI 1.36-4.70, p=0.003). There was a reduction in lung volume as measured by forced vital capacity (FVC), but there was no increase in bronchial hyperresponsiveness (BHR) (OR 0.87; 95% CI 0.35-2.21, p=0.78). Thus although the symptoms of asthma are increased there were no increases in BHR, despite significantly reduced lung volumes. The increase the medication usage is unlikely to have normalised the BHR, as there were ongoing symptoms suggestive of asthma. In chapter 3, data on 5993 children in 7 population studies were analysed and the association between BMI percentile and symptoms of cough, wheeze, ix diagnosis of asthma, medication usage for asthma, atopy, lung function and bronchial responsiveness was studied. After adjusting for atopy, sex, age, smoking and family history, BMI was a significant risk factor for wheeze ever (OR=1.06; 95%CI 1.01-1.10, p=0.008) and cough (OR=1.09; 95%CI 1.05-1.14, p=0.001) but not for recent asthma (OR=1.02; 95%CI 0.98-1.07 p=0.43), or bronchial hyperresponsiveness (OR=0.97; 95%CI 0.95-1.04 p=0.77). In girls, a higher BMI was significantly associated with higher prevalence of atopy (x2 trend 7.9, p=0.005), wheeze ever (x2 trend 10.4, p=0.001), and cough (x2 trend 12.3, p<0.001). These were not significant in boys. With increasing BMI in children, there was no reduction in lung volume, no increase in airway obstruction and no increase in bronchial responsiveness. In chapter 4, the hypothesis that obesity per se is associated with bronchial responsiveness was tested. Six obese women without asthma were compared to 6 non-obese women without asthma with high dose methacholine challenges to assess the bronchial responsiveness. There was no increase in bronchial responsiveness, and no difference in the position or shape of the high dose methacholine curve despite the fact that these women had reduced lung volumes associated with their obesity. In chapter 5, the hypothesis whether reduced lung volume per se would cause a change in greater mechanical effect, ie more marked airway narrowing in both non-asthmatic and asthmatic subjects was tested. Lung volumes and methacholine challenges were undertaken in the supine and erect position on different days. As expected in normal subjects there was a small reduction in lung volume on lying down, this was associated with an increase in the measure of bronchial reactivity DRR. In contrast, in asthmatics, there was no acute fall in lung volume and there were variable changes in the index of reactivity suggesting non-homogeneity in the lung function abnormality. This suggests changes in bronchial reactivity can occur without any relationship to lung volume change. These negative results suggest that lung volume changes that may occur in obesity are unlikely contributors to the apparent increase in asthma symptoms. In chapter 6, the hypothesis that the supposed increase in asthma symptoms in the obese were due to the effects of gastro-oesophageal reflux were assessed in 147 obese subjects graded for gastro-oesophageal reflux severity using manometry and gastroscopy. This study showed that subjects with increased gastro-oesophageal reflux did not have subjective increases in asthma prevalence, obstructive sleep apnoea, or snoring however they had a clear worsening of gas transfer as measured by carbon monoxide transfer suggesting a greater level of parenchymal disease. The overall results are that there is an increase of diagnosis of asthma, increase in symptoms of asthma and medication usage for the treatment of asthma in the obese. Objectively despite reductions in lung volume, there is no increase in bronchial responsiveness in this group suggesting that these symptoms are not related to true asthma, but to alternative co-morbidities associated with obesity such as gastro-oesophageal reflux. Notably gastrooesophageal reflux was not associated with increased asthma prevalence or airway obstruction. However it was associated with reduced gas transfer suggesting parenchymal disease. This suggests that the increase in symptoms of wheeze and shortness of breath in the obese should not be attributed to asthma in the absence of variable airflow limitation that is reversible spontaneously or with treatment, or with an increase in the existing bronchial hyperresponsiveness (BHR) to a variety of stimuli.

Perfluorinated compounds, non-degradable xenobiotics in many consumer products, can cause developmental toxicity in animals, and human exposure is associated with asthma symptoms. We tested the hypothesis that sustained chronic exposure to perfluorooctanoic acid (PFOA), fluorotelomer alcohol (FTOH) or perfluorooctanesulfonic acid (PFOS) induces lung dysfunction that exacerbates allergen-induced airway hyperresponsiveness (AHR) and inflammation. Mice were exposed to the chemicals from early gestation day to adulthood. Some pups were sensitized and challenged with ovalbumin. Serum PFOA was analyzed by liquid chromatograph-tandem mass spectrometry. Lung function was measured using a small animal ventilator. We assayed inflammatory cells in the lung, performed PCR for lung cytokines, and examined bronchial goblet cell hyperplasia by histology. Here we show that either PFOA or FTOH exposure can induce AHR, but neither one predisposes for exaggerated allergic lung inflammation or AHR. FTOH or PFOS exposure appears to suppress allergic lung inflammation, but does not affect allergic lung dysfunction.

Doctor of Philosophy(PhD) / Over the last 20 years both asthma and obesity have increased in prevalence. What is the link? There are data to suggest that increasing obesity is a risk for the increase in prevalence of asthma. A number of mechanisms have been postulated including the effects of reduced lung volume on bronchial reactivity and mechanical changes with lower lung volumes. Other possibilities include other obesity-induced co-morbidities including gastro-oesophageal reflux. The aim of this thesis was to evaluate the link between asthma and obesity in both adult and childhood populations and to undertake experimental studies to examine the effects of changes in lung volume on bronchial reactivity. In chapter 1, the literature is reviewed. The current literature suggests that there is a link between diagnosis of asthma, new onset of asthma, symptoms of shortness of breath and wheeze. In chapter 2, data on 1997 adults in 3 population studies were analysed and the association between body mass index (BMI) and symptoms of shortness of breath and wheeze, diagnosis of asthma, medication usage for asthma, lung function and bronchial responsiveness were studied. This study showed that obesity was a risk for recent asthma (OR 2.04; 95%CI 1.02-3.76, p=0.048), symptoms of shortness of breath and wheeze (OR 2.6; 95%CI 1.46- 4.70, p=0.001), and medication usage for asthma (OR 2.53; 95%CI 1.36-4.70, p=0.003). There was a reduction in lung volume as measured by forced vital capacity (FVC), but there was no increase in bronchial hyperresponsiveness (BHR) (OR 0.87; 95% CI 0.35-2.21, p=0.78). Thus although the symptoms of asthma are increased there were no increases in BHR, despite significantly reduced lung volumes. The increase the medication usage is unlikely to have normalised the BHR, as there were ongoing symptoms suggestive of asthma. In chapter 3, data on 5993 children in 7 population studies were analysed and the association between BMI percentile and symptoms of cough, wheeze, ix diagnosis of asthma, medication usage for asthma, atopy, lung function and bronchial responsiveness was studied. After adjusting for atopy, sex, age, smoking and family history, BMI was a significant risk factor for wheeze ever (OR=1.06; 95%CI 1.01-1.10, p=0.008) and cough (OR=1.09; 95%CI 1.05-1.14, p=0.001) but not for recent asthma (OR=1.02; 95%CI 0.98-1.07 p=0.43), or bronchial hyperresponsiveness (OR=0.97; 95%CI 0.95-1.04 p=0.77). In girls, a higher BMI was significantly associated with higher prevalence of atopy (x2 trend 7.9, p=0.005), wheeze ever (x2 trend 10.4, p=0.001), and cough (x2 trend 12.3, p<0.001). These were not significant in boys. With increasing BMI in children, there was no reduction in lung volume, no increase in airway obstruction and no increase in bronchial responsiveness. In chapter 4, the hypothesis that obesity per se is associated with bronchial responsiveness was tested. Six obese women without asthma were compared to 6 non-obese women without asthma with high dose methacholine challenges to assess the bronchial responsiveness. There was no increase in bronchial responsiveness, and no difference in the position or shape of the high dose methacholine curve despite the fact that these women had reduced lung volumes associated with their obesity. In chapter 5, the hypothesis whether reduced lung volume per se would cause a change in greater mechanical effect, ie more marked airway narrowing in both non-asthmatic and asthmatic subjects was tested. Lung volumes and methacholine challenges were undertaken in the supine and erect position on different days. As expected in normal subjects there was a small reduction in lung volume on lying down, this was associated with an increase in the measure of bronchial reactivity DRR. In contrast, in asthmatics, there was no acute fall in lung volume and there were variable changes in the index of reactivity suggesting non-homogeneity in the lung function abnormality. This suggests changes in bronchial reactivity can occur without any relationship to lung volume change. These negative results suggest that lung volume changes that may occur in obesity are unlikely contributors to the apparent increase in asthma symptoms. In chapter 6, the hypothesis that the supposed increase in asthma symptoms in the obese were due to the effects of gastro-oesophageal reflux were assessed in 147 obese subjects graded for gastro-oesophageal reflux severity using manometry and gastroscopy. This study showed that subjects with increased gastro-oesophageal reflux did not have subjective increases in asthma prevalence, obstructive sleep apnoea, or snoring however they had a clear worsening of gas transfer as measured by carbon monoxide transfer suggesting a greater level of parenchymal disease. The overall results are that there is an increase of diagnosis of asthma, increase in symptoms of asthma and medication usage for the treatment of asthma in the obese. Objectively despite reductions in lung volume, there is no increase in bronchial responsiveness in this group suggesting that these symptoms are not related to true asthma, but to alternative co-morbidities associated with obesity such as gastro-oesophageal reflux. Notably gastrooesophageal reflux was not associated with increased asthma prevalence or airway obstruction. However it was associated with reduced gas transfer suggesting parenchymal disease. This suggests that the increase in symptoms of wheeze and shortness of breath in the obese should not be attributed to asthma in the absence of variable airflow limitation that is reversible spontaneously or with treatment, or with an increase in the existing bronchial hyperresponsiveness (BHR) to a variety of stimuli.

No description available.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. This thesis investigated the role of Syk in the development of the asthma phenotype in acute and chronic mouse models of allergic airways inflammation. Airway hyperresponsiveness (AHR) to methacholine and inflammation increased significantly in HDM-induced compared with the saline control mice. We demonstrated that in vivo inhibition of Syk by selective Syk inhibitors, and genetic deletion of Syk using conditional Syk knockout mice attenuated AHR despite of inflammatory cell influx in the lung. Histological analysis showed airway remodeling in the chronic model, which was attenuated to some degree by deletion of Syk. This study identified a role of Syk in airway hyperresponsiveness and remodeling without significantly affecting leukocyte recruitment in HDM model of airways disease. My results support the improvement of therapeutic strategies in asthma by targeting the Syk pathway.

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.