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Eosinophilia is such a prominent characteristic of many parasitic infections that it is often used for a presumptive diagnosis. Trichinosis is one of the parasitic diseases in which the eosinophil response may attain considerable magnitude. [...]

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Exposure to sufficiently high allergen levels of some allergens appears to protect against the development of allergic diseases, and has also been used to treat established allergic diseases. A reduced prevalence of sensitization and, in some cases, asthma was reported among individuals living in homes with high allergen levels. Furthermore, allergic patients treated with high allergen doses during immunotherapy experienced improved allergic symptoms. The mechanisms for how high allergen exposure may prevent disease are not known and thus, a model in which these mechanisms could be studied in detail is warranted. The objective of this thesis was to model and further characterize the phenomenon of high allergen dose-dependent protection against the development of allergic responses, using a mouse model of epicutaneous sensitization. The impact of exposure to a high dose of cat dander in established allergic disease was also evaluated. Epicutaneous exposure to a high dose of cat dander (150 μg) prevented airway inflammation and airway hyperresponsiveness in genetically different strains of mice, including Th2 prone BALB/c mice. The protective effects against airway inflammation appeared to wane between 10 and 120 days after exposure. When the high dose of cat dander was applied with a sensitizing dose of peanut, peanut induced anaphylaxis was prevented. Exposure to a high dose of another allergen, ovalbumin, was also able to prevent the development of allergic airway disease. In contrast, the high dose of cat dander did not improve established states of allergic disease. Thus, in our mouse model, a high dose of cat dander prevented the development of allergic responses and reflected characteristics of the phenomenon reported in humans. As such, this model may be useful for investigating mechanisms and potential prophylactic options for allergic diseases. As the high dose of cat dander either had no effect or worsened established allergic responses in our model, it is possible that the dose was not high enough to improve disease in sensitized mice. It is also possible that established disease cannot be modified by epicutaneous exposure. / Thesis / Master of Science (MSc)

Co-culture of eosinophils and BEAS-2B cells was found to increase the release of cytokine IL-6 and chemokines MIG, MCP-1, IL-8 and IP-10 and up-regulate the corresponding genes expression in BEAS-2B cells or eosinophils. Interaction of eosinophil-BEAS-2B cells could also elevate adhesion molecules ICAM-1, VCAM-1, ICAM-3, and CD49d expression on the surface of BEAS-2B cells, and CD18 and ICAM-3 on eosinophils, and up-regulate ICAM-1 gene expression in BEAS-2B cells. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha could induce or further induce ICAM-1 expression on eosinophils and BEAS-2B cells upon their interaction. Moreover, activities of both NF-kappaB and p38 MAPK in BEAS-2B cells were markedly elevated after co-cultured with eosinophils. / Freshly isolated eosinophils from human peripheral blood and confluent BEAS-2B cells were co-cultured together in tissue culture plate for a pre-determined time period. Cytokines including interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, IL-12p70, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma and chemokines regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein (MCP)-1, IL-8, and interferon inducible protein (IP)-10 in culture supernatant were evaluated by protein array and quantified by cytometric bead array (CBA) kit of Th1/Th2 cytokines, inflammatory cytokines, and human chemokines using flow cytometry and enzyme linked immunosorbent assay (ELISA) kit. / In order to investigate the immunopathological mechanism in allergic asthma of eosinophils interacting with bronchial epithelium in inflammation site, a in vitro system of co-culture of human bronchial epithelial cells and eosinophils was set up to mimic the inflammatory reaction. / In summary, co-culture of epithelial cells, BEAS-2B cells, and eosinophils could activate NF-kappaB and p38 MAPK signal transduction pathways to induce inflammatory cytokine IL-6, and chemokines IL-8, MCP-1, MIG and IP-10 release in culture supernatant, and up-regulated the expression of surface adhesion molecules ICAM-1, VCAM-1, ICAM-3 and CD49d protein on BEAS-2B, and CD18 and ICAM-3 on eosinophils. (Abstract shortened by UMI.) / In this study, co-culture of a human epithelial cell line, BEAS-2B cells, and peripheral eosinophils was adopted as an in vitro model to investigate the effect of interaction of epithelial cells and eosinophils in airways on pathophysiology of asthma. / Wang Chengbin. / "July 2005." / Advisers: Wai kei Lam; Chun kwok Wong; Yaping Tian. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3723. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 119-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

過敏性疾病的患病率一直上升，約30%至40%的世界人口受到一個或多個過敏疾病影響。然而，現時仍然缺乏有效治療過敏性疾病的方法，大多數傳統療法只能改善臨床症狀，卻沒有針對導致過敏性炎症的主要因素。嗜酸性粒細胞浸入和積累於局部炎症部位以及延緩了的細胞凋亡是過敏性炎症的標誌，而嗜酸性粒細胞亦被認為是過敏性炎症的主要效應細胞。因此，針對嗜酸性粒細胞，抑制其活性和衍生產物，是一個有潛力和有效地治療過敏性炎症的策略。發展針對嗜酸性粒細胞的療法需要詳細了解嗜酸性粒細胞介導炎症反應的機制。 / 在第三章中，我們把嗜酸性粒細胞與皮膚成纖維細胞一起培養，建立了一個體外的皮膚炎症模型，用以研究嗜酸性粒細胞在過敏性皮炎發病病理中的作用。我們發現，嗜酸性粒細胞和成纖維細胞的共培養顯著誘導促炎性的細胞因子IL-6和濕疹相關趨化因子CXCL1，CXCL10，CCL2和CCL5的釋放。這些炎症介質的釋放在能導致皮膚瘙癢的細胞因子IL-31和內源性警報因子IL-33的刺激下進一步加強。 IL-31和IL-33可顯著引起嗜酸性粒細胞和成纖維細胞釋放CXCL8，而當兩種細胞共培養後，CXCL8的釋放進一步增強。在共培養系統中，嗜酸性粒細胞是釋放CCL5的主要來源，而成纖維細胞則是釋放IL-6，CXCL1, CXCL8，CXCL10和CCL2的主要來源。嗜酸性粒細胞和成纖維細胞之間的直接相互作用是CXCL1，CXCL10，CXCL8和CCL5的釋放所必需的。在IL-31和IL-33的刺激下，共培養系統中嗜酸性粒細胞和成纖維細胞的細胞表面粘附分子ICAM-1的表達上調。而嗜酸性粒細胞和成纖維細胞間的互作用，以及IL-31和IL-33對兩種細胞的激活，是經由p38、ERK、JNK，NF-κB和PI3K-AK訊息傳遞徑路所調節的。另外，我們亦發現1,25-二羥維生素D3能顯著抑製共培養系統在IL-31或IL-33激活下IL-6，CCL2和CXCL8的釋放。 / 在第四章中，我們探討在嗜酸性粒細胞和支氣管上皮細胞BEAS-2B的共培養系統中，兩種細胞的細菌相關模式識別受體成員NOD1和NOD2激活機制。我們發現NOD配體iE-DAP和NOD2配體MDP能激活嗜酸性粒細胞內的ERK和NF-κB信號傳導通路，而MDP可以激活嗜酸性粒細胞和BEAS-2B細胞的ERK信號傳導通路。當嗜酸性粒細胞和BEAS-2B細胞共培養時，iE-DAP和MDP均可激活嗜酸性粒細胞和BEAS-2B細胞的ERK和NF-κB信號傳導通路，而且與單獨培養的細胞相比，激活的程度更強。此外，我們還探討了iE-DAP和MDP對 BEAS-2B細胞和嗜酸性粒細胞的核轉錄因子的激活作用。我們發現iE-DAP和MDP可以激活嗜酸性粒細胞和BEAS-2B細胞的某些轉錄因子，包括因子NF-κB、STAT-3和NFAT等，而多數被激活的轉錄因子都跟過敏性炎症和哮喘的發病有關。除了體外研究，我們還建立了過敏性肺部炎症的小鼠模型，用以研究iE-DAP和MDP的生理效應。iE-DAP和MDP均可激活第二型輔助性T細胞相關的免疫球蛋白E生產，以及誘導嗜酸性粒細胞趨化因子CCL5。 / 上述的研究結果表明IL-31與IL-33在過敏性皮炎的發病病理中發揮關鍵作用，通過不同的信號傳導機制激活嗜酸性粒細胞與成纖維細胞的相互作用。另外，我們探討了在嗜酸性粒細胞和BEAS-2B細胞中，調節NOD1和NOD2激活的信號傳導機制，加上動物實驗，所得的結果有助了解細菌感染在哮喘發作中所發揮的影響。這些關於細胞之間和細胞內調控機制的研究結果，進一步增強我們對嗜酸性粒細胞介導炎症的理解，並為發展更高專一性、效用和更少副作用的消炎藥提供了新的標靶和方向。 / The prevalence of allergic diseases has been steadily increased, with about 30-40% of the world population being affected by one or more allergic conditions. Effective treatments on allergic diseases are still lacking, as most of the traditional therapies aim at clinical improvement without targeting the factors that primarily promote the allergic inflammation. Infiltration, delayed apoptosis and accumulation of eosinophils at the local inflammatory sites are the hallmarks of allergic inflammation and eosinophils are considered as the principal effector cells in allergic inflammation. Anti-eosinophils therapeutics inhibit eosinophil activity and eosinophil-derived products are thus potential strategies in treating allergic inflammation effectively. Elucidation of the detailed mechanisms of eosinophil-mediated inflammation is therefore necessary for the development of anti-eosinophils therapeutics. / In Chapter 3, we established an in vitro skin inflammation model by co-culturing eosinophils with dermal fibroblasts in order to study the pathophysiology of eosinophils’ involvement in the pathogenesis of atopic dermatitis (AD). We revealed that co-culture of eosinophils and fibroblasts significantly induced release of pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with pruritogenic cytokine IL-31 and endogenous alarmin IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was upregulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated ERK, JNK, p38 MAPK, NF-κB and PI3KAkt pathways. 1,25-dihydroxy vitamin D3 exerted in vitro suppressive effect on the release of IL-6, CCL2 and CXCL8 release from IL-31 or IL-33 activated co-culture of eosinophils and fibroblasts. / In Chapter 4, we revealed the mechanisms underlying bacterial-related pattern recognition receptor members NOD1 and NOD2 activation in eosinophils and bronchial epithelial cells BEAS-2B from the co-culture system and animal model experiment. NOD1 ligand iE-DAP and NOD2 ligand MDP could activate ERK and NF-κB pathways in eosinophils, while MDP stimulation could activate ERK in both eosinophils and BEAS-2B cells. When the eosinophils and BEAS-2B cells were co-cultured together, both iE-DAP and MDP could induce ERK and NF-κB activation in the two cells, and the activation was enhanced when compared with that in cells cultured alone. Besides, we also investigated the transcription factors activation in eosinophils and BEAS-2B cells by iE-DAP and MDP. iE-DAP and MDP could also activate a panel of transcription factors in eosinophils and BEAS-2B cells, including NF-κB, STAT-3 and NFAT etc, the nuclear transcription factors commonly involved in allergic inflammation and related to the pathogenesis of asthma. Apart from in vitro study, we also established an in vivo allergic pulmonary inflammation mice model to study the physiological effects of iE-DAP and MDP. Both iE-DAP and MDP could activate the Th2 related IgE production and induce eosinophil chemokine CCL5. / The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms. The intracellular signaling mechanisms regulating NOD1 and NOD2 activation in eosinophils and bronchial epithelial cells and animal experiment were also revealed and give implications of the role of bacterial infections in the exacerbation of asthma. Such information further enhances our understandings on both the intercellular and intracellular mechanisms of eosinophil-mediated inflammation, and gives implications for new targets for the development of anti-inflammatory drugs with higher specificity, potency, and less side effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Leung, Ming Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 136-153). / Abstracts also in Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / 摘要 --- p.VI / PUBLICATIONS --- p.IX / ABBREVIATIONS --- p.XI / TABLE OF CONTENTS --- p.XIV / Chapter Chapter 1 --- : General Introduction / Chapter 1.1 --- Allergy --- p.1 / Chapter 1.1.1 --- Definition and characteristics of allergy --- p.1 / Chapter 1.1.2 --- Allergic diseases and their prevalence --- p.2 / Chapter 1.1.3 --- Allergic inflammation --- p.2 / Chapter 1.2 --- Biology of eosinophils --- p.8 / Chapter 1.2.1 --- Development of eosinophils --- p.8 / Chapter 1.2.2 --- Cellular characteristics of eosinophils --- p.9 / Chapter 1.2.3 --- Eosinophils surface and intracellular markers --- p.10 / Chapter 1.2.4 --- Eosinophil-derived mediators --- p.13 / Chapter 1.2.5 --- Accumulation and activation of eosinophils at inflammatory sites --- p.16 / Chapter 1.3 --- Adhesion molecules in allergic inflammation --- p.19 / Chapter 1.3.1 --- Selectins --- p.19 / Chapter 1.3.2 --- Integrins --- p.20 / Chapter 1.3.3 --- Immunoglobulin gene super family --- p.21 / Chapter 1.4 --- Cytokines in allergic inflammation --- p.23 / Chapter 1.4.1 --- Pro-inflammatory cytokines --- p.23 / Chapter 1.4.2 --- Anti-inflammatory cytokines --- p.25 / Chapter 1.5 --- Chemokines in allergic inflammation --- p.27 / Chapter 1.6 --- Signal transduction in allergic inflammation --- p.29 / Chapter 1.6.1 --- Intracellular signaling mechanisms --- p.29 / Chapter 1.6.2 --- Transcription factors activation --- p.33 / Chapter 1.7 --- Perspective treatments --- p.36 / Chapter 1.7.1 --- Inhibition of pro-inflammatory cytokines --- p.36 / Chapter 1.7.2 --- Inhibition of chemokines --- p.37 / Chapter 1.7.3 --- Small interfering (si)RNA against transcription factor --- p.37 / Chapter 1.7.4 --- Signaling pathway inhibitors --- p.38 / Chapter 1.8 --- Aim of study --- p.39 / Chapter Chapter 2 --- : Materials and Methods / Chapter 2.1 --- Materials --- p.42 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Purification of human eosinophils --- p.60 / Chapter 2.2.2 --- Cell culture --- p.61 / Chapter 2.2.3 --- Cell surface and intracellular immunofluorescence staining --- p.63 / Chapter 2.2.4 --- Western blot --- p.64 / Chapter 2.2.5 --- Allergic asthmatic mice model --- p.64 / Chapter 2.2.6 --- Statistical analysis --- p.65 / Chapter Chapter 3 --- : Activation of Eosinophils Interacting with Dermal Fibroblasts by Pruritogenic Cytokine IL-31 and Alarmin IL-33: Implications in Atopic Dermatitis / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.1.1 --- Atopic dermatitis --- p.66 / Chapter 3.1.2 --- Eosinophils in AD --- p.68 / Chapter 3.1.3 --- IL-31 --- p.68 / Chapter 3.1.4 --- IL-33 --- p.70 / Chapter 3.1.5 --- Vitamin D --- p.71 / Chapter 3.1.6 --- Hypothesis and aim of study --- p.73 / Chapter 3.2 --- Results --- p.74 / Chapter 3.2.1 --- Surface expression of receptors for IL-31 and IL-33 on human eosinophils and dermal fibroblasts --- p.74 / Chapter 3.2.2 --- Cytokine and chemokine release upon the interaction of eosinophils and dermal fibroblasts activated by IL-31 and IL-33 --- p.77 / Chapter 3.2.3 --- Source of the release of cytokines and chemokines in the co-culture system upon IL-31 and IL-33 stimulation --- p.80 / Chapter 3.2.4 --- Effect of transwell inserts on cytokine and chemokine release in IL-31 and IL-33-treated co-culture --- p.83 / Chapter 3.2.5 --- Effect of IL-31 and IL-33 on adhesion molecule expression on eosinophils and dermal fibroblast in co-culture system --- p.86 / Chapter 3.2.6 --- Intracellular signaling pathways involved in the interaction of eosinophils and dermal fibroblasts under IL-31 and IL-33 stimulation --- p.88 / Chapter 3.2.7 --- Surface expression of vitamin D receptor on human eosinophils and dermal fibroblasts --- p.96 / Chapter 3.2.8 --- Suppressive effects of 1,25-dihydroxy vitamin D₃ (calcitriol) on cytokine and chemokine release from IL-31 and IL-33-treated co-culture --- p.97 / Chapter 3.3 --- Discussion --- p.100 / Chapter Chapter 4 --- :Intracellular Signal Transduction Mechanisms of NLR Activation in Human Eosinophils and Bronchial Epithelial Cells: Implication for Bacterial Infection in Allergic Asthma / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Allergic asthma --- p.107 / Chapter 4.1.2 --- Eosinophils in allergic asthma --- p.108 / Chapter 4.1.3 --- NOD-like receptors --- p.109 / Chapter 4.1.4 --- Hypothesis and aim of study --- p.110 / Chapter 4.2 --- Results --- p.112 / Chapter 4.2.1 --- Intracellular signaling pathways involved in the interaction of eosinophils and BEAS-2B cells under iE-DAP and MDP stimulation --- p.112 / Chapter 4.2.2 --- Transcription factors activation in eosinophils and BEAS-2B cells under iE-DAP and MDP stimulation --- p.119 / Chapter 4.2.3 --- In vivo activating effect of NOD1,2 ligands on the IgE and chemokine concentration in serum and BALF in allergic asthmatic mice --- p.122 / Chapter 4.3 --- Discussion --- p.125 / Chapter Chapter 5 --- : Concluding Remarks and Future Studies / Chapter 5.1 --- Concluding remarks --- p.130 / Chapter 5.2 --- Future studies --- p.131 / REFERENCES --- p.136

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Hypereosinophilic (HES) syndrome is an umbrella term encompassing several disease subsets that affects humans and veterinary species, ultimately resulting in >1,500 eosinophils/uL circulating in the blood documented over six-months. This eventually culminates in end-organ infiltration and increased patient morbidity and mortality. In mice where the gene Map3k14 encoding NF -kB inducing kinase (NIK) is knocked out, a HES-like syndrome develops that is dependent on Th2 cells and cytokines. NIK is the upstream regulator of the noncanonical NF-kB pathway and is involved in lymphoid organ development, B cell lymphopoiesis, and myelopoiesis. In addition to regulating the noncanonical NF-kB pathway, NIK is also involved in regulation of kB dimers of the canonical NF-kB pathway and can function independent of NF-kB signaling by regulating lipid and glucose metabolism, mitochondrial, and RIP1 binding to influence cell survival and death. Despite previous studies performed in the Nik-/- model, the mechanisms underlying eosinophil development, plasticity, and fitness in conjunction with the bone marrow and splenic microenvironments have not been fully elucidated. In the present work, we reviewed current data exploring the influence of the noncanonical NF-kB pathway and NIK specifically on the development of acute myeloid leukemias (AMLs) and Myelodysplastic Syndrome (MDS) with a focus on how these mechanisms might induce subvariants of HES. We next examined the effect of NIK loss on eosinophilopoiesis within hematopoietic tissues in vivo and in various cell culture environments in vitro via cytology, histology, flow cytometry, FACS, positive cell selection, MTT assay, BrDU assay, and protein microarray analysis. Overall, our findings suggest that NIK influences eosinophil maturation, proliferation, metabolism, survival, and potentially plasticity in vivo and in vitro under different environmental conditions and Th2 cytokine influence. NIK loss was also associated with altered free and bound TNFR1 levels on day 13 in vitro. TNFR1 acts upstream of RIP1 and suggests that these differences may be due to NF-kB independent functions of NIK. Overall, these results provide further insight into the potential mechanisms underlying eosinophilopoiesis in the Nik-/- murine model. This information may prove useful in discovering new treatment options underlying subvariants of HES in both human and veterinary patients. / Doctor of Philosophy / A less well-known albeit important white blood cell (WBC) is the eosinophil. It is essential for combating parasitic infections but is also involved in allergic responses. Hypereosinophilic Syndrome (HES) is an umbrella term encompassing a variety of diseases that affects human and veterinary patients. It results in an overproduction of eosinophils not associated with parasitic infections or allergic responses. Although several variants of the disease exist, diagnosing subsets of HES poses a diagnostic challenge and can impact patient care and prognosis long term. Mice carrying a genetic deletion (Map3k14) encoding a kinase known as NF-kB inducing kinase (NIK) develop HES-like syndrome; these mice are hereafter referred to as Nik-/- mice. HES-like syndrome in Nik-/- mice develops secondary to NIK loss in Th2 lymphocytes (another type of WBC). NIK is an upstream regulator of the noncanonical NF-kB pathway that influences WBC, lymph node, and spleen development. NIK also regulates canonical NF-kB molecules and can function independent of NF-kB signaling by impacting fat and glucose metabolism, binding to mitochondria, and interacting with a kinase known as RIP1 to regulate cell death or survival. Despite previous work studying HES-like syndrome in Nik-/- mice, the specifics of the bone marrow and eosinophil development in response to Th2 cells have not been fully characterized. In the present work, we reviewed data exploring the noncanonical NF-kB pathway and NIK specifically in the development of WBC cancers and how this might manifest as HES. We then studied eosinophil development in the bone marrow, spleen and in culture for both Nik-/- and wild-type mice by assessing cell and tissue morphology, cell surface marker expression, response to Th2 signaling molecules, as well as cell maturation, death, metabolism, proliferation, and cytokine production. Our findings suggest that NIK is essential for eosinophil growth and survival. We also noted differences in a molecule known as TNFR1 in Nik-/- cultures on day 13. This molecule acts upstream of RIP1, suggesting an NF-kB independent function of NIK in regulating eosinophil maturation in response to Th2 molecules. This information may prove useful in discovering new treatments for HES in human and veterinary patients.

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