Microbiology and Immunology Department Theses and Dissertations
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Item Immunological and Social Determinants of Asthma: From Cytokine Signaling to Air Pollution Disparities(2024-10) Cheung, Cherry Cheuk Lam; Kaplan, Mark H.; Cook, Nathan; Cook-Mills, Joan; Yang, KaiAsthma is a chronic respiratory disease characterized by airway inflammation and hyperresponsiveness, impacting 262 million individuals globally. This heterogeneous condition results from a complex interplay of genetic, environmental, and social factors. The pathophysiology involves dysregulated immune responses, particularly through cytokine signaling, and is exacerbated by environmental pollutants and social determinants of health (SDOH). This thesis aims to (1) elucidate novel cytokine signaling pathways involved in asthma, specifically a potential type II IL-9 receptor complex, and (2) evaluate the impact of California's Assembly Bill 617 (AB 617) on reducing air pollution and asthma disparities in disadvantaged communities. The research employs molecular biology techniques, including flow cytometry, proximity ligation assay, and RNA sequencing, to investigate IL-9 signaling in airway epithelial cells. It also involves a policy analysis of AB 617's initial effectiveness in reducing fine particulate matter (PM2.5) levels and asthmaassociated emergency room (ER) visits through environmental monitoring and hospital records. The study identifies a novel type II IL-9 receptor complex composed of IL-9Rα and IL-13Rα1, suggesting new therapeutic targets for asthma management. Policy analysis reveals limited initial success of AB 617 in reducing air pollution and asthma incidence, highlighting the need for enhanced regulatory measures and community engagement. Understanding the molecular mechanisms of IL-9 signaling and addressing environmental and social determinants are crucial for comprehensive asthma management. Integrating scientific research with policy interventions can improve health outcomes and reduce disparities in asthma prevalence and severity.Item Inflammation by Toxoplasma gondii Infection Induces Prostatic Hyperplasia and Accompanying Urinary Dysfunction(2024-08) Stanczak, Emily F.; Arrizabalaga, Gustavo; Bauer, Margaret; Jerde, Travis; Nakshatri, HarikrishnaBenign prostatic hyperplasia is the non-cancerous enlargement of the prostate. This syndrome develops as men age and affects 50% of men by the age of 50 and 80% of men by the age of 80. BPH is associated with a pattern of symptoms and pathology in patients that can be painful, problematic, and lower the quality of life. BPH is a multifactorial disease which may develop due to lifestyle choices, genetics, metabolic disorder, and potentially infection of the organ. Interestingly, previous work from our research group showed that the common parasite T. gondii can infect and establish a chronic infection in the prostate of mice resulting in histological hyperplasia and glandular nodule formation, reminiscent of that observed in men with BPH. This leads to the hypothesis that T. gondii contributes to BPH in humans. In this work, we investigated whether there is a correlation between T. gondii infection and BPH using a cohort of age matched BPH diagnosed and non-BPH diagnosed control donors. My data show that men diagnosed with BPH are significantly more likely to have evidence of T. gondii infection than undiagnosed controls. Additionally, men with antibodies against T. gondii and BPH had significantly more severe pathology in several categories including and most notably in epithelial and glandular nodules seen only in a steroid hormone model of the disease. Moreover, I have determined that mice infected with parasites demonstrated abnormal urination pattern behavior indicating lower urinary tract dysfunction. Based on these results, we conclude that T. gondii can contribute to the development and severity of BPH and BPH pathology in humans. In addition, T. gondii can potentially be used as a model for BPH and BPH-like symptoms and pathology in mice and other model organisms.Item IL-33 Mediated Th2 Effector Functions are Suppressed in Tregs by Bcl6 and Regulated by Sex(2024-08) Lee, Kyu Been; Dent, Alexander; Richer, Martin; Robinson, Christopher; Yang, KaiAllergic airway inflammation (asthma) is a prevalent and uncurable disease worldwide, affecting many individuals’ quality of life. Although asthma does not form from a singular cause, one primary mediator comes from the exposure to environmental allergens and the improper activation of the T cell subset: T helper 2 (Th2) cells. Th2 cells produce pro-inflammatory cytokines and promote the activation and recruitment of various pro-inflammatory cells into the lung, causing greater damage and inflammatory responses in the organ. Th2 cell’s activation is regulated by another T cell subset, Regulatory T (Treg) cells, by expressing anti-inflammatory cytokines and downregulating the inflammatory response. On the contrary, the release of interleukin-33 (IL-33) from damaged lung epithelial cells transitions Tregs into Th2-like Tregs (ST2+ Tregs) which release both pro-and anti-inflammatory cytokines and cannot suppress the inflammatory disease. However, transcriptional repressor protein B cell lymphoma 6 (Bcl6) provides Tregs a stable follicular phenotype and suppresses the ST2+ Treg transition. Preliminary data revealed that Bcl6 repressive function is dependent on mouse sex, in which Tregs of male mice are more resistant to the ST2+ Treg phenotype than those of female mice. However, the removal of Bcl6 also removed the sex-dependent suppression against the ST2+ Treg transition. The project therefore sought to further confirm and answer whether Bcl6 suppressed the ST2+ Treg phenotype in a sex-dependent manner, ultimately leading to a sex-biased asthma prevalence and severity. We utilized quantitative polymerase chain reaction (qPCR) and next-generation sequencing techniques to uncover which genes Bcl6 regulates, how IL-33 affects chromatin accessibility/gene expression, and what relation sex hormones have with Bcl6 in the expression of Th2 cytokines from Tregs. Currently, we have discovered that estrogen-like chemicals in common cell culturing media may be acting on the estrogen receptor of Tregs and causing differential gene expressions based on media conditions. We also determined that Bcl6 is acting independently of mouse sex to suppress Th2 genes in Tregs, contrary to preliminary findings. Overall, we have obtained insight on the role of the estrogen receptor and Bcl6’s mechanism of suppression in relation to sex.Item Combined Inhibition of SREBP and m-TORC1 Signaling Synergistically Inhibits the Proliferation of B Cell Lymphoma(2024-06) Zhu, Zhenhan; Luo, Wei; Capitano, Meagan L; Yuan, XueSterol regulatory element-binding protein (SREBP) signaling plays a crucial role in maintaining sterol homeostasis during B cell activation and the proliferation of germinal center B cells. It is unclear whether this pathway can be targeted to effectively treat B cell lymphoma. We discovered that inhibiting SREBP signaling or its downstream target HMG-CoA reductase (HMGCR) using Fatostatin or Simvastatin effectively restrains the proliferation of B cell lymphoma cells. However, B cell lymphoma cells activate the mTORC1-pS6 pathway in response to statin treatment, suggesting a possible mechanism to counteract statin-induced cell cycle arrest. Combining low dose statin treatment with the mTORC1 inhibitor rapamycin demonstrates a synergistic effect in inhibiting B cell lymphoma proliferation, cell cycle progression and lipid raft generation. These findings emphasize the potential of a combined therapy approach targeting both SREBP and mTORC1 as a novel treatment strategy for B cell lymphoma.Item Innate Immune Roles of Alpha-Defensin 1-3 in Neutralizing Uropathogenic Escherichia Coli(2024-05) Canas Kouaifati, Jorge Jose; Hains, David S.; Kaplan, Mark H.; Nelson, David E.; Dent, Alexander L.; Eadon, Michael T.Urinary tract infections (UTIs) are characterized by microbial colonization of the bladder, ureters or/and kidneys. Host-pathogen interactions compound to drive UTI susceptibility. The host innate immune system is composed of physio-chemical barriers and broad-spectrum potent effector responses that prevent pathogenic host colonization and bystander damage. Uropathogenic Escherichia coli (UPEC) isolates account for the majority of reported UTI cases. Virulence factors that are expressed by CFT073/UPEC heighten pathogenesis by permitting preferential invasion towards kidneys (pyelonephritis). During the acute invasion of the urinary tract tissues, pathogen molecular patterns and host-damage signals represent triggers that lead to host-defense responses. The release of antimicrobial peptides (AMPs) prevents microbial attachment to the epithelium. α-Defensin 1-3 are host-defense AMPs that work by eliciting microbial membrane interactions and inducing immunomodulatory effects. In humans, the DEFA1A3 locus encodes for three α-Defensin peptides (1-3). Chromosome 8 can harbor copy number polymorphism of the DEFA1A3 locus that range from 3-16 copies per diploid genome. Low copy numbers of DEFA1A3 have been associated with increased UTI risk in children (< 5 copies). On the other hand, patients with > 8 per copies per diploid genome have improved antibiotic therapy outcomes. In this thesis, we establish a pipeline to characterize α-Defensin 1-3 mechanisms of action, dissect contributors of expression at the cellular level, and protective DEFA1A3 gene-dose-dependent effects in the pyelonephritis setting. Using a manipulable mouse model expressing transgenic human DEFA1A3 gene copies, we explored dynamics of inducible α-Defensin 1-3 expression in the UPEC-infected kidney. Additionally, in vitro combinations of α-Defensin 1-3 and other host-defense AMPs work in concert to drive diverse cooperative action effects against UPEC. Methods and findings from my research in this thesis improve the current biomedical approaches to study AMP functions. Collectively, my results expand the understanding of DEFA1A3 polymorphic locus as a stratification UTI biomarker and exploration for the pre-clinical evaluation of kidney α-Defensin 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.Item TL1A Priming Induces a Multi-Cytokine Th9 Cell Phenotype That Promotes Robust Allergic Inflammation in Murine Models of Asthma(2024-03) Niese, Michelle Liu; Kaplan, Mark H.; Cook-Mills, Joan M.; Brutkiewicz, Randy R.; Tepper, Robert S.The TNF superfamily member TL1A is a costimulatory molecule that signals through its receptor DR3 on T lymphocytes. The Th9 subset of T lymphocytes secretes the pleiotropic cytokine IL-9 which has functions in allergic airway disease, helminth infections, and tumor immunity. TL1A increases IL-9 production from Th9 cells. However, its role in regulating other functions of Th9 cells is unknown. Here we demonstrate that TL1A increases expression of IL-9 and IL-13 as well as the frequency of IL-9 and IL-13 co-expressing cells in murine Th9 cell cultures, inducing a robust multi-cytokine phenotype. Mechanistically, this is linked to histone modifications allowing for increased accessibility at the Il9 and Il13 loci. We further show that TL1A alters the transcription factor network underlying expression of IL-9 and IL-13 in Th9 cells and increases binding of these transcription factors to Il9 and Il13 loci. IL-9 and IL- 13 expression have been well studied in the context of allergic airway disease (AAD) and both have been shown to exacerbate AAD. We demonstrate that TL1A-priming enhances pathogenicity of Th9 cells in murine models of AAD and that this is largely mediated through the increased expression of IL-9 and IL-13. We lastly show in both chronic and memory recall models of AAD that blockade of TL1A signaling decreases the multicytokine Th9 cell population and attenuates the allergic phenotype. Taken together, these data demonstrate that TL1A promotes development of multi-cytokine Th9 cells that drive allergic airway diseases and that targeting TL1A could be an effective approach for modifying disease.Item A Human Pluripotent Stem Cell-Derived In Vitro Model of the Blood-Brain Barrier in Cerebral Malaria(2024-01) Gopinadhan, Adnan; John, Chandy C.; Nelson, David E.; Bauer, Margaret E.; Absalon, Sabrina; Tran, Tuan M.Blood-brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but the physiological relevance remains uncertain. I aimed to develop a novel in vitro model of the BBB in CM using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) that mimic a near in vivo barrier phenotype. hiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 hours. Barrier integrity was measured using transendothelial electrical resistance (TEER). Localization and expression of tight junction (TJ) proteins, occludin and zona occludin-1 (ZO-1), and endothelial marker, intercellular adhesion molecule 1 (ICAM-1) was determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were also measured. hiPSC-BMECs showed improved barrier integrity and localization of TJ proteins compared to immortalized BMECs. After 6-hours of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and disruption of TJ protein localization compared to co-culture with uninfected RBCs (RBCs), but no change in TJ protein expression was observed by WB in the Pf-iRBCs co-cultures. Expression of ICAM-1 on hiPSC-BMECs co-cultured with Pf-iRBCs was higher compared to co-culture with RBCs. In addition, there was an increase in expression of the angiogenin, platelet factor 4, and phospho-heat shock protein-27 in the Pf-iRBCs co-cultures compared to co-cultures with RBCs. These findings demonstrate the physiological relevance of our hiPSC-BMEC-based in vitro model of the BBB, as determined by elevated TEER and appropriate TJ protein localization. In co-culture with Pf-iRBCs, breakdown in the barrier integrity, changes in TJ protein localization, increase in expression of ICAM-1, and of markers of angiogenesis and cellular stress, all point towards a more relevant in vitro model, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.Item Elucidating the Characteristics and Functionality of the Mouse Mucosal-Associated Mucosal Invariant T (MAIT) Cell Receptor(2023-08) Shrinivasan, Rashmi; Brutkiewicz, Randy R.; Dent, Alexander L.; Tran, Ngoc TungMucosal-associated invariant T cells (MAIT) are a subset of invariant, innate-like T-cells that are abundant in the gut lamina propria, kidney, lungs, and peripheral blood. MAIT cells are stimulated by the recognition of microbial vitamin B-derived metabolites by the MHC class I-like molecule, MR1. Recent studies have implicated MAIT cells in several autoimmune diseases, various cancers, and CNS disorders, making it essential to design animal models that replicate the human disease state. The relatively small population of MAIT cells in mice makes it difficult to isolate and characterize them. The MAIT cell receptor (TCR) is comprised of a Vα7.2-Jα33 rearrangement in humans and TRAV1-TRAJ33 in mice. This project aimed to create a tool to study mouse MAIT cells in detail by generating lentiviral plasmid constructs expressing cDNAs encoding the MAIT cell TCR α and β chains that will be ectopically expressed in TCR-deficient mouse T cells. A bulk TCR analysis of the mouse MR1-restricted MAIT hybridomas 6C2 and 8D12 was performed to confirm variable and joining regions in the TCR α and β chains. This analysis confirmed the proper MAIT cell TCR usage in the MAIT cell hybridomas. As both MAIT cell hybridomas can be stimulated by MR1-presented antigens, we obtained synthetic cDNAs that were generated for the TRAV1-TRAJ33 α chain and TRBV8.2 (TRBV13-2) β chain. These were subcloned into GFP- and mCherry-expressing plasmids and packaged into lentiviruses that will be used for transduction of TCR-deficient mouse T cells. Flow cytometry and ELISAs will ultimately be performed to confirm the functional expression of the MAIT cell TCR. These tools will greatly facilitate the investigation of MAIT cell function in vitro and the ultimate generation of retrogenic mice for the tracking of MAIT cells in vivo.Item Developing In Vitro and In Vivo Models for Lyme Neuroborreliosis (LNB)(2023-08) Alanazi, Fuad Fahad; Yang, X. Frank; Bauer, Margaret E.; Yu, Andy Q.; Relich, Ryan F.; Nass, Richard M.Lyme neuroborreliosis (LNB) is a neurologic disorder caused by infection with Borrelia burgdorferi, resulting in inflammation in the central and peripheral nervous systems. LNB remains poorly understood due to the lack of a suitable experimental model. The non-human primate model for LNB presents significant impediments, such as high costs, specialized training, ethical considerations, and low infection frequency. Finding alternative models is imperative to advance LNB research. This study aims to develop alternative in vitro and in vivo models for LNB. First, we developed an in vitro transwell assay to identify the factors required for the blood-brain barrier (BBB) transmigration of B. burgdorferi. Second, we established a middle-aged mouse model for studying neuroinflammation associated with LNB. Last, we further characterized a Caenorhabditis elegans (C. elegans) model to study B. burgdorferi-associated neuron damage. With these models, we discovered that the Rrp2-RpoN-RpoS pathway in B. burgdorferi is essential for B. burgdorferi to cross the BBB and that the outer surface protein C (OspC) controlled by this pathway plays a vital role in crossing the BBB. We found that B. burgdorferi is detectable in the brains of middle-aged mice but not in younger mice and triggers host immune response, resulting in elevated levels of cytokines such as TNF-alpha, IFN-γ, and IL-9 and reduction in microglia in the infected mice. Lastly, we demonstrated that C. elegans can feed on B. burgdorferi, which may result in neurodegeneration. This provides a powerful tool for screening pathogen and host factors involved in neuroborreliosis. Overall, the in vitro and in vivo models developed in this study will significantly advance LNB research, which may lead to the development of new treatments and improved patient outcomes.Item Distinct Cell Survival and Metabolic Programming Determines Germinal Center Tfh Survival of HIV-1 Infection(2023-07) Syed, Fahim; Yu, Qigui; Dent, Alexander; Yang, Kai; Wan, JunHIV-1 is the causative agent of AIDS in people living with HIV-1 (PLHIV). HIV-1 predominantly targets and kills immune cells that are needed for defense against infections and illnesses. Although therapy can control the spread of HIV-1 in PLHIV and decrease the amount of virus present in the body, some subsets of infected immune cells are able to survive HIV-1 and escape treatment. Any pause in therapy leads to a return to high levels of viral loads due to these surviving infected cells. These subsets of infected immune cells escaping treatment represent a major obstacle to the eradication of HIV-1. One such subset of immune cells, the Germinal Center T follicular helper (GC Tfh) cells, can both survive infection and expand in PLHIV. Using human tonsil tissues, the major site of GC Tfh cells, our lab was able to find two critical factors that influence the GC Tfh cells’ ability to survive and thrive while infected by HIV-1. First, we found that GC Tfh cells have a distinct metabolic profile compared to other types of CD4 T cells found in human tonsils. This was characterized by a preference towards non-glycolytic metabolism even when infected with HIV-1. We found that inhibiting non-glycolytic metabolism resulted in a significant decrease in HIV-1 infected GC Tfh cells. Second, we found that GC Tfh cells sharply upregulate proteins responsible for stopping controlled cell death. We found one of these proteins, BIRC5, was integral to GC Tfh survival of HIV-1 infection. Inhibition of BIRC5 led to overall decreases in surviving infected cells, as well as significant decreases in infected GC Tfh survival. In contrast, inhibition of BIRC5 had no effect on uninfected cells. Our results signify an important advancement in the study of HIV-1 reservoir and will help in developing novel therapeutics to eradicate rather than suppress HIV-1 in PLHIV.