Theses and Dissertations from UMD
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Item INITIAL LOCAL CYTOKINE RESPONSES AGAINST NEISSERIA GONORRHOEAE INFECTIONS IN THE HUMAN CERVIX(2024) Dai, Yiwei; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-specific pathogen that causes gonorrhea, a common sexually transmitted infection. In women, GC initiate infection by colonizing the cervix. Although GC colonization can cause cervicitis, most female infections are asymptomatic. Asymptomatic colonization of the cervix increases the risk of transmission and progression to severe complications, including pelvic inflammatory disease and infertility. Despite its clinical significance, the mechanisms underlying GC asymptomatic colonization remain unclear. Using a human cervical tissue explant model, which can mimic GC infection in vivo, my Ph.D. research examined the early local cytokine responses to GC cervical colonization, a determining factor for asymptomatic and symptomatic clinical outcomes. Luminex and spatial transcriptomic analyses found that cervical tissue explants constitutively secrete and express a broad spectrum of cytokines, with particularly high levels of the IL-1 receptor antagonist IL-1RA, the anti-inflammatory cytokines IL-10, and the multi-functional cytokine IL-6. During the first 24-h inoculation, GC strain expressing an opacity-associated protein binding to the host receptor CEACAMs (MS11OpaCEA) increased the secretion and transcript levels of both pro-inflammatory, like IL-1α/β, and the anti-inflammatory cytokine IL-10, as well as multi-functional cytokines, like IL-6 and CFS3, but MS11 lacking Opa (MS11∆Opa) induced much less. Notable, the cervix secreted IL-1RA at 100-fold higher levels than IL-1α/β. Cervical secreting levels of soluble IL-6 receptors, required for activating IL-6 inflammatory functions, were 10,000-fold less than IL-6. These results support an anti-inflammatory-dominated cytokine environment of the human cervix, and GC further push it in the anti-inflammatory direction. Using isogenic GC strains and inhibitors, the mechanism underlying GC cytokine induction and the impact of GC-induced cytokines on GC infection were examined. My research found that GC-induced inflammatory cytokine production involved NF-κB activation in both epithelial and subepithelial cells. GC-induced IL-10 production depended on the activation of CEACAM-downstream signaling molecule SHP1/2. Reductions in inflammatory cytokines, TNF-α and IL-1β, by an NF-κB inhibitor did not significantly affect GC colonization, epithelial cell-cell junctions, or epithelial shedding. In contrast, neutralizing IL-10 or blocking its receptor reduced GC colonization and increased ectocervical epithelial shedding and disassembly of epithelial cell-cell junctions. Thesis results suggest that IL-10 plays critical roles in strengthening the cervical epithelium and suppressing the epithelial cell-cell junction disrupting function of inflammatory cytokines, and that GC further elevate the local IL-10 level to prevent bacteria from shedding off with epithelial cells, enhancing colonization.Immunofluorescence and spatial transcriptomic approaches were utilized to identify the types of cervical cells contributing to the local cytokine response to GC infection. Cervical epithelial cells and macrophages are two of the major contributors. IL-1RA protein and mRNA were primarily detected at the ectocervical epithelium. IL-6 protein and mRNA were also detected in ectocervical epithelial cells. MS11OpaCEA colonization increased IL-1RA transcript levels, while MS11ΔOpa switched ectocervical epithelial cells from IL-1RA- to IL-8/IL-6-expressing. GC inoculation did not alter the transcriptomic program of CD68+ macrophages adjacent to the ectocervical epithelium, maintaining the tissue-repair signature. However, GC changed the transcriptomic profiles of macrophages at the explant tissue side, exposed to media and inoculated GC, leading to increased expression of either inflammatory M1- or anti-inflammatory M2 signature genes. These results suggest that the human cervix utilizes high levels of epithelial-secreted IL-RA, low levels of soluble IL-6 receptor release, and tissue-repairing macrophages at the subepithelium to control inflammation induced by colonizing GC when the epithelium prevents GC from entering the tissue. Overall, my research results suggest that GC exploit the local cytokine response of the human cervix, dominant by anti-inflammatory IL-1RA, IL-10, and IL-6, to facilitate colonization and desensitize immune detection, promoting asymptomatic colonization.Item INHIBITION OF TYPE ONE INTERFERON SIGNALING THROUGH CROSSTALK WITH TOLL-LIKE RECEPTOR SIGNALING(2024) Shuster, Michael; Briken, Volker V; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interferons (IFNs) are a class of cytokines that play a prominent role in host immunity. Type I IFN is broadly associated with antiviral immunity and susceptibility to bacterial pathogens, but others have shown that type I IFN can be beneficial in some bacterial infections. Additionally, some bacterial infections such as Mycobacterium tuberculosis and Legionella pneumophila can inhibit type I IFN signaling. Questions remain such as how these bacteria inhibit type I IFN signaling as well as if other bacterial pathogens, such as Salmonella enterica, can also inhibit type I IFN signaling. Additionally, type III IFN is a relatively new class of IFN, providing antiviral protection similar to and at times redundant to type I IFN. There are some important non-redundant differences from type I IFN though, such as type III IFN’s broader activity at epithelial surfaces (like those in the lungs) and its reduced proinflammatory effects. The role of type III IFN in bacterial infections as well if bacteria can inhibit this signaling pathway remains poorly understood.Here, we examined if Salmonella enterica can inhibit type I IFN signaling, the specificities of the previously observed inhibition with Mtb infection, and how these bacterial infections are inhibiting this signaling. We demonstrate that Salmonella Typhimurium infection inhibits type I IFN signaling through crosstalk with TLR4 signaling. We establish that TLR4 signaling results in reduced surface level type I IFN receptor, which dampens cellular responsiveness to type I IFN. We show that Mtb does not inhibit type III IFN signaling and that it inhibits type I IFN signaling independently of virulence, specifically EsxA and ESX-5. Additionally, this inhibition of type I IFN signaling seems specific to mouse cells as Mtb-infected human macrophages and dendritic cells did not have inhibited type I IFN signaling. We observed that other TLR signaling pathways result in specifically inhibited type I IFN signaling. Synthesizing a model from our results, there appears to be a mouse-specific crosstalk pathway between TLR signaling and type I IFN signaling, resulting in dampened responsiveness to type I IFN through downregulation of cell surface type I IFN receptor.Item HOST-PATHOGEN INTERACTION DURING CRYPTOCOCCUS NEOFORMANS CNS INFECTION(2024) Chen, Yanli; Shi, Meiqing MS; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen widely distributed in the environment globally. C. neoformans infection initiates from the lung through inhaling the spores. While most healthy individuals can clear the fungus or contain the fungus in the granuloma, immunosuppressed patients and a small group of healthy populations fail in controlling the cryptococcal fungal pulmonary infection. In those cases, C. neoformans transmigrates from the lung to the central nervous system (CNS) and causes fatal meningoencephalitis, which accounts for 112,000 deaths each year worldwide. However, we have a very limited understanding of the transmigration of C. neoformans from the bloodstream to the brain in vivo, and the mechanism involved in the clearance of the organism in the brain remains poorly understood. In this study, we first report a novel approach to quantitatively analyze the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalized by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by the serotype A strain (H99 strain) at a higher rate compared to the serotype D strain (52D strain). Moreover, we found that clearance of C. neoformans in the brain correlates with accumulation and pro-inflammatory M1 polarization of Ly6Chi mononuclear phagocytes and that these phagocytes play a critical role in the clearance of C. neoformans in the brain. Notably, the accumulation of Ly6Chi mononuclear phagocytes coincides with enhanced secretions of TNF and IFN-γ in the brain. TNF receptor (TNFR) signaling, but not IFN-γ receptor (IFN-γR) signaling, mediates the recruitment of Ly6Chi mononuclear phagocytes to the brain in a cell-intrinsic manner. By contrast, IFN-γ induces M1 polarization of Ly6Chi mononuclear phagocytes. Disruption of TNFR or IFN-γR signaling enhances cryptococcal growth in the brain. Thus, Ly6Chi mononuclear phagocytes act as effector cells for cryptococcal clearance in the brain, involving TNFR as well as IFN-γR signaling. Collectively, our study established that 1) internalization of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyze fungal migration into the brain; 2) Ly6Chi mononuclear phagocytes accumulate in the brain during brain infection with C. neoformans and function as effector cells for clearance of C. neoformans in the brain involving TNFR signaling and IFN-γ signaling.Item TLR9 Activation as Immunotherapy in a Murine Model of Metastatic Lymphangioleiomyomatosis(2024) Amosu, Oluwamayowa; Maisel, Katharina; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pulmonary Lymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age, marked by abnormal growth of smooth muscle-like cells leading to cystic lung destruction. Rapamycin, the only approved treatment for LAM, slows disease progression but ~40% of patients have partial or no response to treatment. There is an urgent need for new treatments. Research shows that LAM has hallmarks of cancer, like expression of immune checkpoint receptors, and is responsive to immune checkpoint inhibition in mouse models. This suggests that other anti-cancer strategies could be effective in treating LAM. In this thesis, we investigated toll like receptor (TLR) activation using intranasal administration of CpG, a TLR9 agonist, as LAM immunotherapy. We used a mouse model of metastatic LAM to determine survival after biweekly intranasal CpG therapy (10µg/ 5µg) with and without systemic α-PD-1, rapamycin, or α-CD317 therapy. We used ELISA to measure the cytokine profile and flow cytometry to quantify cell populations and characterize differences in the immune response between CpG-treated and untreated LAM lungs. We found that CpG treatment enhanced median survival from 32 to 60 days in murine LAM. Survival benefit of CpG treatment was inversely dose-dependent and more effective during early stages of disease. CpG-treatment was synergistic with both α-PD-1 checkpoint inhibition and rapamycin, with survival increasing from 60 days (CpG) to 71 days (CpG + α-PD-1) and 100 days (CpG + Rapamycin). Histological analysis showed that CpG treatment decreased the LAM nodule burden but inevitably caused tissue inflammation. Efficacy of CpG treatment in LAM is facilitated in part by plasmacytoid dendritic cells through decreased regulatory T cell numbers, priming of Th17 cells, and increased secretion of inflammatory and cytotoxic cytokines by CD8 T cells. Our findings suggest that adjuvant immunotherapy, like CpG, may offer new treatment strategies for LAM that are compatible with the current standard of care, rapamycin.Item Mathematical Modeling of Cellular Exhaustion to Guide Future Immunotherapy Research(2024) Simmons, Tyler; Levy, Doron; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cellular exhaustion is a dysfunction found in various adaptive immune cells. In chronic settings, like cancer, antigen persistence and prolonged stimulation initiates the development of T cell exhaustion. The exhausted T cell population is a distinct lineage consisting of progenitor exhausted CD8+ T cells and terminally exhausted CD8+ T cells and is characterized by an upregulation of inhibitory receptor frequencies and diminished effector functions. The hypofunctionality of exhausted T cells prevents proper immunity and fails to eradicate the tumor. Recent years have shown a growing interest in targeting T cell exhaustion, attempting to reinvigorate effector functions, as a form of immunotherapy. Though beneficial responses have been reported in clinical settings, patient responses are inconsistent. Complementing the current biological understanding of T cell exhaustion and to advance immunotherapeutic efforts, novel research using mathematical modeling offers valuable insight. Constructing a foundational framework of an exhausted immune response to cancer provides an alternative approach to understanding the tumor-immune system. Presented here is the construction of a mathematical model detailing the development of progenitor and terminally exhausted CD8+ T cell populations in response to a growing tumor. Parameterization and simulation of this model captures biological dynamics observed in experimental and clinical settings. Analysis and conclusions of this model suggest population size and maintenance of progenitor exhausted CD8+ T cells should be a pillar of immunotherapy efforts. Stemming from these conclusions, it was theorized that targeting exhausted CD4+ helper T cells, which, under normal non-chronic conditions, contribute heavily to CD8+ T cell responses, would be a new and effective approach for immunotherapy. To test this hypothesis, the previously constructed model of CD8+ T cell exhaustion was expanded to incorporate CD4+ helper 1 T cells as well as immunosuppressive regulatory T cells. Simulation and analysis of this expanded model further emphasize the need to maintain progenitor exhausted CD8+ T cell numbers. Additionally, model analysis also indicated that the functionality of CD4+ T cells, both regulatory and exhausted CD4+ helper 1 T cells, played a crucial role in tumor persistence. From this work, research regarding CD4+ T cell exhaustion is strongly encouraged. With a better understanding of this dysfunction, CD4+ T cells may be a potentially effective target for future immunotherapy strategies.Item DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.Item MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.Item SYSTEMS IMMUNOLOGY OF IMMUNE IMPRINTS INDUCED BY ACUTE VIRAL INFECTIONS(2023) Liu, Can; Johnson, Philip L.F.; Tsang, John S.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Upon encountering perturbations such as viral infections, the immune system initiates a cascade of molecular and cellular responses. These alterations may persist even after recovery, resulting in enhanced or diminished response to subsequent stimuli compared to the naïve state. Such persistent changes, referred to as immune imprints or long-term non-specific memory, indicate an incomplete resolution from immunological perturbations. The primary focus of this dissertation is to systemically investigate the immune imprints resulting from acute infections and how they shape the baseline immune status to future heterologous challenges.First, we employed cutting-edge single-cell multi-omics and computational approaches to assess the immune response during the COVID-19 disease course and severity correlates at an unprecedented resolution. We identified gene expression profiles – apoptosis in plasmacytoid dendritic cells and IL-15-linked increase of fatty acid (FA) metabolism in CD56dimCD16hi NK cells – as primary correlates of disease severity. This increase of FA signature with disease severity was also concomitant with an attenuated inflammation, indicating a dysfunctional or exhaustion-like state of these NK cells. While the depressed inflammation signature in severe patients was also found in different cell types near hospitalization, it increased temporally at later time points, indicating a critical late-stage juncture in the disease course. Next, we took the opportunity of the period following the first wave of COVID-19 pandemic to study immune imprints in human cohorts who had recovered from COVID-19 before widespread vaccination and reinfection occurred. We demonstrated that individuals who recovered from mild COVID-19, exhibit distinct immune signatures through single-cell transcriptomic profiling. Male recoverees also showed heightened responses to the seasonal influenza vaccine compared to healthy individuals without a history of COVID-19 and female recoverees. These sex dimorphic imprints highlight the interplay between intrinsic factors like sex and non-intrinsic factors such as prior SARS-CoV-2 infection, in shaping an individual's immune system over time. Lastly, we also investigated the immune imprints after acute viral infection using a controlled experimental mouse model of influenza infection. After examining cellular and gene expression profiles in various organs after the infection, we found persistent changes in both adaptive and innate immune components across multiple organs. Moreover, these changes affected subsequent local IL-17 inflammatory response and secondary heterologous vaccinations in anatomically distinct organs. Together, both human and mouse studies here are important pieces toward an improved understanding of long-term immune imprints after perturbations, which can be leveraged to develop more effective and personalized vaccines and disease treatments.Item Immune Modulations of a Helminth Derived Protein(2023) Mekhemadhom Rajendrakumar, Arunraj; Zhu, Xiaoping XZ; Tuo, Wenbin WT; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune responses at the gastrointestinal mucosa modulate nematode parasite infection, initially characterized by the production of epithelium-derived, robust T helper 2 (Th2) type alarmin cytokines, such as interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Subsequently, the immune responses are mediated by releasing the lymphoid cell-derived Th2 immune cytokines, such as IL-13, IL-4, IL-5, IL-9, and parasite-specific antibodies. Studies have shown that parasitic nematode worms can establish a chronic infection in the intestine, even in a robust immune response. This evidence leads us to hypothesize that the nematode evolves to evade or regulate intestinal immunity through specific modulatory mechanisms that interfere with initial intestinal immune responses, allowing the nematode to survive. We used a model nematode, Heligmosomoides polygyrus bakeri (Hpb), to identify nematode-derived proteins with regulatory effects on Th2 immune cytokines during chronic infection. Through high throughput analysis, we found that a Hpb-derived protein could precisely modulate mouse immune response. The presence of the Hpb-derived protein was essential for the parasite's survival as the vaccine conferred a sterilizing immunity. As Th2 cytokines are directly associated with the pathogenesis of several inflammatory and autoimmune diseases, we are understanding how this protein regulates the function of the Th2 cytokines in vitro and in vivo and explore whether the protein could use to treat inflammatory diseases and serve as a vaccine target to control nematode infections.Item LEVERAGING SELF-ASSEMBLY AND BIOPHYSICAL DESIGN TO BUILD NEXT-GENERATION IMMUNOTHERAPIES(2022) Froimchuk, Yevgeniy; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune system has evolved mechanisms to respond not only to specific molecular signals, but also to biophysical cues. Interestingly, research at the interface of biomaterials and immunology has also revealed that the biophysical properties and form of vaccines and immunotherapies impact immunological outcomes. For example, the intermolecular distance between antigen molecules on the surface of nanoparticles can impact formation of T cell receptor clusters that are critical during T cell activation. Despite the importance of biophysical cues in tuning the immune response, the connections between these parameters and immunological outcomes are poorly understood in the context of immunotherapy. Immunotherapies harness an individual’s immune system to battle diseases such as autoimmunity. During autoimmune disease, the immune system malfunctions and mistakenly attacks self-tissue. Immunotherapies can help tailor and guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. To overcome these challenges, experimental strategies are attempting to co-deliver self-antigens and modulatory cues to reprogram dysfunctional responses against self-antigens without hindering normal immune function. These strategies have shown exciting potential in pre-clinical models of autoimmune disease but are unproven in clinical research. Understanding how biophysical features are linked to immunological mechanisms in these settings would add a critical dimension to designing translatable, antigen-specific immunotherapies. Self-assembling materials are a class of biomaterials that spontaneously assemble in aqueous solution. Self-assembling modalities are useful technologies to study the links between biophysical parameters and immune outcomes because they offer precise control and uniformity of the biophysical properties of assembled moieties. Our lab leveraged the benefits of self-assembly to pioneer development of “carrier-free” immunotherapies composed entirely of immune signals. The therapies are composed of self-antigens modified with cationic amino acid residues and anionic, nucleic acid based modulatory cues. These signals are self-assembled into nanostructured complexes via electrostatic interactions. The research in this dissertation utilizes this platform as a tool to understand how tuning the biophysical properties of self-antigens impacts molecular interactions during self-assembly and in turn, how changes in biophysical features are linked to immunological outcomes. Surface plasmon resonance studies revealed that the binding affinity between signals can be tuned by altering overall cationic charge and charge density of self-antigen, and by anchoring the self-antigen with arginine or lysine residues. For example, the binding affinity between signals can be increased by increasing the total cationic charge on the self-antigen, and by anchoring the self-antigen with arginine residues rather than lysine residues. Computational modeling approaches generated insights into how molecular interactions between signals, such as hydrogen bonding, salt-bridges, and hydrophobic interactions, change with different design parameters. In vitro assays revealed that a lower binding affinity between self-assembled signals was associated with greater reduction of inflammatory gene expression in dendritic cells and more differentiation of self-reactive T cells towards regulatory phenotypes that are protective during autoimmunity. Taken all together, these insights help intuit how to use biophysical design to improve modularity of the self-assembly platform to incorporate a range of antigens for distinct disease targets. This granular understanding of nanomaterial-immune interactions contributes to more rational immunotherapy design.