Master of Science (MS), Wright State University, 2019, Microbiology and Immunology

Mouse models of eosinophil-associated diseases have been used to study the mechanisms of disease pathogenesis. In this study, mouse-derived bone marrow cells were used in long-term (6 and 9 months) cell cultures of differentiated eosinophils and macrophages. IL-5 was used to differentiate the stem cells to eosinophils and GM-CSF was used to propagate macrophages from the bone marrow stem cells. The maximum time period for observing the eosinophil cultures was 252 days which is censurably longer than the 18 days culture period observed by others. The results were assessed by describing the microscopic cell morphology by Wright staining, modified Giemsa staining and protein expression by immunofluorescent staining. The GMCSF-stimulated bone marrow cultures produced classically appearing monocyte/macrophages throughout the study and were used to compare the development of the eosinophils over the long-term period of observation. Differentiation of the BM cells was carried out using with growth factors (SCF, FLT3L) and cytokines (IL-5, GM-CSF) over the 252 days period. The most suitable culture plate for long-term of cell growth were thee 60 mm petri dishes. At 252 days, the eosinophils exhibited as bi-lobed nuclear shapes, comparable with human eosinophils. Dendrite-like ramifications were observed on the surfaces of these eosinophils. Long term culture of eosinophils in the presence of IL-5 contributed to formation of eosinophil extracellular traps (EETs) areas. Within the EETs the cells surface of eosinophils developed holes; the nucleus of such cells lost the “ring-like” or “lobular” morphology and appeared in de-condensed fashion. Within the EETs, the plasma membranes of eosinophils developed the protrusions containing cytoplasmic granules. The EETs and protrusions had not been observed previously in the mouse eosinophil models in vitro but were described in human eosinophils. Consequently, the long-term culture of mouse bone-marrow derived eosinophil (open full item for complete abstract)

Committee: Nancy J. Bigley Ph.D. (Advisor); Dawn P. Wooley Ph.D. (Committee Member); Marjorie Markopoulos Ph.D. (Committee Member) Subjects: Immunology; Microbiology

Master of Science (MS), Wright State University, 2008, Microbiology and Immunology

Eosinophils express membrane CD4 protein and can bind HIV-1 glycoprotein (gp) 120. Therefore, eosinophils could serve as host cells for HIV-1 infection in vivo, especially in the late phase of the infection. In culture, HIV-1 infects eosinophil precursors and primary eosinophils. Additionally HIV-1 proviral DNA sequences have been detected in the eosinophils of some HIV-1 positive patients. Since elevated levels of eosinophils occur during HIV-1 infection and parasitic infections, it implies that co-infection of parasites and HIV-1 could cause a much greater increase in the level of potential HIV-1 susceptible eosinophils. Therefore HIV-1 infection of eosinophils could partly explain the rapid spread of HIV-1 and the exacerbation of the disease especially in developing countries where HIV-1 and parasitic infections run concurrently. This study is a critical analysis of the current knowledge on HIV-1 infection of eosinophils focused on highlighting evidence that HIV-1 can productively infect mature human eosinophils in HIV/AIDS patients.

Committee: Dawn Wooley Ph.D (Advisor); Barbara Hull Ph.D (Committee Member); Nancy Bigley Ph.D (Committee Member) Subjects: Biology; Microbiology

Master of Science (MS), Wright State University, 2006, Microbiology and Immunology

Over the past two decades, much research has been done in the field of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS). However, many of the aspects of pathogenesis of HIV infection and its persistence in the body, despite treatment, remain a mystery. Recent evidence suggests that HIV positive patients develop eosinophilia, especially in the later stages of infection and AIDS. Eosinophils are CD4 positive cells that have the potential to be infected by HIV. Studies have shown that an eosinophilic cell line, AML14.3D10, can be productively infected with a T-cell tropic, CXCR4-using (X4) strain of HIV-1. In this study, primary human eosinophils from four healthy volunteers were shown to be susceptible to infection with a T-cell tropic, CXCR4-using (X4) strain of HIV-1, HTLV-IIIB. This data was supported by results from quantitative polymerase chain reaction (Q-PCR), which detected high HIV copy numbers in infected eosinophil samples. In two out of four donors, these copy numbers were comparable to those obtained from infected AML14.3D10, used as a positive control. In all four donors, the number of viral copies detected in infected eosinophils were significantly (p<0.05) higher than those detected in infected peripheral blood mononuclear cells (PBMCs). Donor variability was observed in viral loads detected. No correlation was observed between the viral load and the production of p24. However, infected eosinophils showed higher amounts of p24 production, as compared to infected PBMCs with or without IL-2, in three out of four donors suggesting productive infection. Therefore, it is concluded that primary human eosinophils are susceptible to productive infection by X4 HIV-1.

Committee: Dawn Wooley (Advisor) Subjects: Biology, Microbiology

Master of Sciences, Case Western Reserve University, 2022, Physiology and Biophysics

Gasdermin D (GSDMD) plays a role in pyroptosis, a form of lytic cell death, and IL-1β release during inflammasome signaling which has been characterized previously in macrophages and other mononuclear myeloid leukocytes. This involves the cleavage of GSDMD and IL-1β by Caspase-1. The cleaved GSDMD then localizes to the plasma membrane (PM) where it can oligomerize, eventually forming a pore that allows the release of the cleaved IL-1β. Notably, granulocytic leukocytes, such as neutrophils and eosinophils, are also an important source of IL-1β and play a role in a number of innate immune responses and inflammatory diseases. However, there are only a few studies on the roles of GSDM-family proteins in these types of cells. Our eosinophil studies show, that these cells express the previously described inflammasome components, such as ASC and NLRP3, as well as pro-GSDMD, a precursor of the pore forming N-GSDMD. Furthermore we have demonstrated that when stimulated with bacterial LPS, primary mouse eosinophils can express pro-IL-1β, as previously reported in human and murine neutrophils. We have also established that that during NLRP3 inflammasome activation, primary murine eosinophils rapidly process and release IL-1β without the need of GSDM protein processing, but dependent on Gasdermin D expression. Notably, these cells release IL-1β without GSDMD cleavage by Caspase-1 or accumulation of PM Gasdermin D pores, while dependent on GSDMD expression for IL-1β release. This critical role for GSDMD in IL-1β release is operative during the initial stages of NLRP3 inflammasome activation in eosinophils. During later phases of NLRP3 inflammasome activation, eosinophils release IL-1β via a GSDMD-independent mechanism.

Committee: George Dubyak (Advisor) Subjects: Biology; Cellular Biology; Immunology

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

Respiratory syncytial virus (RSV) is a highly contagious and universally prevalent virus that presents as a respiratory infection, which can range from mild to fatal in severity. An infant's first RSV infection is typically the most severe, but the elderly are also at risk for severe infection. Although life-threatening complications in healthy adults are less common, the burden of illness due to RSV in this population remains substantial. Clinical trials for RSV vaccine candidates began in the 1960's, but to date, there remains no vaccine or therapeutic available. A prophylactic has been developed for RSV, although its use is limited to high-risk pre-term infants during the RSV season. This prophylactic was developed using small animal models of RSV infection. For the work presented herein, RSV pathogenesis and immune response is investigated using the cotton rat. The cotton rat is a less common, but more permissive and predictive model of RSV infection. This document is separated into four chapters. The first chapter is a review of the literature, which provides an overview of RSV, highlights what is known, and indicates areas where there are opportunities to clarify our understanding of the virus, as well as a brief discussion of the strengths and weaknesses of animal models of RSV infection. Chapter two addresses one of the uncertainties in the RSV field, the cellular receptor for the virus. Earlier studies had demonstrated heparan sulfate to be a receptor for RSV, but these studies were performed on immortalized cell lines, which express different proteins on their surface than airway epithelial cells, the target cell for RSV infection. Based on recent work using primary human airway epithelium cells, CX3CR1 was identified as a potential RSV receptor. Adding to that work, chapter two summarizes data that support CX3CR1 as a receptor in vivo using the cotton rat model. Chapter three examines eosinophils in the cotton rat and during RSV infection. This work was (open full item for complete abstract)

Committee: Stefan Niewiesk DVM, PhD (Advisor); Michael Oglesbee DVM, PhD (Committee Member); Mark Peeples PhD (Committee Member); Ian Davis DVM, PhD (Committee Member) Subjects: Immunology; Microbiology; Molecular Biology; Virology

PhD, University of Cincinnati, 2014, Medicine: Immunology

Exaggerated synthesis of type II cytokines subverts host immunity in multiple intracellular infections. This scenario is effectively demonstrated in CCR2-/- animals that have been exposed to the intracellular fungal pathogen Histoplasma capsulatum. The infected mutant mice mount an amplified and sustained IL-4 response that is associated with impaired fungal clearance. We investigated the cellular source of IL-4 and the reason for dysregulated immunity in H. capsulatum-challenged CCR2-/- mice. Our findings reveal eosinophils were the prominent contributors of non-protective IL-4 and depleting this cell population ameliorated fungal infection in CCR2-/- animals. This immune defect was a result of CCL7 engaging chemokine receptors preferentially expressed on eosinophil surface. An unexpected finding in CCR2-/- mice was that elevated IL-4 prompted IL-33 generation in pulmonary macrophages. Dissection of mechanisms that promulgated the latter cytokine revealed IL-4 and H. capsulatum synergistically elicited an IL-33 response in macrophages via STAT6/IRF-4 and Dectin-1 pathways respectively. Furthermore, this IL-4-driven IL-33 response compromised antifungal defenses by driving an alternatively activated phenotype in phagocytes. The molecular interaction between eosinophils and H. capsulatum has not been explored. We discovered that eosinophils phagocytosed H. capsulatum yeasts and this biological process was dependent on the pattern recognition receptor CR3. Internalization of fungi induced a robust IL-4 response that was conserved in both murine and human eosinophils. These observations reveal an unappreciated attribute of eosinophil biology that could potentially trigger a collapse in antifungal immunity in the host. In summary, the investigation in this work provides novel insights into how type II immune responses are shaped and their detrimental effects in the setting of an intracellular fungal infection.

Committee: George Deepe M.D. (Committee Chair); Ian Paul Lewkowich Ph.D. (Committee Member); David Hildeman Ph.D. (Committee Member); Simon Hogan Ph.D. (Committee Member); Judith Rhodes Ph.D. (Committee Member) Subjects: Immunology

PhD, University of Cincinnati, 2012, Medicine: Epidemiology (Environmental Health)

Allergic rhinitis (AR) is an IgE-mediated disease of the upper respiratory tract characterized by symptoms of rhinorrhea, nasal obstruction, itching, and sneezing in response to allergen exposure. Allergic rhinitis is common in childhood, affecting 42% of six year old children, and is associated with an increased risk of asthma. Thus, determining early childhood risk factors may be important for not only AR, but also asthma. The study hypothesis is that size of the specific aeroallergen wheal area in ages one through three is significantly associated with AR at age four. Two specific aims were completed to test this hypothesis. The first specific aim of this study was to determine the size of the skin prick test (SPT) wheal area from 15 individual aeroallergens during early childhood (age one through three) and its association with AR at age four. The second aim was to determine if any individual age-specific aeroallergen wheal area association persisted in a multivariate logistic regression model after controlling for covariates and multiple testing. The analysis was extended further to address a more specific phenotype, allergic eosinophilic rhinitis (AER), defined as AR with nasal eosinophilia. We examined if the individual aeroallergen wheal areas were associated with AER in univariate and multivariate analysis. This study was part of the Cincinnati Childhood Asthma and Air Pollution Study where allergen skin prick tests (SPT) were performed at ages one through four at annual clinical visits. SPT wheal circumferences were traced, recorded on transpore tape, and digitized which allowed accurate estimates of wheal areas by planimetry. Timothy and Alternaria wheal areas at age two were significantly associated to AR at age four (aOR 1.06; 95% CI, 1.02 - 1.11; p = 0.01) and (aOR 1.07; 95% CI 1.0-1.15; p = 0.04), respectively. Fescue, dog, and Penicillium wheal areas at age three were significantly associated with AR at age four (aOR 1.08; 95% CI, 1.02 - 1.15; p = 0. (open full item for complete abstract)

Committee: Grace Lemasters PhD (Committee Chair); David Bernstein MD (Committee Member); Linda Levin PhD (Committee Member); Michell Lierl MD (Committee Member) Subjects: Epidemiology