Among the environmental airborne fungi one of the most common is <i>Alternaria alternata</i>. From a clinical perspective Alternaria has long been associated with IgE-mediated, histamine-dependent mold allergy, allergic rhinitis, chronic rhinosinusitis (CRS) and asthma. Recently it has been proven that an abnormal immunological response to Alternaria most likely contributes to the pathogenesis of upper respiratory airway disorders. In this body of work, we present for the first time results of several sets of experiments including, 1) the analysis of A. alternata spore germination expressed sequence tags (ESTs), 2) the survey of global allergen homologues in fungal genomes, and 3) the first microarray experiment investigating airway epithelial cell responses to this fungus. In the first project, the analyses of the EST dataset offered a first look into the gene content of A. alternata and represents the beginning of future research of this ubiquitous fungus. Annotation and classification of ESTs revealed a number of genes that could be involved in the immunomodulation process of the human immune response toward fungi. We also discovered that the majority of known allergens are expressed during the spore germination phase of A. alternata. For investigating the allergenic potential of fungi we developed a whole genome approach by querying fungal genome sequences (<i>A. alternata, A. brassicicola,</i> and <i>Aspergillus fumigatus</i>) with a database of all known allergenic proteins from a taxonomically diverse group of organisms. Interestingly, we identified homologues of diverse types of allergens in these fungal genomes and also many homologues of allergens from other organisms including those from pollen, insects, and venoms. Finally, we investigated global gene expression changes of human airway cells in response to <i>A. alternata</i> and an ∆alt a 1 deletion mutant. We found that wild type Alternaria spores induced significant changes in gene expression patterns in human airway epithelial cells, especially known immune response genes. Furthermore, results of these analyses revealed that Alt a 1 is a major factor in inducing epithelial inflammatory responses. / Ph. D.

Chronic exposure to the ubiqutious airborne fungus, Alternaria alternata, has long been implicated in the development and exacerbation of human allergy and asthma. Alt a 1 was identified previously by several groups as the major allergen secreted by A. alternata, due to its IgE-specific reactivity with sera from atopic patients. Despite the well-documented clinical importance of Alternaria and its major allergen, little knowledge exists regarding their role and interaction with the innate immune system. Here for the first time we characterize the innate immune response to A.alternata and verify the significance of Alt a 1 in contributing to this response in human airway cells and murine models. Our studies establish a baseline response for both a chronic and single-challenge murine inhalation model with Alternaria spores. Both models demonstrate live conidia induce a robust response, arguably more pathologically relevant compared to studies employing Alternaria extracts. We also elucidate the overall importance of Alt a 1 by utilizing recombinant Alt a 1 protein, A. alternata (Δalt a 1) deletion mutants, and an A.alternata (Alt a 1+) overexpression mutant. Both Alt a 1 protein and A. alternata conidia stimulated production of pro-inflammatory cytokines/chemokines in mice after a single intranasal challenge. Infiltration of effector cells (macrophages, neutrophils, eosinophils, and lymphocytes) into the lungs along with other hallmarks of airway inflammation was observed. In addition, Alt a 1 protein and conidia evoked secretion of pro-inflammatory cytokines in treated human airway epithelial cells while the Alt a 1+ overxpression mutant induced a significantly higher response. In contrast, spores of Δalt a 1 caused an attenuated response in both human cells and murine lungs suggesting that this single protein may play a major role in inducing the innate immune response in airway epithelium at the organismal level. Finally, we identified key biochemical properties of the Alt a 1 protein including a single histidine required for esterase activity and a unique RXLR-like motif which controls Alt a 1's ability to bind external lipids and enter human airway cells. Overall, these results improve our understanding of how Alternaria induces innate immunity and identifies possible therapeutic targets within allergenic proteins. / Ph. D.

Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P). / Ph. D. / Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and allergy to several fungal species including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying how the allergen, Alt a 1, from the fungus, <i>Alternaria alternata</i>, can cause an allergic response in the human airways. In several studies, 90-100% of allergic patients who have allergy to Alternaria, have proteins in their bloodstream that specifically recognize Alt a 1. This indicates that they are allergic to Alt a 1. Though we know that these patients have allergy to Alt a 1, we do not know how this protein causes the characteristic symptoms of allergy, such as a runny nose, watery eyes, hives, and breathing difficulty. Published studies have increasingly made it clear that molecules on the surface of cells that line the airways are important players in the body’s response to allergens. A dust mite allergen, Der p 2, can interact with one such receptor on human cells. The receptor may not be the only way that allergens can cause a response. Studies have shown that allergens can directly enter human cells. For example, a Birch tree allergen, Bet v 1, has been shown to enter human lung cells. This project was focused on two primary aims; (1) Identify how human airway cells response to Alt a 1, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to cause human airway cells to produce several molecules that lead to the characteristic symptoms of allergy, and that this response was dependent on a receptor on human airway cells. We also found that Alt a 1 rapidly enters human airway cells.