Dissertations / Theses on the topic 'Airway eosinophilia'

Eosinophilia bronchitis is a condition characterised by chronic cough and a sputum eosinophilia without the variable airflow obstruction or airway hyperresponsiveness characteristics of asthma. This thesis describes the incidence of eosinophilic bronchitis as a cause of chronic cough and determines whether the presence of a sputum eosinophilia is associated with a favourable response to corticosteroids in eosinophilic bronchitis and chronic obstructive pulmonary disease (COPD). This is the first study to investigate in detail the immunopathology of patients with eosinophilic bronchitis compared to asthma. Understanding differences in the immunopathology will inform our understanding of the development of the disordered airway physiology observed in asthma. We demonstrated that eosinophilic bronchitis is a common cause of chronic cough and that a sputum eosinophilia predicts a good response to corticosteroids in this condition, asthma and COPD. We found sputum, bronchial wash and bronchoalveolar lavage (BAL) eosinophilia and bronchial submucosal evidence of eosinophilic airway inflammation, increased Th2 cytokine expression and basement membrane thickening and increased constitutive intracellular expression of IL-4 from BAL derived T-cells in subjects with eosinophilic bronchitis to the same degree as those with asthma. Thus, the immunopathology of eosinophilic bronchitis is very similar to asthma questioning the importance of these classical pathological characteristic of asthma in the development of abnormal airway physiology. In addition, we quantified the inflammatory cell infiltration of the airway smooth muscle and found a striking increase in the number of mast cells within the airway smooth muscle in asthma compared to eosinophilic bronchitis and normal subjects. Our findings suggest that in asthma the microlocalisation of mast cells within the airway smooth muscle is a key factor in the development of variable airflow obstruction and airway hyperresponsiveness. Thus, specific targeting of the mast cell-smooth muscle interaction may provide a novel approach to the effective treatment for asthma.

Background: Eosinophilic airway inflammation (>3% sputum eosinophils) is a feature of subgroup of subjects with chronic obstructive pulmonary disease (COPD). My objectives were to investigate the clinical characteristics of eosinophilic COPD, its stability over time and extent in bronchial tissue, whether it is related to parasite exposure or atopy and whether its persistence is due to abnormal clearance by macrophages. Methods: Subjects were studied that had participated in previous observational studies. The repeatability of sputum eosinophils was measured between 3 monthly visits for 1 year. The extent of eosinophilic inflammation in bronchial tissue was assessed using immunohistology on bronchial tissue from COPD and control subjects. Positive serology for parasites was tested in serum samples for 4 helminth species. Atopy was assessed in the subjects using serum total Ig-E and skin prick test. Eosinophil efferocytosis by macrophages was investigated in vivo using cytoplasmic area of red hue of macrophages and in vitro using apoptotic eosinophils fed to monocyte-derived macrophages from COPD and healthy controls. The dynamics of eosinophil clearance during exacerbations was explored using the red hue technique. Results: Eosinophilic and non-eosinophilic COPD have similar lung function and exacerbation frequency. In eosinophilic COPD the health status is better and bacterial colonisation is lower. This phenotype is stable over time. Airway tissue eosinophilis are increased in COPD subjects with high blood eosinophils and are positively correlated with features of remodelling. Eosinophilic COPD is not associated with helminth exposure, but is related to elevated total Ig-E. Macrophage efferocytosis of eosinophils is impaired in COPD and is associated with the severity and frequency of COPD exacerbations. Efferocytosis of eosinophils by macrophages is increased following oral corticosteroid therapy at exacerbation. Conclusion: Eosinophilic COPD is a distinct and stable phenotype that persists in the blood, bronchus and sputum. Its persistence is partly related to atopy and impaired clearance by macrophages with the latter associated with COPD exacerbation severity and frequency.

There is no doubt that eosinophils and neutrophils are important cells participating in asthma pathogenesis. The most prominent feature reflecting asthma pathogenesis is late-phase airway inflammation, which occurs a few hours after allergen inhalation. The worldwide published studies on asthma show that most attention is paid to individual, not complex, functions of neutrophils and eosinophils in the airways. Moreover, associations between peripheral blood neutrophil and eosinophil activity and infiltration of these cells in the airways during asthma have not been com¬pletely elucidated yet. There are no data about peripheral blood neutrophil and eosinophil activity during allergen-induced late-phase airway inflam¬mation in asthma patients. Therefore, the aim of this study was to evaluate peripheral blood neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma. We found that an inhaled allergen activates peripheral blood neutrophil and eosinophil chemotaxis, phagocytosis, generation of reactive oxygen species and also reduces apoptosis during late-phase airway inflammation in asthma. Furthermore, altered peripheral blood neutrophil and eosinophil functional activity is related with airway neutrophilia and eosinophilia. Our findings provide new evidence about neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma patients.
Mokslininkai neabejoja, jog eozinofilai ir neutrofilai yra vienos svarbiausių ląstelių, dalyvaujančių astmos patogenezėje, kurią labiausiai atspindi vėlyva kvėpavimo takų uždegimo fazė, išsivystanti praėjus kelioms valandoms po alergeno įkvėpimo. Pasaulinėje literatūroje publikuojami darbai, nagrinėja atskirus kvė¬pavimo takų neutrofilų ir eozinofilų aktyvumo pokyčius. Ypač mažai darbų apie periferinio kraujo neutrofilų ir eozinofilų funkcijas bei jų ryšį su šių ląstelių pagausėjimu kvėpavimo takuose, sergant astma. Taip pat nėra tyrimų, vertinančių periferinio kraujo uždegimo ląstelių (neutrofilų ir eozi¬nofilų) funkcijų alergeno sukeltos vėlyvos fazės kvėpavimo takų uždegimo metu. Todėl šio tyrimo tikslas buvo įvertinti periferinio kraujo neutrofilų ir eozinofilų funkcinį aktyvumą alergeno sukeltos vėlyvos fazės kvėpavimo takų uždegimo metu sergant astma. Tyrimo metu nustatėme, kad įkvėptas alergenas aktyvina periferinio kraujo neutrofilų ir eozinofilų funkcijas - chemotaksį, fagocitozę, reaktyvių deguonies formų susidarymą, degranuliaciją bei silpnina apoptozę vėlyvos fazės kvėpavimo takų uždegimo metu. O šių ląstelių aktyvumo pokyčiai yra susiję su kvėpavimo takų neutrofilija ir eozinofilija. Moksliniame darbe pateikiami rezultatai suteikia naujų duomenų apie sergančiųjų alergine astma periferinio kraujo neutrofilų ir eozinofilų funkcinių savybių ypatumus ir parodo jų pokyčius alergeno sukeltos vėlyvos fazes kvėpavimo takų uždegimo metu.

Tissue eosinophilia is a prominent feature of allergic inflammatory diseases that may be in part mediated through the regulation of eosinophil survival at inflammatory site. T helper 2 type cytokines, such as interleukin (IL)-5, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), are the important mediators of allergic inflammation leading to prolonged survival of eosinophils. Due to the proposed central role of eosinophils in asthma and other allergic airway diseases, there is considerable interest, to determine the mechanisms that regulate eosinophil functions and accumulation. IL-12 and IL-18 attract considerable attention for their immunomodulatory roles on T helper cell subsets, and their potential to affect on eosinophil function.
The general aim of this study was to determine the effect of IL-12 and IL-18 on eosinophil functions. Results from this thesis demonstrate that eosinophils express functional receptors for IL-12 and IL-18. Acting alone or in synergy, IL-12 and IL-18 induced eosinophil apoptosis, in vitro. The apoptotic effect of IL-12 was reversed by IL-5, suggesting that IL-5 and IL-12 have counter-regulatory effects on eosinophil survival. Our regulation studies demonstrated that Phorbol-Myristate-Acetate (PMA) induced optimal expression of IL-18 and IL-12 receptors by eosinophils. IL-18 receptor expression by eosinophils was markedly increased following stimulation with interferon (IFN)-gamma, Tumor Necrosis Factor (TNF), or IL-12. Up-regulation of IL-18 receptor upon IL-12 stimulation was particularly important, which may explain IL-12 and IL-18 synergy on eosinophil apoptosis. We also investigated IL-12 and IL-18 expression by eosinophils. Eosinophils did not express IL-18. However, there was constitutive IL-12 expression by eosinophils. Release of IL-12 was also confirmed in eosinophil supernatants, which suggested an autocrine mechanism of IL-12 action on eosinophils.
Extending our understanding of the role of IL-12 and IL-18 in allergic inflammation, we have defined a novel pathway by which IL-12 and/or IL-18 may actually regulate eosinophils functions, and exert an inhibitory effect on eosinophil survival. Our findings provide critical new insights into mechanisms regulating eosinophil survival. To gain an even more detailed understanding of regulatory signals mediating eosinophil survival, we need to define the mechanisms involved in IL-12 and IL-18 signalling. We have just begun to identify the molecules involved, and these include IL-12 and IL-18 receptors. Activation via IL-12 and/or IL-18 receptor-mediated mechanisms may provide a novel strategy for reducing the numbers or inhibiting the function of these cells in allergic diseases or other diseases characterized by increased numbers of, or mediators from, eosinophils.

Although the aetiology of chronic cough in guidelines is clearly stated as asthma and related syndromes, gastro-oesophageal reflux disease, and upper airways disease, the inflammatory mechanisms underlying these conditions differ. Recent studies on asthma have increasingly focused on its molecular phenotypes instead of clinical characteristics. Predominantly in this thesis I hypothesize that by dividing cough patients into the clinical characteristics of eosinophilic and neutrophilic groups will enhance our ability to recognise the type of airway inflammation, and consequently will lead us to more targeted treatment approaches. To investigate this hypothesis I conducted a randomized, single centre, open label, controlled, clinical trial to examine the outcome of anti-inflammatory therapy with either montelukast or prednisolone in 50 patients with chronic cough. Furthermore, I studied the epidemiology of 137 chronic cough patients attending the Hull cough clinic. Results from the clinical study demonstrated that patients with FeNO≤20ppb had twice the number of coughs compared with patients with FeNO≥30ppb. This was reflected on quality of life as assessed by the LCQ and HARQ. Confirming this finding I found in the epidemiological study, that patients attending the hull cough clinic with FeNO≤25ppb scored significantly higher in HARQ compared with FeNO≥25ppb. In the clinical trial study I have shown that FeNO was a good marker for eosinophilic inflammation. There was a high degree of correlation with FeNO, blood and sputum eosinophilia thus confirming phenotypic identity. Whether the FeNO can be used to identify the different characteristics between eosinophilic and non-eosinophilic coughs needs further investigation. Cough patients in both low and high FeNO groups have shown a similar response to montelukast despite anticipating little or no effect in those without eosinophilic inflammation. These results suggest that response to montelukast may not be predicted by presence of eosinophilic biomarkers alone but may be act by effecting localised leukotriene mediated inflammation.

Elucidation of the inflammatory mechanisms of asthma has highlighted the role of the eosinophil as a major effector cell in asthma pathogenesis due to the arsenal of toxic granule proteins and other biological mediators secreted by this cell. Apoptosis induction in eosinophils and their consequent phagocytic removal from the inflamed lung would prevent the release of their detrimental cell constituents and would facilitate inflammation resolution. This thesis first established that airway epithelial cells (AEC) could specifically recognise and ingest apoptotic and not freshly isolated peripheral blood eosinophils. AEC also differentiated between apoptotic neutrophils and apoptotic eosinophils with the former not being recognised or ingested. Colon, mammary and retinal-pigmented epithelium exhibited the same selective phagocytosis suggesting that this was a ubiquitous epithelial phenomenon. Exposure of the AEC to the pro-inflammatory cytokines IL-1 and TNFa, or the glucorcorticoid dexamethasone enhanced the phagocytic capacity of these cells in a time and concentration dependent manner. Enhancement was observed both in terms of the number of cells within a monolayer phagocytosing apoptotic eosinophils and also with respect to the number of cells phagocytosed by these phagocytic AEC. CD44 ligation on the surface of A549 was also revealed to enhance phagocytic ability. The receptors utilised for apoptotic eosinophil recognition and phagocytosis were avb3, avb5, b5, CD36, PSr and unidentified lectins, which recognise the amino sugars, glucosamine, n-acetyl-glucosamine and n-acetyl-galactosamine but not their parent sugars. Neither cytokine nor glucorcorticoid of phagocytosis was associated with an increase in the expression of these receptors. Preliminary data was also found suggesting that cell-cell interactions between freshly isolated eosinophils and small airway epithelial cells (SAECs) that were co-cultured for 24h resulted in eosinophil apoptosis induction that was above that of constitutive apoptosis and only partially abrogated by IL-5 addition to the co-culture medium.

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ Eosinophils obtained from inflammatory tissues and secretions display an altered phenotype in comparison to peripheral blood eosinophils, with increased surface expression of major histocompatibility complex (MHC) proteins and adhesion molecules (Hansel et al., 1991), and migration across the microvascular endothelium may also increase their capacity to generate an oxidative burst (Walker et al., 1993; Yamamoto et al., 2000). Eosinophils are phagocytic cells, and have been shown to present simple (no requirement for intracellular processing) and complex antigens to MHC-restricted, antigen-specific T lymphocytes (Del Pozo et al., 1992; Weller et al., 1993). Furthermore, eosinophils express the costimulatory molecules required for effective antigen presentation (Tamura et al., 1996), and ligation of costimulatory molecules on the eosinophil cell surface can induce the release of eosinophil derived cytokines (Woerly et al., 1999; Woerly et al., 2002). Therefore the eosinophil may also regulate immune responses.¶ To date, no studies have demonstrated the ability of eosinophils to modulate activated T lymphocyte function via presentation of relevant antigen in the context of MHC class II (MHC-II), concomitant with Th2 cytokine release. In the experiments described in this thesis, murine eosinophils have been observed to rapidly migrate to sites of antigen deposition within the airways mucosa of naïve mice, suggesting a potential role for this granulocyte in the primary response to inhaled antigen. However, human allergic diseases are often diagnosed after the establishment of allergic responses, and symptom development. Therefore, a murine model of allergic airways disease (AAD) was used to investigate the ability for eosinophils to participate as antigen presenting cells (APCs), and thereby modulate activated T lymphocyte function both in vitro and in vivo. Detailed histological analysis of the pulmonary draining lymph nodes following antigen challenge in sensitised mice revealed a rapid infiltration of eosinophils into this tissue, which preceded the accumulation of eosinophils in bronchoalveolar lavage fluid (BALF). This suggested that eosinophils were preferentially translocating to the draining lymph nodes following antigen challenge, and that the subsequent accumulation of these cells in the BALF was a consequence of continued antigen delivery to the lower airways.¶ Eosinophil trafficking to lymphoid tissue via the afferent lymphatics was substantiated using electron microscopy of lymph node sections and the intravenous (i.v.) transfer of fluorescently labeled eosinophils, which did not traffic to lymph nodes via the blood. During the resolution of AAD, eosinophils were noted for their persistence in the pulmonary draining lymph nodes. These observations suggested a continued modulation of T cell function by lymph node dwelling eosinophils during AAD resolution, particularly in light of recent observations for draining lymph node T cell proliferation following instillation of antigen-pulsed eosinophils into the allergic mouse lung (Shi et al., 2000).¶ To further investigate the antigen presenting capacity, eosinophils were obtained from the BALF of mice with AAD, and their surface expression of MHC class II (MHC-II) proteins and costimulatory molecules confirmed using flow cytometric analysis. The ability to acquire and process complex antigen both in vitro and in vivo was also confirmed using naturally quenching fluorescenated ovalbumin (OVA), which is degraded into fluorescent peptides by the action of intracellular proteases. Thus, eosinophil expression of the surface molecules necessary for effective antigen presentation was confirmed, as was their ability to process complex antigen. Further investigations revealed that eosinophils can present complex OVA antigen to CD4+ T lymphocytes obtained from the allergic mouse, and to in vitro derived OVA-specific Th2 cells. In the presence of exogenous antigen, eosinophils co-cultured with T lymphocytes were able to induce Th2 cytokine production, and demonstrated an ability for eosinophils to modulate T lymphocyte function in vitro.¶ The ability for eosinophils to act as antigen presenting cells in vivo was also investigated. Eosinophils obtained from the antigen-saturated lungs of OVA sensitised and challenged mice were transferred to the peritoneal cavities of naïve host mice. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a pulmonary eosinophilia similar to that of OVA sensitised and challenged mice. To validate this finding, the experimental procedure was altered to accommodate the use of non-allergy derived eosinophils, which were pulsed with OVA in vitro, prior to transfer into naïve recipients. When subsequently challenged with aerosolised OVA, eosinophil recipients developed a peripheral blood and pulmonary eosinophilia, and stimulation with OVA induced IL-5 and IL-13 cytokine production from pulmonary draining lymph node cells. Notably, the AAD induced by transfer of antigen pulsed eosinophils did not induce detectable OVA-specific IgG1, which may be attributed to the lack of soluble antigen required for B cell antibody production.¶ During the course of these investigations, an OVA T cell receptor (TCR) transgenic mouse (OT-II) was procured with a view to defining the interaction between eosinophils and activated T lymphocytes (Barnden et al., 1998). Despite having specificity for the OVA323-339 peptide, an immunodominant epitope that skews naïve T cell responses towards Th2 cytokine release (Janssen et al., 2000), T lymphocytes from the OT-II mouse preferentially secreted IFN-γ in response to stimulation with either OVA peptide or OVA. These mice were further characterised in a mouse model of AAD, and found to be refractory to disease induction and progression, which may be attributed to significant IFN-γ secretion by transgenic CD4+ T lymphocytes during antigen sensitisation. Indeed, these cells were noted for their ability to attenuate pulmonary eosinophilia when transferred to OVA sensitised and challenged wild type mice, although serum OVA-specific IgG1, peripheral blood eosinophilia levels and airways response to methacholine challenge remained intact.¶ Knowledge of the biased Th1 phenotype in naïve OT-II provided a unique opportunity to investigate the fate of T lymphocytes bearing high affinity OVA-specific TCRs following neonatal antigen exposure to soluble OVA. In a previous study, subcutaneous (s.c.) administration of soluble OVA to wild type neonatal mice was suspected to have deleted OVA-specific T cells from the T cell repertoire (Hogan et al., 1998a). Using flow cytometry and TCR specific antibody, the delivery of s.c. OVA to OT-II neonates did not alter transgenic T cell populations in adult mice. Instead, it was surprising to find a skewing towards the Th2 phenotype and loss of IFN-γ secretion following OVA sensitisation and challenge in adult mice. A mechanism for this reprogramming of the transgenic T cell from the Th1 to a Th2 phenotype following OT-II neonatal exposure to soluble OVA is proposed, and further experimentation may validate this hypothesis.¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia

Background: According to recent studies in children, IgE sensitization not only against perennial allergens, but also against food allergens, is related to asthma risk and increased airway inflammation. During the last decade, a new technique for IgE determination based on allergen components has become available, but its use in epidemiological studies has been limited. Aims: To investigate the relationship between the pattern of IgE sensitization to allergen components and the prevalence of asthma, airway inflammation and hyperresponsiveness in a population-based setting. To examine the relationship of IgE sensitization to allergen extract, and airway inflammation, airway hyperresponsiveness and blood eosinophilia in asthmatics. To examine the natural history of IgE sensitization to food allergens in adults. To compare extract-based and component-based IgE measurements in relation with new-onset respiratory disease and airway inflammation and hyperresponsiveness. Methods: The present thesis is based on cross-sectional and longitudinal analyses of the adult, the population-based study ECRHS (European Community Health Survey) and a cross-sectional, observational study of young subjects with asthma. IgE sensitization was examined by means of both extract-based and component-based tests. Airway inflammation was assessed by exhaled NO and airway hyperresponsiveness with methacholine test. Results: IgE sensitization to food allergens independently related to increased airway inflammation in both a population-based study and a study of asthmatics. Furthermore, a relation was found with increased blood eosinophils in asthmatics. The decrease in prevalence of IgE sensitization against food allergens during a 9-year follow-up was larger than the decrease of aeroallergens. Subjects with IgE sensitization to both cat extract and components showed more frequent airway inflammation, greater bronchial responsiveness and higher likelihood of developing asthma and rhinitis than subjects with IgE sensitization only to cat extract. Conclusions: The presence of IgE antibodies against food allergens was independently associated with airway and systemic inflammation. Both aeroallergens and food allergens should be examined in order to understand the signaling of local and systemic inflammation in asthma. Prevalence of IgE sensitization to food decreased in adults to a larger extent than IgE sensitization against aeroallergens. Measurement of IgE sensitization to cat allergen components appears to have a higher clinical value than extract-based measurement

Indiana University-Purdue University Indianapolis (IUPUI)
More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is defined as a 'chronic inflammatory disorder of the airways, which involves many cells and cellular elements". Eosinophilic airway responses have been well characterised in asthma, and recently non-eosinophilic forms of asthma have been identified. The non-eosinophilic asthma phenotype is characterised by clinical symptoms of asthma and airway hyperresponsiveness occurring in the absence of raised sputum eosinophils. The non-eosinophilic pattern of inflammation has been reported across whole spectrum of asthma severity including mild asthma, persistent asthma and severe refractory asthma. The aim of this thesis was to investigate the characteristics and inflammatory mechanisms of non-eosinophilic asthma. The recognition of non-eosinophilic asthma using induced sputum eosinophil counts was examined. The role of proteolytic enzymes and innate immune activation as potential mechanisms of non-eosinophilic asthma were also examined in subjects with stable persistent symptomatic asthma treated with inhaled corticosteroids and healthy controls. A definition of non-eosinophilic asthma based upon a normal sputum eosinophil proportion was found to be reproducible. This was a heterogeneous inflammatory phenotype containing a group with increased neutrophils (neutrophilic asthma) and a group with normal levels of neutrophils and eosinophils termed paucigranulocytic asthma. Eosinophilic asthma was characterised by the presence of high levels of active matrix metalloproteinase-9 and low levels of neutrophil elastase. Neutrophilic asthma was characterised by high levels of neutrophil elastase and almost no active matrix metalloproteinase-9. Subjects with neutrophilic asthma also had evidence of chronic bacterial colonisation of the airways, high levels of airway endotoxin and innate immune activation with increased expression of the toll-like receptors 2 and 4 and increased surfactant protein A in sputum. Airway inflammation is heterogenous and can be classified into phenotypes based upon abnormal levels of sputum neutrophils and eosinophils. More than 50% of symptomatic persistent asthma is non-eosinophilic in nature, with more than 20% of these subjects having a neutrophilic airway inflammation and the remainder exhibiting a paucigranulocytic asthma. Innate immune activation is an important inflammatory mechanism in neutrophilic asthma, which may explain the increased expression of pro-inflammatory cytokines and proteolytic enzymes in neutrophilic asthma. Over 30% of subjects with persistent asthma have a persistent eosinophilia despite receiving high doses of inhaled corticosteroids. The findings of this thesis have important implications for the management, treatment and monitoring of asthma and support the inclusion of a measure of airway inflammation in the clinical assessment of asthma to guide therapy and further investigation into airway disease.

博士
國立陽明大學
臨床醫學研究所
107
Despite COPD (chronic obstructive pulmonary disease) and asthma are usually considered two distinct diseases, a substantial portion of patients present with features of both diseases. COPD with eosinophilic airway inflammation may represent a unique phenotype of asthma-COPD overlap, with shared features of COPD and asthma. A convenient test or tool is urgently needed to detect such population. Hence, we examined the role of exhaled nitric oxide (eNO) and an enhanced bronchodilator response in identifying COPD patients with sputum eosinophilia. The first part of the thesis, we prospectively enrolled 90 COPD patients without past medical history of asthma or allergic diseases, whose eNO, lung function and cellular profile/Eosinophil cationic protein/Immunoglobulin E (IgE) of induced sputum were measured. Eosinophil cationic protein and IgE in venous blood were also determined. Comparison between subjects with and without sputum eosinophilia (>3%) was made. Patients with sputum eosinophilia (29/90=32%) had significant higher levels of eNO (29 vs. 18 ppb, p=0.01) compared to those without it. The sputum eosinophil level was significantly correlated to the eNO level (r=0.485, p<0.001). The eNO level at the cut-off of 23.5 ppb had the maximum sum of a sensitivity (62.1%) and a specificity (70.5%). The unadjusted and adjusted odds ratio (OR) of a higher eNO level (>23.5 ppb) in prediction of sputum eosinophilia is 3.909 (CI=1.542-9.910, p =0.004) and 4.329 (CI=1.306-14.356, p=0.017), respectively. The last part of the thesis looked into the relationship of bronchodilator reversibility and eosinophilic airway inflammation in COPD. We retrospectively analyzed 264 stable COPD patients without a past history of asthma, showing 82 (31.1%) patients had sputum eosinophilia and FEV1 reversibility was weakly correlated to the sputum eosinophil level (r=0.162, p=0.008). Patients with FEV1 increment > 0.4L & >15% had a higher sputum eosinophil level (6.11 vs 1.02%, p=0.049) whereas the level did not differ if stratified by FEV1 increment >0.2L & >12%. Very positive FEV1 reversibility (>0.4L & >15%) in prediction of sputum eosinophilia remained significant after adjustment of age, baseline FEV1 and FVC (OR: 4.262, p=0.029). The ROC analysis showed AUC was 0.58 (p=0.034) and FEV1 increment > 0.4L & >15% had a positive predictive value of 63.6% and an overall accuracy of 70.1%. In conclusion, eNO can predict presence of eosinophilic airway inflammation in COPD patients whereas an enhanced bronchodilator response can barely meet the need.

碩士
長庚科技大學
健康產業科技研究所
106
Licochalcone A, a flavonoid, was isolated from the root of Glycyrrhiza glabra. Licochalcone A was reported to have anti-malarial, anti-tumor, anti-bacterial, antiviral and anti-inflammatory effect. The aim of this study was to investigate whether licochalcone A suppressed eosinophil infiltration and airway hyperresponsiveness (AHR) in asthmatic mice, and decreased the inflammatory response and oxidative stress in tracheal epithelial cells. BALB/c mice were sensitized and challenged with ovalbumin to induce allergic asthma in mice. These asthmatic mice were given various doses of licochalcone A by intraperitoneal injection. Additionally, activated human tracheal epithelial cells (BEAS-2B cells) and adenocarcinomic human alveolar basal epithelial cells (A549 cells) were treated with licochalcone A, and evaluated for levels of proinflammatory cytokines and chemokines. We found that licochalcone A significantly decreased AHR, and T-helper type 2 (Th2) cell over expression, and suppressed goblet cell hyperplasia, eosinophil infiltration, and inflammatory response in the lung tissue of asthmatic mice. Licochalcone A also reduced the levels of Th2 cytokines and chemokines in bronchoalveolar lavage fluid, and suppressed OVA-IgE production in serum. Furthermore, licochalcone A treatment of activated BEAS-2B cells and A549 cells decreased production of proinflammatory cytokines and eotaxins, as well as suppressed ICAM-1 expression and thus adhesion of eosinophils to inflammatory BEAS-2B cells and A549 cells in vitro. Our findings suggest that licochalcone A can improve allergic asthma in mice, and therefore has therapeutic potential in humans.

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is a common chronic inflammatory disease of the airways that affects over 2.2 million people in Australia. Asthma is a heterogeneous inflammatory disease typically characterised by T helper lymphocyte type 2 (Th2)-mediated eosinophilic inflammation, exaggerated responses to innocuous stimuli, mucus hypersecretion leading to airways obstruction and airway remodelling. These physiological changes result in wheezing, chest tightness, and breathing difficulties. However, it has been established that eosinophilic inflammation is only present in 50% of asthmatic patients. Around 30% of non-eosinophilic asthmatics have neutrophilic rather than eosinophilic inflammation, which is a key feature of neutrophilic asthma. Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that is commonly found in the upper respiratory tract of about 75% of healthy individuals. It is normally asymptomatically carried in people, however it may cause otitis media and is a common cause of community-acquired pneumonia. NTHi has also been linked to a number of chronic airway diseases. It has been detected in patients with bronchiectasis, chronic bronchitis and is commonly associated with chronic obstructive pulmonary disease (COPD) exacerbations. It has also recently been associated with neutrophilic asthma, however, the role of NTHi in neutrophilic asthma has not been investigated. Using murine models of NTHi infection and allergic airways disease (AAD), we investigated the relationship between infection and AAD. We showed that NTHi infection induced features of neutrophilic asthma; reduced Th2-mediated eosinophilic inflammation, reduced airways hyper-responsiveness (AHR) compared to eosinophilic AAD, and importantly, significantly increased Th17 responses and neutrophilic inflammation. In the first study it was demonstrated that the combination of infection and AAD reduced the expression of MHC II and CD86 on dendritic cells (DCs), suggesting that infection induced changes in presentation of antigen to naïve T-cells and subsequent adaptive responses. Infection also induced Interleukin (IL)-17 production from innate cells and Th17 cells. Critically, we show that inhibiting IL-17 significantly reduced neutrophilic inflammation in the airways. This highlights the crucial role of IL-17 in infection-induced neutrophilic AAD. The second study showed that the induction of AAD during infection delayed bacterial clearance from the lungs compared to infection alone controls. In contrast to Th2-mediated eosinophilic inflammation, this model of infection-induced neutrophilic AAD was resistant to dexamethasone treatment. All features of infection-induced neutrophilic AAD, including eosinophil and neutrophil influx, antigen-specific IL-5, IL-13 and Interferon (IFN)-γ, NTHi-specific IL-17, and AHR were unchanged with steroid treatment. This study also demonstrated that neutrophil and macrophage activation and function was inhibited in neutrophilic AAD. This lack of innate immune response may enable chronic bacterial infection. The final study investigated clarithromycin, a macrolide, and combination therapy with dexamethasone, as possible treatment strategies for neutrophilic asthmatics. This study demonstrated that clarithromycin alone significantly reduced neutrophil influx and IL-17 responses, but increased Th2-mediated eosinophilic inflammation. However, the combination of clarithromycin and dexamethasone suppressed all key features of AAD, including eosinophilic and neutrophilic inflammation, ovalbumin (OVA)-specific IL-5, IL-13, and IFN-γ, NTHi-induced IL-17, and AHR. These novel findings further the understanding of the potential role of NTHi in the development of neutrophilic asthma. We have identified some mechanisms of how infection may lead to features observed in neutrophilic asthma, and importantly, possible treatment strategies for neutrophilic asthmatics, and perhaps, other neutrophilic airway diseases with evidence of infection.

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is an inflammatory disease that manifests in the airways. There are an estimated 300 million people worldwide currently suffer from asthma. Common asthma symptoms include dyspnea and wheezing. These are consequences of the reversible airflow obstruction associated with airway inflammation. The symptoms can be mild or can be as severe as life threatening depending on nature of underlying inflammation. Although heredity plays a role in the disease pathogenesis, the high and rising prevalence of asthma, particularly in recent decades highlights a strong influence of the environment. To this end, epigenetic phenomena including alteration of DNA methylation and chromatin structure are likely contributors to the pathogenesis of asthma as well as a plausible source of phenotype heterogeneity. Especially subtle alteration of DNA methylation patterns which occur early in life may impact on disease development. However, the exact role of epigenetic mechanisms in the pathogenesis of asthma and inflammatory phenotypes of asthma are not well understood. This thesis investigates; 1) Alterations in infant peripheral blood DNA methylation profiles associated with pre-natal exposure to maternal asthma, 2) The role of chromatin structure by analysing histone acetyl-transferases (HAT) and histone de-acetylases (HDAC) activity of peripheral blood monocytes in inflammatory phenotypes of adult asthma, 3) Alterations in the DNA methylation profile of peripheral blood monocytes associated with inflammatory phenotype of adult asthma. The primary findings of this thesis are: 1) Maternal asthma during pregnancy is associated with alterations in peripheral blood DNA methylation in infants’. 2) Inflammatory phenotypes of asthma are associated with differential DNA methylation in peripheral blood monocytes. Gene network analyses of these differentially methylated genes revealed distinct molecular pathways, suggesting possible implications in the disease pathogenesis. 3) Neutrophilic asthma is associated with lower HDAC activity and higher HAT activity of peripheral blood monocytes compared to both eosinophilic and paucigranulocytic asthma. Collectively, the findings of this thesis emphasised the significance of epigenetic factors playing a role in the development of asthma and inflammatory phenotypes of asthma. An association of peripheral blood methylation profiles of infants with maternal asthma suggests a potential inheritance of the disease susceptibility. The characteristic alterations of DNA methylation in blood monocytes suggest an underlying epigenetic basis for the inflammatory phenotypes while the differences in HAT/HDAC activity in monocytes further emphasise a role for the epigenome in the development of inflammatory phenotypes. The findings of this thesis warrant further investigation and may help us get one step closer to understanding the role of epigenetics in airway inflammation in asthma.

The eosinophil is a leukocyte whose intracellular mediators are considered to play a central role in the pathogenesis of allergic diseases, including allergic asthma, allergic rhinitis and atopic dermatitis, and which is also involved in immunological responses to parasites. Eosinophil differentiation and maturation from bone marrow progenitors is regulated by interleukin-5 (IL-5), which may be secreted by T helper 2 (Th2) T lymphocytes, and is consistently upregulated in allergic conditions. Eotaxin is a potent chemoattractant for circulating and tissue eosinophils, and the production of this chemokine promotes eosinophil infiltration and accumulation within sites of allergic inflammation.¶ ...¶ In conclusion, eosinophils residing in the allergic lung have the capacity to interact with activated T cells, both within this tissue and the draining lymph nodes. Despite their relative inefficiency as antigen presenting cells (Mawhorter et al., 1994), eosinophils may participate en masse in the serial triggering of activated TCRs, and provide appropriate costimulatory signals that modulate T lymphocyte function. Through the elaboration of Th2 cytokines and stimulation of T cell proliferation, antigen presenting eosinophils may transiently prolong or exacerbate the symptoms of allergic diseases. Alternatively, eosinophils presenting relevant antigens may inhibit T cell activity via degranulation, and such activity has recently been observed in a parasite model (Shinkai et al., 2002). Finally, experiments in the OT-II mouse have provided valuable information to suggest that therapies designed to modulate eosinophil numbers in allergic tissues through the secretion of opposing cytokines such as IFN-γ, may be of limited benefit. The results shown here suggest that airways dysfunction remains intact despite significantly reduced pulmonary eosinophilia