Dissertations / Theses on the topic 'Allergic Airways Disease'

The system adverse effects of inhaled corticosteroids were investigated in dose-response studies. By measuring cortisol suppression, it was shown that a more potent inhaled corticosteroid (fluticasone propionate (FP)) exhibits greater systemic bioactivity (2-fold at highest licensed doses) than a weaker steroid (triamcinolone acetonide (TAA)). No differences were detected between inhaled corticosteroids of similar potency (TAA and flunisolide), even when using sensitive and novel measures e.g. low dose ACTH stimulation and early morning urine cortisol excretion. The latter test may prove to have clinical implications for monitoring patients, as it was shown to be more sensitive than dynamic or basal serum cortisol measures. However, the lung delivery of a corticosteroid has a greater effect on systematic bioactivity than its dose or potency, as the systematic activity of FP via two different inhaler devices was shown to vary more than 4-fold. Studies comparing oral prednisolone with inhaled FP, showed FP to exhibit dose-related suppression of serum cortisol in a 1:8.5mg ratio compared to prednisolone. Interestingly, the effects of FP on markers of bone metabolism were less marked than adrenal suppression, compared to the effects of prednisolone. Intra-nasal FP also produced significant urinary cortisol suppression, whereas other intra-nasal corticosteroids (TAA, budesonide (BUD), beclomethasone, mometasone) had no significant effects on 24 hour cortisol, bone or blood markers. Furthermore, the addition of intranasal to inhaled FP resulted in more patients with sub-normal cortisol values. A meta-analysis of all dose-response studies assessing different inhaled corticosteroids was also performed with results supporting this data. When assessing therapeutic effects of inhaled corticosteroids, it was shown that a lower dose of BUD was required to optimise symptoms, lung function and exhaled nitric oxide (eNO), compared to other measures of inflammation; serum markers (ECP), and bronchial hyperreactivity to adenosine monophosphate (AMP) and methacholine.

Allergen-reactive CD4+ T cells are implicated in the pathogenesis of allergic disease. Peptide immunotherapy (PIT) involves therapeutic administration of short immunodominant peptides from within the protein allergen to which CD4+ T cell responses are directed. This approach can induce tolerance of allergen-reactive CD4+ T cells, while negating the risk of severe allergic reactions associated with whole allergen specific immunotherapy. PIT therefore holds promise as a diseasemodifying treatment for allergic patients. However, further information regarding the mechanisms of action of PIT are required to aid translation to the allergy clinic. Chicken ovalbumin (OVA) is a commonly used model allergen in mouse models of allergic airways inflammation (AAI). Trackable, T cell receptor transgenic T cells recognizing the immunodominant 323-339 peptide of OVA (pOVA) allow mechanistic investigation of PIT in response to pOVA. This thesis investigated the hypothesis that strong, systemic T cell responses induced by intravenous administration of soluble pOVA will induce i) tolerance to pOVA and ii) linked suppression to any additional OVA T cell epitopes, hence improving OVA-induced AAI. Contrary to the hypothesis, intravenous pOVA PIT did not improve disease in a C57BL/6 model of OVA-induced AAI. Models of OVA-induced allergic sensitisation and AAI were therefore developed incorporating trackable CD4+ pOVA-reactive T cells (OT-II cells). pOVA PIT induced tolerance of these cells in an allergic sensitisation setting, but had limited impact on the overall OVA response. Yet, in a model of AAI driven solely by Th2 polarised CD4+ OT-II cells, pOVA PIT did improve disease. It was concluded that, in non-transgenic C57BL/6 mice, CD4+ T cells responding to additional epitope(s) within OVA were important in driving disease and that these T cells were not subject to linked suppression following pOVA PIT. Using a panel of overlapping peptides constituting the sequence of OVA, a novel CD4+ epitope within OVA was characterised. The effects of PIT using pOVA in combination with a peptide containing this additional epitope on OVA-induced AAI were then assessed. Findings from this project therefore hold importance for future mechanistic work surrounding PIT in allergic disease.

Asthma is a chronic inflammatory disease of the airways, resulting in significant morbidity and premature deaths. Rhinoviruses, agents of the common cold, have been identified as the most frequent viruses inducing asthma exacerbations. However the mechanisms involved in this process, especially in moderate-to-severe asthmatics, are not clear. IL-17, a protein secreted by Th17 and IL-17+γδ T cells, is found to be up-regulated in asthmatics and correlates with severity of disease. Observations from a clinical study of rhinovirus infection showed increased induction of IL-17 in nasal lavage fluid from asthmatics following experimental rhinovirus infection compared to healthy controls. Using mouse models, this thesis addresses the interactions of rhinovirus with IL-17 in the context of allergic airways disease. Primary infection of C57BL/6 mice did not induce IL-17 responses as seen in human subjects; this may be explained by the apparent lack of induction of IL-23 following primary infection in mice. Supplementation of rhinovirus infection with recombinant IL-17 showed significant, selective increases in neutrophil chemokines with subsequent increases in both neutrophil recruitment and activation, as defined by myeloperoxidase activity. In an existing Th2-driven model of allergic airways disease, rhinovirus was seen to selectively expand Th2 responses while not increasing allergen-induced Th17, IL-17+γδ T cells or IL-17. However, rhinovirus infection in a Th17-driven model of allergic airways disease showed a significant increase of neutrophil numbers with a concomitant increase in airway hyperresponsiveness. This work augments existing knowledge on rhinovirus exacerbation of Th2-mediated asthma with the interactions between Th17 and rhinovirus, which may play a significant role in moderate-to-severe asthma exacerbations. This supports current research on therapeutic targeting of the IL-17 pathway in asthmatics.

Allergic asthma is a chronic inflammatory disease of the lung and deficiencies in pro-resolving mechanisms may contribute to the persistence of inflammation. The overall aim of this project was to establish a resolution model of house dust mite (HDM) induced allergic airway disease (AAD) and identify mediators of resolution. In our model, features of disease, induced by HDM at peak disease 4 hours, airway hyper-reactivity (AHR), Th2 lymphocytes and eosinophils remained significantly elevated 7 days after last challenge, resolving to baseline by 13 days. The levels of FoxP3+ regulatory lymphocytes also follow this pattern. However, as disease waned there was an elevation in the levels of alveolar macrophages and up regulation of the homeostatic molecule CD200R up to 13 days. Exposure to a single i.n administration of HDM in the resolved airways resulted in a rapid increase in Th2 inflammation and AHR suggesting that after resolution of HDM inflammation there is altered immune homeostasis in the lung. The pro-resolving lipid Lipoxin A4 was induced in the lung by HDM exposure and remained detectable during resolution. Depletion of alveolar macrophages during the resolution phase of allergen challenge resulted in delayed clearance of Th2 lymphocytes, airway neutrophils and interstitial macrophages. Conversely, adoptive transfer of alveolar macrophages during resolution resulted in reduced numbers of lung tissue leukocytes, specifically neutrophils and interstitial macrophages. This suggests a cross talk between these macrophage subsets and a novel interaction for pulmonary homeostasis. The anti-inflammatory peptide Annexin A1 is highly expressed by alveolar macrophages and mice deficient in Annexin A1 had enhanced AHR and Th2 immunity response to HDM. Blocking the Annexin A1 receptor FPR2 enhanced AHR and lung inflammation. Conversely, therapeutic administration of an Annexin A1 mimetic improved AHR and Th2 immunity. These studies demonstrate that Annexin A1: FPR2 pathway may be important in HDM disease and that resolution of allergic airways disease is an active process resulting in altered homeostasis of the lung.

Asthma is a chronic disease characterised by variable airflow obstruction, bronchial hyperresponsiveness and airways inflammation. At an immunological level Th2 inflammation and the presence of activated eosinophils and mast cells are key features of asthma. ST2, the receptor for the novel cytokine IL-33, is expressed upon Th2 lymphocytes and mast cells but its role in clinical and experimental asthma remains unclear. IL-33 has been shown to induce local and systemic eosinophilia when administered to the peritoneum of mice. In this thesis I have set out to test the hypothesis that the activation of mast cells by IL-33 acting on cell surface ST2 plays a critical role in allergic airways inflammation. I began by studying the function of ST2 on mast cells in vitro. I found that ST2 was expressed at an early stage of development, and correlated closely with the expression of the stem cell factor receptor (c-kit), a marker present on mast cells from a progenitor stage. Despite this mast cells generated form ST2 gene deleted mice proliferated and matured normally. When mast cells were activated by IL-33, acting in an ST2-dependent manner, pro-inflammatory cytokines and chemokines were released that have potential roles in asthma, specifically IL-6, IL-13, MIP-1α and MCP-1. To extend these findings I looked at the role of ST2 in allergic airways inflammation. I first optimised and validated an ovalbumin and adjuvant based ‘short’ twelve day model of murine asthma and demonstrated that ST2 gene deletion results in attenuated eosinophilic inflammation. In addition to being ST2 dependent it is possible that this adjuvant based short model is mast cell dependent, unlike longer adjuvant based models which are mast cell and ST2 independent. Therefore I went on to study an adjuvant-free model of asthma which has been demonstrated to be mast cell dependent. In this adjuvant-free model of asthma the airway inflammation was attenuated in ST2 gene deficient mice compared with wild type mice, while AHR was unaffected. There was an associated reduction in IgE production and thoracic lymph node recall Th2 cytokine responses. I then examined the effect of ST2 activation in the lungs. When IL-33 was administered directly to the airways of naïve mice it induced the features of experimental asthma. There was extensive eosinophilic inflammation within the lung tissue and airspaces. The Th2 cytokines IL-5 and IL-13, and the eosinophil chemoattractant chemokines eotaxin-1 and eotaxin-2 were detected at increased concentrations. Significant airways hyperresponsiveness was also generated. Using ST2 gene deleted mice I demonstrated that these effects were ST2 specific. Although I have shown that mast cells are activated by IL-33 in vitro, I used mast cell deficient mice to demonstrate that the eosinophilic inflammation generated by IL-33 is unaffected by the absence of mast cells. These data show that IL-33 can induce in the lungs the cardinal pathological characteristics of asthma, and that it appears to act upstream of other important mediators such as IL-13 and the eotaxins. Furthermore the IL-33 receptor ST2 is required in an adjuvant free model of asthma, which is more akin to human disease. Placing these findings in the context of recent evidence that IL-33 is released by structural cells in response to damage or injury suggests that IL-33 may play a key role in initiating the immunological features of clinical asthma. As a consequence of this position in the hierarchy of inflammation IL-33 offers a promising direct target for novel biological therapies in asthma.

Asthma is a chronic inflammatory airway disease and both environmental and genetic factors contribute to disease onset. As asthma predominantly develops during childhood it is important to investigate asthma pathogenesis in the context of the maturing immune system and developing lung. A neonatal house dust mite (HDM)-induced model of allergic airways disease (AAD) was used to assess the impact of age, diet, pollution and maternal allergy on the development of neonatal AAD. To investigate the effect of age, age at first allergen challenge was altered. Allergen exposure during the first week of life caused robust AAD, but exposure between 14-21 days resulted in a hyporesponsiveness phenotype. This period corresponded to a nadir in pulmonary interleukin (IL)-13 producing T cells and innate lymphoid type 2 (ILC2) cells. Maternal asthma is a risk factor for the development of childhood asthma. HDM exposed offspring born to allergic mothers had reduced airway compliance 8 weeks of age and mounted a faster IgE response than offspring from non-allergic mothers. Serum vitamin D levels have been correlated with asthma control in adults and children. Vitamin D insufficiency in utero resulted in pulmonary Th2-skewing and a decrease in T regulatory cells irrespective of HDM or PBS exposure. Restoration of vitamin D reduced eosinophilia and IgE levels in HDM exposed mice. Increased air pollution has been correlated with asthma exacerbations. Exposure of pregnant female mice to diesel exhaust particles resulted in increased inflammation in the lungs of offspring, but did not exacerbate AAD. The data presented in this thesis strongly support a case for in utero and early life exposures as a crucial determinant in immune programming in early life, with likely additional influences on lung development.

Rates of allergic airways disease are steadily rising in developed countries, arguing for an environmental etiology. Epidemiological studies have pointed to a role for the infant gut microbiota in immune system development that could alter allergic disease susceptibility. To investigate whether changes in gut microbiota impact disease severity in murine models of asthma and hypersensitivity pneumonitis (HP), we administered clinical doses of antibiotics to mice during different periods in their development. Classically, allergic asthma is induced by T helper type 2 (Th2) inflammatory responses. In contrast, HP develops via Th1/Th17-mediated mechanisms. Consistent with their polarized immune phenotypes, these two diseases were exacerbated after different antibiotic exposures. Mice receiving perinatal vancomycin developed more severe asthma relative to control animals, as demonstrated by increased Th2-driven airway inflammation, antigen-specific IgE and lung pathology. The data presented here suggest that increased asthma severity in this model of allergic airways disease is mediated by mechanisms involving elevated IgE levels and reduced regulatory T cell populations. This effect was not observed in mice given streptomycin, nor when either antibiotic was administered to adult mice. Conversely, the severity of HP was unaffected by vancomycin, but increased after streptomycin treatment; this was demonstrated by exacerbated airway inflammation of the Th1/Th17-type, as well as increased IFNγ and IL-17A cytokine production and lung pathology. Microbial community analysis reveals that antibiotic treatment has profound effects on the gut microbiota; these effects were highly specific to the type of antibiotic used and the length of administration. Bacteroidetes dominated the intestinal flora after streptomycin treatment, while vancomycin drastically reduced diversity and promoted the overgrowth of a distinct group of Firmicutes. The extensive use of antibiotics in our society warrants a closer look at the effects of different antibiotics on the composition of the microbiota and how this may impact the prevalence of diseases like asthma and HP. The work in this thesis presents an interesting dichotomy, where contrasting shifts in gut flora appear to have opposite consequences depending on the immunological nature of the disease.

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

Asthma is a multi-faceted chronic inflammatory disease accompanied by loss of airway epithelial integrity leading to remodeling of the airways. Perturbations to the lung redox environment, including alterations in glutathione (GSH) content, have been reported in asthma. GSH can be conjugated to protein cysteines, controlling protein function in an oxidant-dependent process known as protein S-glutathionylation (PSSG). The thioltransferase, glutaredoxin-1 (Glrx1), deglutathionylates proteins under physiological conditions, restoring sulfhydryl groups of target proteins. Glrx1 is emerging as a critical player in settings of allergic airway disease, but its function in regulating epithelial cell responses to asthma-relevant cytokines has not been examined. Furthermore, the role of Glrx1 in controlling the extent of airway remodeling in response to house dust mite (HDM) in vivo is still not well understood. Interleukin-17A (IL-17A) is a potent cytokine that stimulates epithelial cells to produce pro-inflammatory mediators, in part by activating the nuclear factor kappaB (NF-κB) pathway, a key regulator of inflammation. We demonstrate that interleukin-17A (IL-17A) induces rapid activation of both classical and alternative NF-κB, while simultaneously resulting in protein oxidation and PSSG. In particular, we show IL 17A induces S-glutathionylation of RelA (RelA-SSG) and IKKα (IKKα-SSG), which is enhanced following siRNA-mediated knockdown of Glrx1. We also demonstrate that absence of Glrx1 leads to increased nuclear content of RelA and RelB and enhanced production of NF-κB-driven pro-inflammatory genes, KC and CCL20 while decreasing IL-6 expression. Finally, we show that siRNA-mediated knockdown of IKKα attenuates nuclear RelA and RelB and dampens pro-inflammatory gene production. Together, these data indicate a crucial role for the Glrx1/PSSG axis in controlling RelA-SSG, IKKα-SSG and epithelial cell responsiveness to IL-17A. Mice lacking Glrx1 were previously shown to display enhanced resolution of allergic airway disease induced by ovalbumin (Ova) challenge. In this study, we determined the role of Glrx1 in a HDM model of allergic airway disease. Wild type (WT) mice and Glrx1 deficient (Glrx1-/-) mice demonstrated similar total lung cell counts, but Glrx1-/- mice displayed fewer neutrophils than WT mice. Conversely, mice overexpressing Glrx1 specifically in CCSP positive cells in the lung (Epi-Glrx1) showed attenuated total lung cell counts and lung eosinophils compared to control mice. Immunohistological analysis of remodeling markers revealed that Glrx1-/- mice displayed increased HDM-induced mucus metaplasia, α smooth muscle actin (αSMA) positivity and collagen staining compared to WT mice. Evaluation of total lung collagen showed that Glrx1-/- mice had significantly higher collagen content compared to WT mice. In Epi-Glrx1 mice, attenuation of mucus metaplasia, αSMA content and collagen staining was observed compared to control mice. Furthermore, Epi-Glrx1 mice also demonstrated significantly impaired collagen production compared to control mice. We also demonstrate that Glrx1 absence results in decreased expression of the epithelial cell marker, E-cadherin, and increased expression of αSMA, a mesenchymal marker. Together, these studies demonstrate a critical role for Glrx1 in controlling epithelial cell responses to IL-17A and in mediating in vivo collagen production in response to chronic allergen exposure.

Epidemiological studies, genetic analyses, as well as clinical and experimental data indicate a potential for the innate immune pattern-recognition receptor, the toll-like receptor 4, and its bacterial ligand, lipopolysaccharide, to initiate, exacerbate, or conversely prevent or treat allergic airway disease, depending on conditions and mechanisms that are still being elucidated. Animal studies suggest that many of these pro- or anti-inflammatory events are driven by activation of a signaling pathway downstream of TLR4 which relies upon the intracellular adaptor protein 'myeloid differentiating factor 88' (MyD88), whereas the other major TLR4-activated signaling pathway controlled by the adaptor 'Toll, IL-1 receptor and Resistance protein (TIR) domain-containing adaptor inducing interferon-β' (TRIF), has not been explored in the context of allergic asthma. Here, we investigated the role of TLR4 and TRIF activation in different murine models of experimental allergic asthma. In Chapter 2, we studied its role in the context of exposure via the airways to an extract of a natural aeroallergen, the birch tree pollen, hypothesizing that this pathway might function to prime allergic sensitization and/or to facilitate the amplification of the allergic response. In Chapters 3 and 4, we examined the role of TLR4 and TRIF activation in the context of exposure to a mucosal adjuvant consisting of TLR4 and TLR2 bacterial ligands, intended for potential use in vaccination or allergen-specific immunotherapy, in order to determine whether this pathway was relevant to the inhibition of allergic airway disease development. In this latter context, we also examined cellular mechanisms that may be relevant to the prevention of allergic airway disease, namely the immunomodulatory effect of this adjuvant on the T cell response. We find that in both contexts, the TLR4-TRIF-dependent signaling pathway inhibits aspects of allergic airway disease and thus appears to have protective and potentially immunotherapeutic effects against the development of allergic asthma. We also determined that TLR4 activation and, to a significant degree, TRIF-dependent signaling, can influence the CD4+ T cell response by enhancing the expression of a T cell-expressed co-stimulatory molecule, the inducible co-stimulatory molecule (ICOS), and expanding CD4+ICOS+ cells which may contribute to mediating the TLR4-TRIF-dependent inhibition of allergic airway disease development. Thus, we identify TLR4-TRIF-dependent signaling as a potentially important pathway in allergic asthma and advance current knowledge regarding the adjuvant effect of TLR4 ligands upon T cells.
Les études épidémiologiques, les analyses génétiques ainsi que les données cliniques et expérimentales indiquent un rôle potentiel pour le récepteur toll-like-4 (TLR4) et son ligand de source bactérienne, le lipopolysaccharide, dans l'initiation, l'exacerbation ou au contraire dans l'inhibition ou le traitement de maladies allergiques respiratoires dépendamment de conditions et de mécanismes qui demeurent encore à être élucidés. Des études réalisées chez l'animal suggèrent que plusieurs de ces événements pro- ou anti-inflammatoires sont induits par l'activation d'une voie de signalisation en aval de TLR4 qui dépend de la protéine adaptatrice 'myeloid differentiating factor 88' (MyD88), alors qu'une autre voie majeure activée par TLR4 et sous le contrôle de l'adaptateur 'Toll, IL-1 receptor and Resistance protein (TIR) domain-containing adaptor inducing interferon-β' (TRIF), n'a pas été encore étudiée dans le contexte de l'asthme. Dans cette étude, nous avons analysé le rôle de l'activation de TLR4 et TRIF dans différents modèles murins d'asthme. Au chapitre 2, nous avons étudié sont rôle suite à une exposition des voies aériennes à un extrait d'un aéroallergène naturel, le pollen de bouleau, dans l'hypothèse que cette voie de signalisation puisse être sollicitée afin d'initier la sensibilisation allergique et/ou faciliter l'amplification de la réponse allergique. Aux chapitres 3 et 4, nous avons examiné le rôle de l'activation de TLR4 et TRIF suite à une exposition à un adjuvant muqueux composé de ligands bactériens de TLR4 et TLR2, potentiellement destinés à un usage en vaccination ou dans le cadre d'une immunothérapie allergène-spécifique, afin de déterminer si cette voie de signalisation était pertinente pour contrer le développement de maladies allergiques respiratoires. Dans ce contexte, nous avons également examiné les mécanismes cellulaires qui pourraient s'avérer pertinents dans la prévention des maladies respiratoires d'origine allergique, à savoir l'effet immunomodulateur de cet adjuvant sur la réponse des lymphocytes T. Nous avons trouvé que, dans les deux cas, la voie de signalisation TLR4-TRIF bloque plusieurs aspects des maladies respiratoires d'origine allergique et ainsi semble avoir un effet protecteur et potentiellement immunothérapeutique contre le développement de l'asthme. Nous avons également déterminé que l'activation de TLR4 et, dans une large mesure, la voie de signalisation dépendante de TRIF, peut influencer la réponse des cellules T CD4+ en augmentant d'une part l'expression d'une molécule co-stimulatrice exprimée par les cellules T, the inducible co-stimulatory molecule (ICOS), et d'autre part en augmentant la population de cellules CD4+ICOS+ qui contribuent à médier l'inhibition dépendante de TLR4-TRIF sur le développement des maladies allergiques respiratoires. Ainsi, nous avons identifié la voie de signalisation dépendante de TLR4-TRIF comme étant une voie potentiellement importante dans l'asthme et fait avancer les connaissances actuelles concernant l'effet adjuvant des ligands TLR4 sur les cellules T.

Allergic airway disease, i.e. allergic rhinitis (AR) and asthma, is a common health problem. The prevalence is increasing in most countries of the world. Traffic-related air pollution has been found to induce and enhance allergic airway disease, but the underlying mechanisms are not known. Oxylipins are fatty acid metabolites, of which several have been linked to asthmatic airway inflammation. Oxylipin profiles have previously been investigated in bronchoalveolar lavage (BAL), mainly reflecting the peripheral lung, but not in bronchial wash (BW), which better reflect the proximal airways. The airway epithelium is covered by a respiratory tract lining fluid (RTLF) The RTLF contains antioxidants to protect from oxidative stress, which may be caused by exposure to air pollution. Previous studies have reported diminished levels of the antioxidant ascorbate (vitamin C) in the RTLF of patients with asthma. Little is known about the regulation of vitamin C in the lung. The aim of this thesis was to investigate airway inflammatory responses to diesel exhaust exposure in patients with AR and allergic asthma; to evaluate oxylipin profiles in different regions of the lung in patients with allergic asthma; and to study the distribution of vitamin C transporters in the airways of patients with allergic asthma. Diesel exhaust (PM10 100 μg/m3 for 2 h) induced a neutrophilic airway inflammation in healthy individuals evaluated 18 h after exposure. Patients with AR and asthma did not respond with an enhanced airway inflammation. However, a small increase in myeloperoxidase was found in BAL from patients with AR, as well as decreases in epithelial tryptase and BW stem cell factor. This indicates that other mechanisms than classical inflammation are responsible for the increased sensitivity to traffic-related air pollution in patients with allergic airway disease. Oxylipin baseline profiles differed between peripheral and proximal airways in both allergic asthmatics and healthy individuals. Total oxylipin concentrations, and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway, were elevated in BW from asthmatics compared to healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. This suggests that lung compartment-specific sampling should be considered in future studies. Sodium dependent vitamin C transporter 2 (SVCT2) was, for the first time, found present in the human lung epithelium, localised mainly within goblet cells. A negative correlation between SVCT2+ goblet cells and vitamin C suggests that these cells may play a hitherto unknown function in ascorbate re-uptake and recycling at the air-lung interface.

An abundance of clinical data has demonstrated that a subset of asthmatic patients present with a neutrophilic infiltrate that is anticipated to be implicated in the pathology of the disease. However, this assumption is based largely on circumstantial evidence and the exact role of neutrophils in the pathophysiology of asthma has not been extensively explored. The aim of this thesis therefore was to elucidate the role of neutrophils in asthma by utilizing the house dust mite (HDM) murine model of allergic airway disease (AAD). We demonstrated that administration of HDM to different inbred mouse strains yielded very different inflammatory and airway remodelling profiles culminating variable airway hyperresponsiveness (AHR). Pertinently, whereas HDM-exposed 129/S2 mice exhibited a hyper-eosinophilic response, Balb/c mice presented with a mixed granulocytic response with a prominent neutrophilic component, which was TLR-4 independent. Specific depletion of neutrophils in HDM-exposed Balb/c mice was not protective but actually resulted in increased Th2 inflammation, with elevated ILC2s, Th2 CD4+ T cells, Th2 cytokines and serum IgE and IgG1 antibody responses. This elevated type 2 inflammation in neutrophil depleted animals was dependent on augmented Th2 cytokine production by ILC2s and a monocytosis that gave rise to enhanced antigen presentation. G-CSF was substantially elevated in neutrophil depleted mice and directly promoted the Th2 cytokine production by ILC2 and drove the monocytosis by acting on progenitors in the bone marrow. This data demonstrates that neutrophils may play a regulatory role in restricting Th2 inflammation rather than being strictly pathological. The enzyme leukotriene A4 hydrolase (LTA4H) regulates two distinct pathways that reciprocally regulate neutrophil recruitment. Classically, LTA4H functions intracellularly to generate the pro-inflammatory lipid mediator and potent neutrophil chemoattractant leukotriene B4 (LTB4). Accordingly, pharmaceutical companies have developed LTA4H inhibitors for the treatment of asthma, but they have failed to demonstrate efficacy in the clinic. Rationalizing the failure of these inhibitors, my lab have previously demonstrated a secondary extracellular anti-inflammatory role for LTA4H in degrading neutrophil chemoattractant Proline-Glycine-Proline (PGP). We have now demonstrated that mice lacking the LTB4 receptor, BLT1, showed reduced inflammation and AHR upon HDM exposure relative to wild type controls. Conversely, HDM treated lta4h-/- mice displayed an exacerbated airway resistance relative to littermate controls, despite being incapable of generating LTB4 and displaying reduced Th2 inflammation. This exacerbated airway resistance in lta4h-/- mice was attributable to a novel, neutrophil-independent, role for PGP in the context of AAD in driving pathological airway remodelling, with a pronounced epithelial hypertrophy and mucus hypersecretion apparent.

Regulatory T cells (Treg) are critical to the maintenance of immune tolerance, partly by controlling the unwanted activation of effector T cells (Teff) and thereby enhancing the resolution of autoimmune and allergic inflammation. Recent data suggest that Treg can specialize to better control different types of inflammation by using transcriptional machinery which controls differentiation and function of Teff. This thesis addresses questions related to the efficacious use of Treg, notably their ability to adopt distinct phenotypic profiles under different inflammatory contexts and their need to recognize antigen in the inflamed organ. Two differentially mediated mouse disease models were used in this project, namely Th1/Th17-mediated experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis and Th2-mediated allergic airways inflammation (AAI) as a model of asthma. A new model of rMOG-induced AAI was developed to specifically answer the questions on the importance of cell phenotype versus antigen-reactivity for the effective Treg-mediated suppression. It was demonstrated that Treg from the inflamed CNS in EAE had an upregulated expression of Th1 master regulator T-bet and Th1-associated chemokine receptor CXCR3, whereas Treg derived from the inflamed lung in AAI had an increased expression of Th2 master regulator GATA-3, lacked expression of T-bet and displayed decreased levels of CXCR3. This specialized and activated phenotype was restricted to tissue-derived Treg. The importance of appropriate Treg phenotype for effective suppression was suggested by the observed inability of CNS-derived Treg to inhibit AAI. A different Treg subset, TGF-β-induced Treg (iTreg), was shown to express high levels of T-bet and CXCR3, but not GATA-3 upon induction in vitro. iTreg effectively suppressed both Th1 and Th2 types of inflammation and the antigenreactivity was key to this. This thesis demonstrates that Treg are capable of acquiring a distinct phenotype corresponding with a CD4+ T cell response driving inflammatory disease and identifies antigen-reactivity as key to the efficacious suppression of inflammation. It also highlights substantial phenotypic differences between iTreg and naturally-occurring Treg which could be associated with different modes of suppression.

Nuclear Factor kappaB (NF-êB) is a transcription factor whose activation is increased in settings of allergic asthma. At least two parallel NF-êB pathways exist: the classical pathway, which plays a role in inflammation and cell survival, and the alternative pathway, which regulates lymphoid cell development and organogenesis. The classical NF-êB pathway regulates inflammatory responses derived from lung epithelial cells; however, the role of the alternative pathway in lung epithelial cells remains unclear. We demonstrate that both classical and alternative NF-êB are activated in lung epithelial cells in response to multiple pro-inflammatory agonists, and siRNA-mediated knockdown of alternative NF-êB proteins largely attenuates pro-inflammatory cytokine production. Furthermore, simultaneous activation of both pathways leads to cooperative increases in pro-inflammatory responses, indicating a potential role for both classical and alternative NF-êB in the regulation of epithelial-derived pro-inflammatory responses. NF-êB activation in the epithelium modulates allergic inflammation in mouse models of allergic airway disease, however, its role in the context of an allergen relevant to human asthma remains unknown. In order to address the impact of inhibition of NF-êB in the epithelium in vivo, we utilized a House Dust Mite (HDM)-induced model of allergic airway disease. We demonstrate that HDM exposure activates classical and alternative NF-êB in both murine lung epithelium and human bronchial epithelial cells. Furthermore, following exposure to HDM, airway hyperresponsiveness, neutrophilic inflammation, and remodeling are attenuated in transgenic CC10-NF-êBSR (airway epithelial specific inhibitor of classical and alternative NF-êB) mice in comparison to wild type mice. Our data also demonstrate that specific knockdown of the alternative NF-êB protein, RelB, in the lung partially protects against HDM-induced pro-inflammatory responses, indicating that both classical and alternative NF-êB are important in HDM-induced responses. NF-êB proteins are modified by the redox-dependent post-translational modification, S-glutathionylation, under conditions of oxidative stress. S-glutathionylation of IKKâ, an upstream kinase in the NF-êB pathway, is known to decrease its catalytic activity; however, it is unknown how S-glutathionylation of IKKâ occurs. GSTP is an enzyme that catalyzes protein S-glutathionylation under conditions of oxidative stress and has been associated with the development of allergic asthma. We aimed to determine whether GSTP regulates NF-êB signaling, S-glutathionylation of IKKâ, and pro-inflammatory cytokine production. We demonstrate that siRNA-mediated knockdown of GSTP modulates NF-êB activation, NF-êB transcriptional activity, and pro-inflammatory cytokine production in response to LPS, a component of a bacterial cell wall. Furthermore, we demonstrate that GSTP associates with IKKâ in response to agonist stimulation and dampens IKKâ-induced pro-inflammatory cytokine production, surprisingly, independent of its catalytic activity. We also show that GSTP associates with other proteins of the NF-êB pathway, indicating a potential dual mechanism for repression of NF-êB-induced signaling. These studies collectively demonstrate that classical and alternative NF-êB contribute to epithelial-derived inflammatory responses, and GSTP may be a novel target by which NF-êB can be regulated.

Research Doctorate - Doctor of Philosophy (PhD)
This thesis presents studies on novel therapeutic interventions for the treatment of allergic asthma providing proof of concept through extensive investigations in mouse models of human disease. The current increase in the incidence of asthma worldwide along with inability of current medications to treat the primary causes of the disease indicates that novel therapeutic approaches are required. This will improve the quality of life and disease burden concerns of the community. I investigated two alternate therapeutic approaches in an effort to identify new candidate targets with significant therapeutic potential. The first research chapter (see chapter 3) presents an initial study on the role of miRNA in the development of allergic airways disease (AAD). This study also demonstrates proof-of-concept for the use of modified, cholesterol conjugated complementary sequences termed antagomirs to specifically inhibit the expression of miR-126 in the airways and to alleviate AAD. The second research chapter (see chapter 4) presents a comparative study in which treatment of allergic mice with an antagomir suppressing miR-145 is compared to mice treated with the current gold standard therapy, systemic glucocorticoids. Here it is demonstrated that the novel therapeutic approach of selectively inhibiting the upregulation of miR-145 in the airway wall is as potent as treatment with systemic dexamethasone to alleviate AAD. The third research chapter (see chapter 5) presents a study where miR-126 was inhibited in a chronic model of AAD. The findings of this study confirm an important role of miR-126 in the regulation of allergic airways inflammation but suggest that in this model miR-126-independent mechanisms promote the development of tissue remodelling, hallmark features of chronic asthma. These results suggest that targeting a single miRNA may not be sufficient to reduce all aspects of AAD. The fourth research chapter (see chapter 6) presents a study of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induced signalling pathway in AAD and rhinovirus (RV)-induced exacerbation of AAD. Here I identify a novel role for TRAIL induced Midline-1 (Mid1) driven polyubiquination and silencing of the Protein Phosphatase 2a (PP2a). Furthermore, blocking this signalling pathway through either the silencing of Mid1 with siRNA or the synthetic reactivation of PP2a using the small molecule AAL(S) was capable of alleviating AAD and RV-induced exacerbation. This study provides proof-of-concept that modulation of the TRAIL induced signalling pathway may provide therapeutic benefit in the treatment of AAD. Together these studies have investigated novel and relevant targets for therapeutic intervention in AAD. By targeting immuno-regulatory systems such as miRNAs and TRAIL regulated signalling cascades at the initial site of allergen exposure –the airway surface, these approaches have the potential to successfully modulate the complex aberrant immune response that initiates and underpins allergic asthma.

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 a chronic inflammatory disorder of the airways characterised by clinical symptoms such as wheeze, shortness of breath, airflow obstruction, mucus hypersecretion and airway hyper-responsiveness (AHR). Numerous factors contribute to the pathogenesis of asthma, including respiratory infections, allergens, pollution and smoking. These factors can trigger asthma exacerbations, which are defined as a worsening of clinical symptoms. Asthma exacerbations are a major cause of hospitalisation and occur in all asthmatic patients regardless of disease severity. Although mild-to-moderate asthma can generally be well-controlled with glucocorticoid (GC) treatment, exacerbations are often difficult to treat and new effective approaches are needed. Viral respiratory infections, such as respiratory syncytial virus (RSV), are associated with exacerbations in patients with pre-existing asthma. Although allergic asthma is critically regulated by increased CD4+ T-helper (Th) type 2 (Th2) responses and type 2 innate lymphoid cells (ILC2s), viral infections are thought to exacerbate asthma by enhancing allergic inflammation and/or through activation of innate immune responses. Clinical studies have identified a complex range of immune responses during viral-induced exacerbations involving Th2, Th1 and Th17 responses, and activation of host innate immunity including macrophages. However, the disease mechanisms and the role of innate immune responses in particular, which underlie viral-induced exacerbations remain poorly understood. The aim of this PhD project was to establish a mouse model of RSV-induced exacerbations, which presents hallmark features of viral-induced exacerbations in humans. This model was then used to investigate the immunological mechanisms underpinning disease, and to identify new potential approaches for treatment. Chapter 2 describes the development and characterisation of our RSV-induced exacerbation model of AHR and airway inflammation in a mouse model of pre-existing allergic airways disease (AAD). Exacerbation was associated with activation of innate host immune responses. Notably, exacerbation only occurred on the background of AAD, indicating the importance of an underlying type 2 environment for pathogenesis. Further, RSV-induced exacerbation failed to respond to GC treatment. In this study, key functional roles for tumour necrosis factor-alpha (TNFα), monocyte chemoattractant protein (MCP)-1 and pulmonary macrophages in exacerbation were demonstrated through intervention studies. Further, increased TNFα and MCP-1 expression was observed in neutrophilic asthmatic patients, a subpopulation that often poorly respond to GC treatment. Chapter 3 investigated the role of the pro-inflammatory cytokines interferon-gamma (IFN-γ) and interleukin (IL)-27 as key regulators in the model of RSV-induced steroid-resistant exacerbation. RSV-induced exacerbation increased expression of IFN-γ and IL-27, which was resistant to GC treatment. Neutralisation of either IFN-γ or IL-27 completely suppressed RSV-induced AHR. This study further highlighted the role of these molecules in conjunction with macrophages in the induction of RSV-induced exacerbation. In Chapter 4, the effectiveness of a new anti-inflammatory bromodomain and extra terminal (BET) inhibitor (I-BET) was assessed for the suppression of steroid-resistant AHR. Two steroid-resistant airways disease models; a previously-described short-term model induced by IFN-γ and lipopolysaccharide (LPS) administration to the lung (two factors linked to steroid-resistant asthma) and our RSV-induced exacerbation model (Chapter 2) were used. I-BET treatment effectively suppressed AHR and airway inflammation in both models, by inhibiting the activation of pulmonary macrophages. These findings suggest that inhibition of BET proteins may be a novel therapeutic pathway to treat asthma exacerbations, by targeting macrophages. Thus these investigations indicate that in the context of underlying pre-existing allergic airways inflammation (found in asthma), RSV infection stimulates innate immune responses, increasing inflammation and AHR. In particular, key roles for TNFα, MCP-1, IFN-γ, IL-27 and pulmonary macrophages in the pathogenesis of RSV-induced exacerbation were demonstrated. Further, the new anti-inflammatory reagent, I-BET, was shown to significantly suppress all key features of RSV-induced steroid-resistant exacerbation (e.g inflammation and AHR) and IFN-γ/LPS-induced steroid-resistant AHR. These observations provide evidence for an important role of BET proteins in the regulation of steroid-resistant AHR and airway inflammation. Therefore, these studies provide evidence that targeting innate immune activation could be a therapeutic approach for the treatment of viral-induced asthma exacerbation.

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013
As células NKT invariantes Foxp3+ demonstram propriedades imunossupressoras após indução da expressão de Foxp3, tendo por isso sido designadas “células NKT reguladoras Foxp3+”. Estas células partilham vários marcadores fenotípicos com as células T reguladoras, tais como a expressão de CD25, GITR e CTLA-4, não perdendo, contudo, as suas características de células iNKT, nomeadamente a expressão de PLZF. Muito embora estas células tenham sido identificadas in vivo em nódulos linfáticos cervicais de ratinhos protegidos de encefalomielite autoimune experimentalmente induzida (EAE), após administração de α-galactosilceramida, o seu estudo tem sido feito recorrendo à conversão in vitro de células iNKT. Assim sendo, para que se verifique a conversão para células que expressem Foxp3, as células iNKT são isoladas por citometria de fluxo e colocadas em cultura com TGF-β, IL-2 e anti-CD28, na presença de anti-CD3 imobilizado em placa de cultura. Apesar do facto das células iNKT Foxp3+ terem já sido caracterizadas, várias características fenotípicas permanecem ainda por estudar. Além disso, a possibilidade de estas células exercerem um impacto semelhante ao apresentado pelas células T reguladoras em algumas patologias nunca foi avaliada. Um bom modelo para testar esse possível impacto é o modelo murino de indução da doença alérgica das vias respiratórias através da administração de ovalbumina (OVA). Neste modelo está descrito que a transferência adoptiva de células T Foxp3+CD25+CD4+ por via sistémica tem a capacidade para suprimir as principais características da doença. Assim sendo, este trabalho tem como objectivos: (1) caracterizar a população de células iNKT Foxp3+ e (2) avaliar se estas exercem um impacto protector in vivo num modelo de ratinho da doença alérgica das vias respiratórias induzida por OVA. Para optimizar o processo de obtenção de células iNKT Foxp3+, células iNKT isoladas de fígado e de baço de ratinhos C57BL/6J foram purificadas por citometria de fluxo e colocadas em cultura com TGF-β, IL-2 e anti-CD28 na presença de anti-CD3 imobilizado em placa durante 4 dias. Após conversão, foi possível demonstrar que não existem diferenças fenotípicas significativas entre células iNKT Foxp3+ de fígado e de baço, dado que ambas as populações expressam de igual forma os marcadores de superfície Nrp-1, CTLA-4, GITR, CD103, PD-1 e NKG2D, não expressando NK1.1 nem CD62L. Foi também demonstrado que células iNKT recentemente saídas do timo apresentam uma maior propensão para conversão em células que expressem Foxp3. Os ensaios efectuados basearam-se num estudo recente que demonstrou que células NKT recentemente saídas do timo apresentam uma expressão característica de neuropilina-1 (Nrp-1), um receptor transmembranar comummente expresso pelas células T reguladoras. Embora tanto as células iNKT Nrp-1+ do fígado como as do baço apresentem maior propensão para conversão que as células Nrp-1-, esta conversão é superior nas células do baço. No que diz respeito aos receptores de quimiocinas, este estudo demonstrou que as células iNKT Foxp3+ expressam CXCR3 e CXCR6, não expressam CCR7 e expressam pouco CXCR5. Este padrão de expressão é semelhante ao já descrito na literatura para as células iNKT, podendo indicar que estas células migram para locais de infecção e inflamação. Vários modelos murinos têm sido descritos como uma excelente ferramenta para estudar a doença alérgica das vias respiratórias, já que estes apresentam um conjunto de características típicas da asma alérgica em seres humanos. Foi demonstrado que estes animais desenvolvem infiltrados inflamatórios nas vias respiratórias, onde se observa em secções de pulmão e em lavados broncoalveolares, eosinófilia e hiperplasia de células calciformes. Estes animais também apresentam um aumento na concentração sérica de IgE e IgG1 específica para alergénio, bem como citocinas Th2 e hiperplasia das células calciformes. Os modelos de imunização activa têm por base a administração de um antigénio, tanto numa pré-imunização, como numa re-exposição por via intra-nasal, de maneira a mimetizar a resposta alérgica a estímulos exógenos. Na fase de re-exposição, o contacto com o alergénio desencadeia uma resposta inflamatória nas viaas respiratórias. O uso de antigénios proteicos como alergénios permite um melhor controlo e reprodutibilidade do modelo, dado que é possível controlar a administração de uma determinada concentração numa determinada localização. No caso particular destes modelos de doença alérgica das vias respiratória, o antigénio mais comummente utilizado é a ovalbumina de ovo de galinha. No entanto, o facto de que mesmo pequenas variações no protocolo, como por exemplo a via de imunização, o adjuvante utilizado, a dose de antigénio administrada, e o background genético dos animais, podem influenciar os resultados do estudo, fazem com que seja crucial uma caracterização prévia do modelo a utilizar. Para analisar o desenrolar das respostas imunes características do protocolo escolhido de indução da doença alérgicas das vias respiratórias através de OVA, ratinhos Thy1.1 foram sensibilizados por via intraperitoneal com 10μg de OVA-Alum, aos dias 0, 7 e 14, e re-estimulados por via intranasal, aos dias 21, 22 e 23 com 50μg de OVA em solução salina. Como grupo de controlo, foram também incluídos no estudo ratinhos Thy1.1 não sensibilizados. Ao dia 24 todos os animais foram sacrificados por injecção letal, tendo sido recolhido sangue, pulmões, nódulos linfáticos e lavado broncoalveolar. Desta análise foi possível concluir que neste modelo de doença alérgica das vias respiratórias induzida por OVA se verifica um aumento na percentagem de eosinófilos presentes no lavado broncoalveolar, e infiltrado inflamatório junto das vias respiratórias. É igualmente visível um aumento das concentrações séricas de ambas as imunoglubulinas testadas. No entanto, este modelo não demonstra qualquer alteração nas concentrações de citocinas Th2 no pulmão. A possibilidade da ocorrência de uma resposta Th1 foi excluída pela ausência de IgG2a específica para OVA no soro dos animais sensitizados. Finalmente, para testar o possível impacto de células iNKT reguladoras Foxp3+, procedeu-se à transferência de células iNKT CD25+Foxp3+, células T CD4+CD25+Foxp3+ ou células iNKT Foxp3- por via intratraqueal para ratinhos Thy1.1 onde a doença alérgica das vias respiratórias foi induzida pelo protocolo descrito. A partir destes procedimentos foi possível verificar que, como esperado, ratinhos que receberam células T reguladoras apresentam um decréscimo na percentagem de eosinófilos presentes no BAL e menores infiltrados inflamatórios no pulmão, sendo também visível uma diminuição na concentração sérica de IgE. No caso de ratinhos para os quais foram transferidas células iNKT Foxp3+, estes demonstraram um ligeiro decréscimo quer na percentagem de eosinófilos presentes no BAL, quer no infiltrado inflamatório junto das vias respiratórias. Pelo contrário, a transferência de células iNKT Foxp3- não exerceu qualquer efeito sobre o número de eosinófilos, aumentando significativamente a concentração sérica de IgE. A presença das células transferidas foi confirmada pela sua presença nos nódulos linfáticos drenantes (nódulos linfáticos do mediastino). Em conclusão este estudo mostra que as células NKT invariantes Foxp3+ do fígado e do baço apresentam capacidade de conversão e fenótipo semelhante, partilhando muitas das suas características fenotípicas com as células T reguladoras. Estas células expressam também CXCR3 e CXCR6, mas não expressam CCR7 e poucas expressam CXCR5. Ao analisar o impacto das células NKT invariantes Foxp3+ na doença alérgica das vias respiratórias através de transferências adoptivas, foi possível concluir que estas parecem possuir a capacidade de diminuir ligeiramente a eosinofilia característica da doença. No entanto, esta é a única característica da doença onde estas células parecem ter algum impacto. O facto do modelo de doença alérgica das vias respiratórias utilizado não permitir o correcto estudo de respostas Th2, provavelmente devido ao facto da estirpe utilizada apresentar uma tendência para respostas Th1, sugere que mais experiências são necessárias, baseadas numa diferente estirpe de ratinho, para esclarecer de modo claro esta questão.
Foxp3+ invariant NKT cells are a subset of iNKT cells that display immunosuppressive properties. These cells share many phenotypic hallmarks with regulatory T cells, while retaining its NKT cell characteristics. Despite the fact that Foxp3+ iNKT cells have been already characterized, many phenotypical characteristics remained unstudied. Moreover, a possible impact of those cells in the prevention of inflammatory pathologies has never been addressed. A good model to test this issue is OVA-induced allergic airways disease in mice, where Foxp3+CD25+CD4+ T cells have been described to suppress some manifestations of the disease. Therefore, this work aimed to further characterize the Foxp3+ invariant NKT cell population and evaluate its impact in vivo on a mouse model of OVA-induced allergic airways disease. To obtain as many Foxp3+ invariant NKT cells as possible converted in vitro, sorted iNKT cells were cultured for 4 days with TGF-β, IL-2 and anti-CD28 in the presence of plate-bound anti-CD3. Upon conversion it was possible to show that there are no significant phenotypical differences between liver and splenic Foxp3+ iNKT cells, as both populations have similar expression of CD25, Nrp-1, CTLA-4, GITR, CD103, PD-1 and NKG2D, although differ in CD62L and NK1.1 expression. It was also shown that recent thymic emigrant iNKT cells are more prone to conversion into Foxp3-expressing iNKT cells. Regarding chemokine receptors, as iNKT cells, Foxp3+ iNKT cells express CXCR3 and CXCR6, do not express CCR7 and express almost no CXCR5, in the same way as Foxp3- NKT cells. Upon adoptive cell transfers of Foxp3+ iNKT cells, OVA-sensitized mice showed a small decrease airway eosinophila.

Research Doctorate - Doctor of Philosophy (PhD)
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and their cellular expression is differentially regulated at various developmental and functional stages. The objective of my PhD research was to assess 1) whether miRNAs are differentially regulated in an ovalbumin-induced model of allergic airways disease, 2) whether corticosteroid (dexamethasone) treatment alters miRNA expression, 3) whether miRNAs play a functional role in disease development, 4) whether miRNAs are differentially regulated in Th cell differentiation and 5) whether miRNAs play a functional role in Th cell differentiation and function. To address these questions, we performed miRNA profiling on the lungs of allergic mice and compared these profiles to lung profiles from dexamethasone-treated mice and non-allergic controls, using miRNA microarray analysis and real-time PCR. We generated distinct miRNA signatures and identified 29 miRNAs that showed significantly altered expression in allergic lungs. Analysis of predicted miRNA targets revealed novel target genes with altered mRNA expression and demonstrated synergistic miRNA regulation within allergic lungs. Using antagomirs, we inhibited the function of two specific miRNAs (mmu-miR-155-5p and mmu-miR-449a-5p) in the airway wall and investigated the effect on hallmark features of allergic airways disease. While antagomir administration successfully reduced expression of targeted miRNAs, it failed to induce alterations to disease phenotype, suggesting multiple miRNAs regulate changes associated with allergic disease. We further show that antagomir delivery to the lung achieves only variable efficacy across different cell types. While antagomir delivery efficiently reduced specific miRNA expression in myeloid cells, lymphocytes are only partially targeted, suggesting that therapeutic targeting of miRNA function in lymphocytes may require a different approach. We further performed miRNA profiling in naive and effector Th cells in vitro and identified a global up-regulation of miRNA expression in activated Th cells. Using antagomirs, we inhibited the function of several miRNAs and again found that antagomir delivery to Th cells proves inadequate for suppression of target miRNA expression.

Research Doctorate - Doctor of Philosophy (PhD)
Rhinovirus (RV) infections are common ailments and are the most common precipitant of asthma exacerbations. This thesis contains investigations of novel mechanisms in RV infections and RV-induced exacerbations of house dust mite (HDM) driven allergic airways disease (AAD). The role of TLR7, CCL7, IRF-7, TRAIL and the off-target effect of long-acting β₂-agonists (LABAs) on PP2A are characterised in the host response to RV with a combination of in vivo and in vitro approaches. In chapter 2, TLR7 signalling is identified as crucial for antiviral responses to RV and for dampening allergic Th2 responses, protecting against RV-induced exacerbations of allergic airways disease. Likewise, high levels of allergic signalling through IL-5 suppresses TLR7 mediated antiviral responses. In chapter 3, results from the mouse lung transcriptome response to RV infection guided the investigation of two of the most up-regulated genes, CC-motif ligand 7 (CCL7) and interferon regulatory factor 7 (IRF-7). By inhibiting CCL7 or IRF-7 in naïve mice, the antiviral response and inflammation was suppressed following RV infection. Inhibiting CCL7 during infection of allergic mice also reduced inflammation. In chapter 3, tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is shown to be pro-inflammatory and pro-viral during RV infection. TRAIL is up-regulated in the lung during the course of RV infection. TRAIL-deficient mice were protected against inflammation and airways hyperresponsiveness (AHR). RV-titre was reduced in TRAIL-deficient mice and manipulation of TRAIL in vitro had direct effects on viral titre. In chapter 4, the LABA Salmeterol demonstrated anti-inflammatory effects by directly activating PP2A, and suppressing AHR independently of β-2 adrenoreceptors. In summary, I have taken multiple approaches to identify novel mechanisms of the host response to RV infection and RV-induced exacerbations of allergic airways disease to identify novel therapeutic targets that may treat the underlying inflammatory mechanisms of asthma exacerbations.

Research Doctorate - Doctor of Philosophy (PhD)
Activation of innate immune responses in individuals with asthma by respiratory viral and bacterial infections or colonization with pathogenic bacteria can cause disease exacerbation. Exacerbations have a negative impact on quality of life and are characterised by persistent airway inflammation, worsening of disease symptoms and poor responsiveness to standard corticosteroid therapy. How activation of innate host defence pathways by bacterial infection triggers steroid-resistant inflammatory pathways and disease exacerbation is poorly understood. Better disease models are urgently required to identify mechanisms underlying disease exacerbation. We hypothesised that bacterial infection (mimicked by lipopolysaccharide (LPS) exposure) would exacerbate pre-existing allergic airways disease (AAD) in a mouse model, causing steroid-resistant airways inflammation and airway hyperresponsiveness (AHR). Mice were initially sensitized and subsequently challenged with nebulised ovalbumin (OVA) to induce AAD. LPS was then administered into the lung, in the presence or absence of dexamethasone (DEX) to assess steroid sensitivity. Disease outcomes were assessed by quantifying lung function (airways hyperresponsiveness; AHR), inflammatory cell infiltration, tissue cytokine levels and microarray profiling. LPS administration induced steroid-resistant AHR and increased inflammatory cytokine expression (including interleukin (IL)-27, interferon (IFN)γ, macrophage inflammatory protein (MIP)-1α and tumour necrosis factor α (TNFα)), while CD4⁺ T-helper 2 (Th2) cytokines (IL-5 and IL-13) were not altered compared to OVA-treated mice. Neutrophil and macrophage numbers were also increased in the bronchoalveolar fluid (BALF) following LPS administration. Targeted depletion of alveolar macrophages with 2-chloroadenosine (2-CA) significantly suppressed AHR. Further, IL-13 was required for exacerbation, as LPS failed to exacerbate AHR in IL-13 deficient mice or following administration of IL-13 blocking antibodies. Microarray profiling of lung samples revealed that microRNA (miR)-135b-5p expression was markedly increased following LPS administration in mice with pre-existing AAD and expression was only partially suppressed by corticosteroid treatment. Inhibition of miR-135b-5p function by antagomir treatment suppressed LPS-induced exacerbations AHR and markedly reduced inflammatory cell infiltration. In summary, we developed a novel mouse model of LPS-induced steroid-resistant exacerbation of AAD, which mimic critical features of infection-induced exacerbation of asthma. Our findings highlight key roles for pulmonary macrophages, IL-13 and miR-135b-5p in the development of disease symptoms. Targeting these pathways may be a useful treatment for acute bacterial-induced exacerbation of asthma.

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is a chronic inflammatory disease of the airways, associated with debilitating reversible airflow obstruction. The majority of healthcare costs from asthma-related hospitalisations are attributed to exacerbations by respiratory viruses, with rhinoviruses (RV) being the most commonly detected. This thesis presents original research papers detailing investigations to elucidate the mechanisms underlying RV-induced exacerbations of allergic airways disease (AAD). The first manuscript (see Chapter 2) details the elucidation of a novel TRAIL signalling pathway where the TRAIL-regulated gene product Midline-1 (MID1), which inhibits protein phosphatase 2A (PP2A), was found to promote AAD through increased homing of myeloid dendritic cells (mDCs) to the airway via CCL20 release. Notably, inhibition of MID1 or reactivation of PP2A abolished airway hyperresponsiveness (AHR) and attenuated airways inflammation and mucus hypersecretion in mouse models of AAD and RV-induced exacerbation. The second manuscript (see Chapter 3) investigates the importance of Toll-like receptor (TLR) 7-elicited interferon (IFN) responses during RV infection in an asthmatic setting. We show that following exposure to house dust mite (HDM), mice deficient in TLR7 display exaggerated eosinophilic inflammation and attenuated anti-viral responses when challenged with RV. TLR7 expression in the lungs of mice was found to be suppressed by interleukin-(IL)-5-induced eosinophilia, while human asthmatics with eosinophilic but not neutrophilic airways inflammation also showed reduced TLR7 and IFN expression. The third manuscript (see Chapter 4) revisits established therapeutic agents, long-acting β₂ agonists (LABAs), in light of recently described interactions with PP2A. This study extends those findings by reporting that administration of salmeterol, or other β₂ agonists, protected mice against HDM- and RV-induced lung inflammation as effectively as the corticosteroid dexamethasone. Salmeterol but not dexamethasone mediated this via increased PP2A activity, the inflammatory phenotype recapitulated when PP2A was targeted by siRNA. Taken together, these studies have identified new targets for the therapeutic intervention of asthma and RV-induced exacerbation.

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is a chronic inflammatory disease of the airways that affects over 300 million people worldwide. The disease is characterised by episodes of breathlessness, coughing, wheezing and airway hyperresponsiveness (AHR). Asthma results from a dysregulation in immunity that is underpinned by a cohort of effector T cell populations including T helper (Th)1, Th2, Th17 and natural killer T (NKT) cells. These effector T cells produce numerous inflammatory cytokines and chemokines that induce eosinophil influx, mucus hypersecretion and AHR. Antigen presenting cells play key roles in priming these responses. Regulatory T cells (Tregs) are essential for suppression of aberrant immune responses and maintenance of immune homeostasis. Both the number and function of Tregs is impaired in asthmatics, compared to healthy individuals. This reinforces the importance of Tregs in regulating a balanced immune response. Microbial agents have been associated with increased or decreased risk of asthma. Microbial agents that have been associated with decreased asthma risk are under intense investigation for their potential utilisation in therapeutic strategies for asthma. Streptococcus pneumoniae vaccination has been associated with decreased asthma-related hospitalisations in children and the elderly. Furthermore, early mouse studies observed that S. pneumoniae infection attenuated blood eosinophilia during parasitic infection. More recent studies have shown that both live and ethanol killed S. pneumoniae suppress the development of allergic airways disease (AAD) in mice, including eosinophil recruitment to the lungs, Th2 cytokine release, mucus hypersecretion and AHR. Therefore S. pneumoniae has the potential for development into a novel immunotherapy for asthma. To examine this concept we first investigated the capacity of human S. pneumoniae vaccines, which were developed to prevent S. pneumoniae infection, to suppress AAD in mouse models (Chapter 2). In the next study, and in order to determine which components were required for S. pneumoniae-mediated suppression of AAD, S. pneumoniae components were tested for their capacity to suppress AAD (Chapter 3). Two potential S. pneumoniae-based immunotherapies were identified: the conjugate vaccine and the combination of type 3 capsular polysaccharide and pneumolysin (T3P+Ply). These S. pneumoniae immunotherapies suppressed the development of AAD when administered before, during and after sensitisation. Importantly, S. pneumoniae immunotherapy also attenuated established AAD. This demonstrated that S. pneumoniae immunotherapy has potential for therapeutic use in the prevention and/or treatment of asthma. To determine the mechanisms involved in S. pneumoniae-mediated suppression of AAD a number of investigations were performed. Tregs were shown to be induced by S. pneumoniae immunotherapy. Furthermore, anti-CD25 antibody-mediated depletion of Tregs reversed the effect of immunotherapy. Hence, Tregs were required for immunotherapy-mediated suppression of AAD. In the third study, Tregs were shown to be induced in a biphasic manner to suppress immune responses and AAD through a broad range of mechanisms (Chapter 4). Together, these studies have identified potential and novel S. pneumoniae immunotherapies for asthma and determined the mechanism of action that underpins suppression of AAD.

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

Asthma is a chronic condition characterized by variable airflow obstruction, bronchial hyper-responsiveness, airway inflammation and remodeling. In spite of tremendous advances, the regulatory mechanisms controlling these pathological features have not yet been completely addressed. From an immunological perspective, type 2 inflammation and eosinophilic infiltration are the most striking hallmarks of asthma. At physiological level, structural changes such as increase in smooth muscle mass take the center stage which is usually associated with clinical measures of asthma. There might be some regulatory mediators capable of tuning airway inflammation and remodeling under homeostatic conditions but abrogated in asthmatic conditions. Semaphorin 3E (Sema3E) is an axon guidance molecule that is ubiquitously expressed and plays diverse roles in structural and inflammatory cells such as regulation of cell migration, proliferation and angiogenesis. However, its role in clinical and experimental asthma remains unclear. In this thesis, I have set out to uncover the expression and function of Sema3E in allergic asthma. It is generally hypothesized that Sema3E is down-regulated in allergic asthma which orchestrates the function of inflammatory (dendritic cells and neutrophils) and structural (airway smooth muscle) cells. Replenishment of Sema3E, which is suppressed under asthmatic conditions, could confer protection against allergic asthma by modulation of cellular functions. I began by comparing the expression of Sema3E between allergic asthmatics and healthy subjects. A remarkable down-regulation of Sema3E under asthmatic patients was observed which was further confirmed in a mouse model of the disease. Decreased expression of Sema3E was specifically demonstrated on bronchial epithelial cells obtained from asthmatic patients at both mRNA and protein levels. To address the function of Sema3E in allergic asthma in vivo, I extended my studies to mouse models of the disease and demonstrated that Sema3e gene deletion results in exacerbated allergic asthma pathology induced by allergen exposure. To investigate the translational relevance of my findings, I performed treatment of an asthmatic mouse model with exogenous Sema3E in which its intranasal administration attenuated airway inflammation, remodeling and hyper-responsiveness. The mechanism underlying Sema3E’s role in pathogenesis of allergic asthma was extensively studied indicating a crucial role of this mediator in modulation of dendritic cells and neutrophils functions. Our data demonstrated that both dendritic cells and neutrophils express the Sema3E high affinity receptor, PlexinD1, which makes them responsive to Sema3E treatment. Then, I studied expression and function of PlexinD1 on human airway smooth muscle (ASM) cells. I found that PlexinD1 surface expression was reduced on ASM cells from asthmatic patients. Treatment of ASM cells with Sema3E inhibited their proliferation and migration as the characteristic feature of airway remodeling. Suppression of Rac1 GTPase activity and phosphorylation of Akt/PI3K and ERK/MAPK were found as signaling mechanisms underlying Sema3E’s inhibitory effects. Together, these findings show that Sema3E thereby appears as a novel regulatory mediator, upstream of pro-allergic events, suggestive of a new approach to attenuate allergic asthma deficits.
May 2016

Indiana University-Purdue University Indianapolis (IUPUI)
Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. The majority of the T cells in tissues such as the lung are tissue-resident memory (Trm) cells, defined as cells that maintain long-lasting presence in the tissue and have rapid functional recall following challenge. Allergen-specific CD4 T helper cells that secrete the cytokine IL-9 have been shown to be a necessary component of asthma pathogenesis. However, the precise characterization and function of IL-9-secreting CD4+ cells (Th9 cells) are unknown. Here we demonstrate that IL-9 production is progressively lost in Th9 cells over several rounds of culture and that environmental cues dictate the instability or effector function of the Th9 phenotype. We show Th9 cells are long-lived tissue-resident cells with the capacity to rapidly respond to secondary allergen challenge causing allergic airway disease (AAD). We found in a memory model of Aspergillus fumigatus challenge, Th9 cells maintain tissue residency throughout a 12-week period of antigen-free rest. Additionally, we demonstrated increased frequency of IL-9-producing cells and quantity of IL-9 upon rechallenge, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. The protection afforded by IL-9 antibody blockade was not seen in a chronic model asthma-like disease demonstrating IL-9 has a specific role in allergic memory responses. Interestingly, IL-9-secreting cells have a polyfunctional multi-cytokine phenotype demonstrating a highly pathogenic state that we reproduced in culture. These observations suggest that IL-9 from Trm cell populations and Th9 cells play a novel role in allergen recall responses and are potential therapeutic targets for patients suffering from chronic intermittent asthma.
2022-05-05

Indiana University-Purdue University Indianapolis (IUPUI)
Rationale: Tissue remodeling and complement activation are asthma hallmarks. Type V collagen [col(V)], a cryptic antigen, becomes exposed during lung remodeling. IL-17 is key to anti-col(V) immunity, and regulates complement activation. We have reported that col(V)-induced tolerance down regulates IL-17 and prevents immune-mediated lung diseases. Objectives: Determine a role for anti-col(V) immunity in asthma. Methods: Serum anti-col(V) antibodies were measured in asthma patients, and immunohistochemistry utilized to detect interstitial col(V) in fatal asthma. Balb/c mice were tolerized with col(V) prior to sensitization with ovalbumin (OVA), and subsequent OVA intranasal challenge. Airway hyper-responsiveness (AHR) to methacholine was measured; and RT-PCR utilized to determine local Il17 transcripts. Bronchoalveolar lavage levels of C3a¸ C5a and OVA-specific IgE were measured; and immunohistochemistry utilized to detect expression of complement regulatory proteins, expression, CD46/Crry and CD55, in lung tissue. Results: Compared to normal subjects, anti-col(V) antibodies were increased in asthmatics; and interstitial col(V) was over expressed in fatal asthma. OVA-induced AHR up regulated anti-col(V) antibodies systemically, and increased OVA-specific IgE and C3a in BAL, and parenchymal Il17 transcripts. Col(V)-induced tolerance abrogated AHR, down regulated OVA-induced T cell proliferation, as well as total and OVA-specific IgE, C3a, IL-17 expression and tracheal smooth muscle contraction. Crry/CD46 and CD55, key to preventing complement activation, were down regulated on goblet cells in murine allergic airway disease. Conclusions: Anti-col(V) immunity correlates with asthma pathogenesis, and col(V)-induced tolerance may be a novel therapeutic for asthma. Decreased expression of Crry/CD46 and CD55 on goblet cells may in part account for complement activation in asthma.

Phthalate plasticizers are found in consumer products and home furnishing materials. Phthalate urinary metabolites are detected in nearly every sample in population-based studies indicating widespread exposure. Prior epidemiologic studies have associated vinyl flooring, a proxy for phthalate exposure, or house dust concentrations of phthalates with eczema and asthma in children. However, these studies lack adequate exposure measurements, consideration of the early life period, and prospective designs. In light of these gaps in the literature, we designed epidemiologic analyses to address our overarching hypothesis that early life exposure to a mixture of phthalates will have associations with adverse allergic and respiratory health outcomes in children. We tested this hypothesis in five self-contained manuscripts that characterize sources of exposure to phthalates in early life, demonstrate the application of new statistical methods for estimating effects of these highly correlated biomarkers, and test the association between early life exposure to phthalates and eczema and airway inflammation in children. Participants were enrolled from the longitudinal birth cohort of the Columbia Center for Children's Environmental Health (CCCEH) in New York City. Phthalate metabolites were measured in prenatal and child urine samples at the Centers for Disease Control and Prevention. Questionnaires and visual inspections were combined with phthalate measurements from personal and indoor air sampling and urinary metabolite concentrations to examine sources and patterns of phthalate exposure associated with personal care product use and flooring materials in the home. The use of perfume and personal care products was associated with higher exposure to the metabolite of diethyl phthalate (DEP) but not di-n-butyl phthalate (DnBP). Vinyl flooring in the home was associated with higher indoor air and urinary metabolite concentrations for butylbenzyl phthalate (BBzP) but not di(2-ethylhexyl) phthalate (DEHP). Because some phthalates share exposure sources and have multiple metabolites, the urinary biomarker concentrations can be highly correlated. Using a reanalysis of the association between prenatal phthalate metabolites and reduced gestational age, we demonstrate that simple Bayesian models can estimate effects for highly correlated exposure measures without the instability of conventional modeling approaches. We found that prenatal concentrations of the metabolite of butylbenzyl phthalate were associated with the report of early-eczema but not atopy among children in the cohort. In a cross-sectional analysis, children's urinary concentrations of metabolites of diethyl phthalate and butylbenzyl phthalate were both associated with higher fractional exhaled nitric oxide, a marker of airway inflammation. These findings suggest several important sources of exposure to phthalates and demonstrate new methods for highly correlated exposures that have not been widely applied in the environmental health sciences. The association of biomarkers of exposure to butylbenzyl phthalate and eczema extend the findings of previous studies. Our results include the first report of an association between phthalates and airway inflammation in children.

Research Doctorate - Immunology/Microbiology
Asthma is a chronic inflammatory disease of the airways that is characterised by activation of CD4+ T-helper 2 type (Th) cells and eosinophils. The cause of this aberrant Th2 response is unknown but lack of early life infection is thought to play a significant role. The timing of infection and the type of pathogen may be critical to programming the immune response to a protective Th1, or destructive Th2, phenotype. The immune responses to infection with Salmonella typhimurium and Mycobacterium bovis Bacille Calmette Guerin (BCG) have been identified as targets for reprogramming or preventing the development of asthma. However, the role of these infections in contributing to a Th2-Th1switch or suppression of this response remains limited. In this investigation ovalbumin (OVA) T-cell receptor (TCR) transgenic (Tg) mice in combination with these bacterial strains expressing OVA have been used to specifically track the affects of each infection as well as OVA exposure on the T-cell response and the development of allergic airways disease (AAD) in the mouse model. BCG infection as an adult and a neonate prior to OVA challenge induced significant reductions in eosinophils in broncho-alveolar lavage fluid (BALF) and lung tissue compared to sham-infected mice that received OVA challenge. However, high levels of both Th1 (interferon gamma (IFN-γ)) and Th2 (interleukin (IL)-4, IL-5, IL-13) cytokines from supernatants of cultured peri-bronchial lymph node (PBLN) cells and splenocytes were found in all groups examined. Further studies tracking the development of the immune system after BCG infection at birth without OVA exposure revealed significant decreases in lung tissue eosinophils and decreased immunoglobulin (Ig) G1, IgG2a and IgE levels from serum compared to sham-infected controls. This coincided with decreased numbers of CD4+ and CD8+ T-cells in the spleens and PBLN cells. Levels of cytokines in splenocytes and PBLN cell cultures failed to show significant trends toward either a polarised Th1 or Th2, leaving a mixed Th1/Th2 phenotype. Infection with S.typhimurium lowered eosinophil levels in BALF, and mucous secreting cell (MSC) and eosinophil number in lung tissue after challenge with 23 OVA, compared to sham-infected mice challenged with OVA. In mice infected as neonates and adults prior to OVA challenge increased levels of IFN-γ from splenocyte culture supernatants were found, compared to sham-infected OVA challenged controls. Decreased levels of IL-5 from splenocyte culture supernatants was found in neonates but not adult mice infected with S.typhimurium prior to OVA challenged compared to sham-infected OVA challenged controls. High levels of both Th1 and Th2 cytokines were present in splenocyte and PBLN culture supernatants from all groups tested, indicating a mixed Th1/Th2 phenotype rather than a profound switch to Th1 immune response. Further studies showed that infection with S.typhimurium at birth without OVA exposure causes changes to the development of the neonatal immune system resulting in decreased eosinophil numbers in BALF and lung tissue, decreased levels of serum IgG1 and IgG2a, and a shift from Th2 to a mixed Th1/Th2 cytokine profile. These changes were found in samples examined up to 9-weeks post infection. This investigation demonstrates that infection with BCG or S.typhimurium can alter the immune system resulting in attenuation of various immunological and patho-physiological features of asthma. Infection with BCG or S.typhimurium as a neonate appears to produce the most pronounced modification in the subsequent immune responses to OVA. These findings provide important insights into possible modified vaccination regimes at birth and during childhood, which may have the potential to prevent the development of asthma and allergic inflammatory disorders in adulthood.

碩士
國立陽明大學
環境衛生研究所
92
Asthma prevalence is increasing in many countries, and childhood asthma has also become one of common chronic diseases and the most important health issue in children. Many hypotheses have been raised to explain the phenomena, and “hygiene hypothesis” is one of them. It states that lack of infections in early childhood stimulates the development of atopy, but which infections have protective effect need to be clarified. A follow-up study based on kindergartens, and the observing period is from age 3 to 6. In follow-up period, we collected data that asthma including medical history of children and their parents, some environmental factors such as housing status, and dietary habits. To investigated the associated between asthma and the concentrations of endotoxin and house dust mite. In our study, the recover rate of first questionnaire investigate was 63.8 %, secondary was 80.9 %. In follow-up period, incidence of childhood asthma, allergic rhinitis, and atopic dermatitis was 3.85 %, 20.83 %, and 5.71 %, respectively. The prevalence of asthma and allergic rhinitis were male higher than female (p < 0.01).Children that parents with a history of asthma have higher prevalence of asthma than other children (p < 0.01, p < 0.01). Children that parents with a history of allergic rhinitis have higher prevalence of allergic rhinitis than other children(p < 0.01, p < 0.01).Use the airconditioner and an air cleaner in childhood bedroom, the concentrations of endotoxin in sofa is lower. In the dietary habits of child, we observed that the higher frequencies of seafood without fish was positively associated with childhood allergic rhinitis symptom (OR = 1.43), the higher frequencies of deepsea fish was positively associated with childhood asthma symptom (OR = 1.75). In our study, we observed a few of risk factors that the childhood asthma affected by housing environment and dietary factors. Male gender, mother with a history of asthma, father with a history of asthma were the main risk factor. Results showed asthma in age 3~5 children were more correlated with allergic history of parents and atopy.