Microbiology and Immunology Department Theses and Dissertations
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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.Item The Role of IL-9 in Inflammatory Diseases: Allergic Asthma, Lung Cancer, and Urinary Tract Infections(2023-06) Pajulas, Abigail Lacanlale; Kaplan, Mark H.; Cook-Mills, Joan; Dent, Alexander; Zhou, BaohuaAmong the cytokines regulating immunity, interleukin 9 (IL-9) has gained considerable attention for its role in inflammation, immune tolerance, and tumor immunity. IL-9 has a broad array of functions and acts on multiple cell types to regulate immune responses. IL-9 receptor is expressed on both non-hematopoietic cells and hematopoietic cells in the innate and adaptive immune system. IL-9 demonstrates a remarkable degree of tissue-specific functionality that varies by tissue site and the context of the inflammatory milieu. In this dissertation, we investigate the biological activities of IL-9 and identify distinct IL-9-responsive cell type in the immune pathogenesis of disease models including allergic airway disease, lung cancer, and urinary tract infection. When examining airway hyperreactivity, we found IL-9-dependent mast cell function was critical. Using adoptive transfer models and newly generated mice with an inactivation of the Il9 gene restricted to T cells generated by CD4-cre/LoxP-mediated targeting, we demonstrate that T cell secreted IL-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration during allergic airway inflammation. In IL-9-mediated pro-tumor responses, interstitial macrophages, but not mast cells, respond to T cell IL-9 to enhance B16 metastatic tumor growth. In the context of urinary tract infection, IL-9 contributes to protection against E. coli bladder infection potentially by enhancing CCL20 production in epithelial cells to recruit macrophages and neutrophils. Altogether, IL-9 can exert cell type-specific effects that identify its roles in immunity and disease. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial, and where it has the potential to complicate clinical outcomes.Item Identifying Immunological Signatures in Blood Predictive of Host Response to Plasmodium Falciparum Vaccines and Infections Using Computational Methods(2023-05) Senkpeil, Leetah Celine; Tran, Tuan M.; Sullivan, William J.; Yu, Andy Q.; Zhang, JieMalaria infects more than 240 million people every year, causing more than 640,000 deaths in 2021 alone. The complex interactions between the Plasmodium parasites that cause malaria and host immune system have made it difficult to identify specific mechanisms of vaccine-induced and naturally acquired immunity. After more than half a century of research into potential immunization methods, reliable immune correlates of malaria protection still have yet to be identified, and questions underlying the reduced protective efficacy of malaria vaccines in field studies of endemic populations relative to non-endemic populations still remain. In this thesis, I use computational methods to identify biological determinants of whole-parasite vaccine-induced immunity and immune correlates of protection from clinical malaria. Our systems analysis of a PfSPZ Vaccine clinical trial revealed that innate signatures were predictive of increased antibody response but also a decrease in the cytotoxic response required for sterilizing immunity. Conversely, these myeloid signatures predicted protection against parasitemia for subjects receiving a saline placebo, suggesting a role for myeloid-lineage cells in clearing pre-erythrocytic parasite stages. Based on these findings, I created a structural equation model to examine the interactions between cellular, humoral, and transcriptomic responses and the effects these have on protection outcome. This revealed a direct positive effect of CD11+ monocyte-derived cells on parasitemia outcome post-vaccination that was mediated by the presence of P. falciparum-specific antibodies at pre-vaccination baseline. Additionally, this model illustrates an indirect role of CD14+ monocyte activation in restricting immune priming by the PfSPZ Vaccine. Together, this data supports our hypothesis that innate immune activation and antigen presentation are uncoupled from cytotoxic cell-dependent immunity from the PfSPZ Vaccine and that this effect may be antibody-dependent.Item The Detection and Analysis of Pathogen-Reactive Immunoglobulins in the Urine of Men With Nongonococcal Urethritis(2023-05) Ryan, John D.; Nelson, David E.; Jordan, Stephen J.; Kaplan, Mark H.; Absalon, SabrinaInflammation of the urethra—urethritis—is commonly diagnosed in men and women who have sexually transmitted infections (STI). Characteristic signs and symptoms of urethritis include urethral discharge and burning pain during urination (dysuria). However, these findings are non-specific and can be elicited by STI for which optimal treatment approaches differ. We wanted to investigate if immunoglobulins (antibodies) in the urine of men with acute urethritis could determine the etiologies of these cases. Previously, we conducted an observational case-control study of biological males to compare the urethral microbiota of participants with unambiguous, laboratory-confirmed urethritis (cases) and participants without urethral inflammation (controls). This revealed that nearly 2 in 5 men with nongonococcal urethritis tested negative for all common STI. We identified atypical urethral pathogens in approximately 1/3 of these STI-negative individuals using shotgun metagenomic sequencing. However, we did not detect microorganisms suspected to be urethral pathogens in the remaining 2/3 of STI-negative participants. We hypothesized that these men with “pathogen-negative” urethritis had persisting inflammation from a recent STI that already cleared spontaneously by the time of testing. We observed that urine IgA antibodies against Chlamydia trachomatis (Ctr) infectious particles were significantly more prevalent among men with pathogen-negative urethritis compared to controls. In contrast, we found that the prevalence of urine anti-Ctr IgA was similar between controls and urethritis cases with atypical infections. However, our efforts to detect antibodies against another common STI, Mycoplasma genitalium (Mgen), were complicated by low abundance in urine and the unexpected prevalence of Mgen-reactive antibodies among controls. Collectively, our results suggest that signs and symptoms of urethritis can continue after the causative STI(s) have been eliminated. Furthermore, male urine represents a practical, non-invasive source of pathogen-reactive antibodies that could be evaluated using point-of-care diagnostic tests to elucidate urethritis etiologies. Importantly, our results also suggest that sexual partners of men with pathogen-negative, nongonococcal urethritis are an unrecognized chlamydia reservoir.Item Characterization of Allergen-Specific Immunoglubulin E Development in a Food Allergy Model and Its Regulation by T Follicular Helper and T Follicular Regulatory Cells(2023-05) Chen, Qiang; Dent, Alexander; Kaplan, Mark; Brutkiewicz, Randy; Zhou, BaohuaFood allergy is a highly prevalent and serious disease regulated by immunoglobin E (IgE) antibodies specific for food allergens.The development of IgE is regulated by T follicular helper cells (TFH) and T follicular regulatorycells (TFR) in the germinal center (GC). We aimed to understandthe regulation of IgEin the GC by TFH and TFR cellsusinga mouse food allergy model. We found that the dosage and timingof allergen delivery into thegut is criticalfor allergen-specific IgE development, in part because the timing of allergen delivery affected the expression of regulatory factors by TFH and TFR cells. We studied FGL2, an inhibitory factor, and found that down-regulation of FGL2 in TFH cells was important for the allergic IgEresponse. Apart from inhibitory factors, TFH cell-derived IL-4 is required for IgE responses. We unexpectedlyfound that TFR cells in food allergy produce comparable amountsof IL-4 to TFH cellsand IL-4–expressing TFR cells promoteallergen-specific IgEin food allergy. The IgE response is highly sensitive to IL-4 levels, suggesting the need for extra IL-4 from TFR cells. However,TFR cells have distinct functionsdepending on the immune environment, since TFR cells repress IgEinanairway inflammation model. We found that TFR cells in airway inflammation have a different gene expression profile from TFR cells in food allergy, whichmay explain their distinct functions. Lastly, previous studies showed that high-affinity IgE driving anaphylactic reactions is produced via IgG1-switchedintermediate B cells. We challenged this paradigm by showing that high-affinity IgE develops in the absence ofIgG1-switchedB cellsin our food allergy model.Overall, our studies reveal that IgE is regulated by novel pathways in food allergy. We hope to exploit these new pathways to develop new specific therapies for food allergy.Item Host Factors That Influence Coxsackievirus B3 Replication and Pathogenensis(2023-04) Dhalech, Adeeba Haroon; Robinson, Christopher M.; Hurley, Thomas D.; Katzenellenbogen, Rachel A.; Richer, Martin J.; Spinola, Stanley M.Enteric viruses are infectious human pathogens that initiate infection in the gastrointestinal tract. They follow a fecal-oral route of transmission and are spread by contamination of food, water, or contact between individuals. Furthermore, enteric viruses also cause significant morbidity, mortality, and economic burdens yearly. Coxsackievirus (CV) is commonly isolated among enteric viruses and is an etiological agent of hand, foot, and mouth disease, hemorrhagic conjunctivitis, and myocarditis. The virus predominantly infects infants and young children and accounts for 11% of the fatality rate in neonates. Despite CV’s impact on human health, there are no treatments or vaccines for CV infections. Using a mouse model to study a key CV, Coxsackievirus B3 (CVB3), our laboratory has found two critical factors that impact CVB3 replication and pathogenesis. First, we have demonstrated that intestinal bacteria enhance intestinal CVB3 replication. We found that certain specific bacteria (Salmonella enterica) and its cell wall components, like lipopolysaccharides (LPS), enhanced CVB3 stability and infectivity in vitro. Additionally, we found that particular constituents of LPS are required for stability to occur. These data suggest that specific bacteria may be integral in maintaining CVB3 infectivity in the intestine. Besides virus-microbiome interaction, CVB3 is also impacted by sex hormones. Using castrated mice models, we observed a sex bias to CVB3 infection, with male mice succumbing to CVB3-induced disease at an increased rate compared to female mice. Our data suggest that testosterone, a predominant male sex hormone, enhanced CVB3 intestinal replication and viral dissemination to organs in male and female mice, but lethality only in male mice. Moreover, testosterone also affected the immune response by reducing the activation of the CD8+ T cells. CD8+ T cells are required to clear the viral infection and are integral in vaccine development. In contrast, we found an enhanced CD8+ T cell response in female mice to CVB3 infection, suggesting a sex-dependent T cell response that may underlie the sex bias in disease. Overall, these data represent an essential advancement in the CV field and will help develop future therapeutics and aid in vaccine design to limit CV infections.