Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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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 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 Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode(2022) Bhanja, Anshuman; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune response is critical in fighting off invading pathogens and potentially harmful foreign substances. B cells detect antigens through the B cell receptor (BCR). The binding of cognate antigen to the BCR induces a signaling response, a critical initiation and regulatory step for B cell activation and differentiation. The actin cytoskeleton has been shown to play essential roles in BCR signaling. When encountering membrane-associated antigens, actin amplifies signaling by driving B cell spreading and BCR clustering, while promoting signal attenuation by causing B cell contraction. This signal attenuation is essential for curtailing the activation of autoreactive B cells. However, the mechanism by which the actin cytoskeleton switches BCR signaling from amplification to attenuation was unknown. My thesis research examined the mechanisms by which actin reorganization transitions B cells from spreading to contracting and B cell contraction switches BCR signaling from amplification to attenuation, using mouse splenic B cells, a functionalized planar lipid bilayer system, and total internal reflection fluorescence microscopy. Our results show that branched actin polymerized by Arp2/3 is required for B cell transition from spreading to contraction after driving B cell spreading. Ubiquitously expressed Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP), but not the haematopoietically specific WASP, activates the branched actin polymerization and generates inner actin foci from lamellipodial actin networks, by sustaining their lifetime and centripetal movement. N-WASP-dependent inner actin foci are necessary for recruiting non-muscle myosin II, creating an actomyosin ring-like structure at the periphery of the membrane contact region to drive B cell contraction. B cell contraction primarily increases the BCR molecular density in individual BCR-antigen clusters, measured by the peak fluorescence intensity. Inhibition of B cell contraction by Arp2/3 inhibitor and B cell-specific N-WASP knockout (cNKO) reduced the increasing rates of BCR molecular density. Increased molecular density caused by B cell contraction leads to decreases in the levels of phosphorylated BCR, the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. However, the levels of total Syk and SHIP-1 have a different relationship with BCR density in individual clusters: Syk does not decrease until a high threshold of BCR density, which can be achieved only by contracting B cells, but SHIP-1 consistently reduces with the increase in BCR molecular density. Inhibiting B cell contraction by cNKO reduces the molecular density of BCR clusters but does not affect the relationship of the Syk and SHIP-1 levels with BCR molecular density in clusters. Taken together, our results suggest that the actin cytoskeleton reorganizes from the lamellipodial branched actin networks to centripetally moving actin foci, enabling actomyosin ring-like structure formation, through N-WASP-activated Arp2/3. Actomyosin-mediated B cell contraction attenuates BCR signaling by increasing receptor molecular density in individual BCR clusters, which causes the dissociation of both stimulatory and inhibitory signaling molecules. My thesis research results reveal a novel negative regulatory mechanism for BCR signaling, an essential checkpoint for generating pathogen-specific and suppressing self-reactive antibody responses.Item MUCIN-MEDIATED AND INTERFERON-DRIVEN DEFENSE MECHANISMS AGAINST INFLUENZA VIRUS INFECTION IN HUMAN AIRWAY EPITHELIUM(2022) Iverson, Ethan; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human airway epithelium represents the primary site of infection for many respiratory viruses, including influenza A virus (IAV). To safeguard this tissue and maintain the functionality of the lung, humans possess a two-layer, extracellular, mucus barrier composed predominantly of individual proteins termed mucins. Additionally, underlying epithelial cells produce interferons upon virus detection that promote the establishment of a local antiviral state through autocrine and paracrine signaling. However, despite these protective measures, IAV continues to cause significant annual morbidity and mortality across the globe. Therefore, we sought to further investigate how specific mucin molecules interact with IAV, and how interferon drives intrinsic antiviral defense in the context of a human airway epithelial (HAE) culture system. By utilizing fluorescently-labeled influenza virus particles we further elucidate the adhesive interactions between mucus and influenza virus while also detailing, for the first time, real-time IAV diffusivity within patient-derived mucus samples. These results reveal that the polymeric structure of mucus greatly influences the mobility of IAV within human secreted mucus. Additionally, we investigate the interaction between influenza virus and tethered mucin 1 (MUC1), finding that MUC1 expression is enhanced by virus-driven inflammation and interferon signaling. Moreover, by establishing a genetically-tractable airway epithelial model, we detail the protective role MUC1 plays in preventing the initial establishment and spread of influenza virus in HAE. Specifically, we find that the loss of MUC1 significantly enhances IAV uptake and spread. Finally, we observe that the directionality of IFN exposure at airway epithelial surfaces impacts the magnitude of protection against IAV and SARS-CoV-2. We then detail the cellular composition of our HAE culture system and define a shared IFN response profile across all HAE component cell types as well as cell type-specific interferon stimulated genes. Together our work provides novel insight into the innate and intrinsic anti-viral properties of the human airway epithelium.Item THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE(2020) Hamidzadeh, Kajal; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are innate immune cells that participate in host defense to invading pathogens. They are powerful producers of cytokines and inflammatory mediators due to their efficient recognition of pathogen associated molecular patterns (PAMPs) via toll like receptors (TLRs). We and others have shown that the activation response to PAMPs is transient. In the present work, we demonstrate that stimulated macrophages produce adenosine and prostaglandin E2, which function as regulators of the macrophage activation response. Macrophages also upregulate receptors for these regulators to terminate inflammation and promote wound healing. We performed high throughput RNA sequencing to characterize the transcriptomes of human monocyte-derived macrophages in response to stimulation with LPS + Adenosine or LPS + PGE2. These cells exhibited a decrease in inflammatory transcripts and an increase in transcripts associated with cell growth and repair when compared to cells stimulated in the absence of these regulators. Macrophages can be generated from precursor cells in response to two different growth factors; M-CSF (macrophage colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor). M-CSF is expressed constitutively in a variety of tissues, while GM-CSF is expressed primarily in the lung, but can be induced in other tissues under inflammatory conditions. We demonstrate that human macrophages differentiated in M-CSF readily adopt an anti-inflammatory, growth promoting phenotype in response to LPS + Adenosine or LPS + PGE2, while macrophages differentiated in GM-CSF do not. This observation suggests that M-CSF derived human macrophages may be better able to alter their activation state in response to surrounding signals in order to maintain homeostasis. GM-CSF derived macrophages, in contrast, may undergo a more prominent activation response that is associated with inflammation and tissue destruction due to their inability to efficiently respond to resolving molecules.Item HIGH-RESOLUTION ANALYSIS OF HIV ENVELOPE-SPECIFIC ANTIBODY RESPONSES TO ACCELERATE RATIONAL IMMUNOGEN DESIGN(2020) Lei, Lin; Li, Yuxing; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recent isolation of HIV broadly neutralizing antibodies (bNAbs) from HIV infected individuals has reinvigorated efforts to develop B cell-based vaccines. As the sole viral target for bNAbs, HIV envelope glycoprotein (Env) has been engineered as soluble trimers to recapitulate bNAbs responses via vaccination. However, Env-based immunogens thus far primarily induce vaccine-matched neutralizing antibody (nAb) responses. This thesis aims to understand the mechanisms restricting the neutralization breadth and to provide strategies for iterative improvements. First, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for guinea pigs, a small animal model desirable in the field for initial immunogenicity analysis. This method allowed us to dissect the antibody responses at the clonal level with high accuracy and efficiency. Secondly, we have delineated the specificity of autologous neutralization elicited by the current generation HIV trimer mimicry, BG505 SOSIP.664. Our results reveal a prominent epitope in the C3/V4 region of the Env targeted by one nAb/B cell clonal lineage. We demonstrate that the nAb responses to this neutralization determinant are prevalent in trimer-vaccinated guinea pigs, rabbits, and non-human primates. In addition, this defined nAb response shares a high degree of similarity with the early nAb response in an HIV- infected pediatric patient, who later developed a bNAb response. This study offers insights into re-designing Env immunogens in the highly immunogenic region to broaden nAb responses. Lastly, we have engineered novel immunogens based on the Env sequence of a virus strain isolated from bNAb VRC01 donor, which can engage the VRC01 germline precursor in vitro. Sequential prime-boost immunizations in a VRC01-germline immunoglobulin (Ig) encoding genes knock-in mouse model with the designed immunogens induced focused VRC01-like serum antibody responses and clustered VRC01-class somatic mutations in the knock-in VRC01-germline Ig genes. In addition, the mAbs recovered from the immunized mice neutralize selected viruses containing the N276 glycan, a critical roadblock impeding the affinity maturation of VRC01-class bNAbs. Our findings demonstrate that, in the transgenic mouse model, our immunogens effectively activate bNAb precursor B cells and guide their affinity maturations required for bNAb function, which has important implications for HIV vaccine development.Item MUCOSAL DELIVERY OF INFLUENZA VACCINE ANTIGENS(2018) Park Ochsner, Susan Soo; Zhu, Xiaoping; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Most pathogens infect humans and animals at mucosal surfaces, yet few mucosal vaccines are available to provide protection at these sites. Though influenza virus initiates its infection in the respiratory mucosal epithelium, currently approved influenza vaccines are administered by parenteral routes, which do not offer effective respiratory immunity. A successful mucosal influenza vaccine should induce both local and systemic immunity, however, the respiratory epithelium is an imposing barrier that prevents vaccine antigens to effectively traverse the airway. The neonatal Fc receptor (FcRn) mediates transport of IgG across the epithelial cell monolayer lining mucosal surfaces. To exploit this antibody transfer pathway for antigen delivery, I produced a soluble fusion protein that fused the monomeric Fc portion of IgG to an influenza hemagglutinin (HA) antigen harboring the T4 fibritin trimerization domain. Intranasal innoculation of the HA-Fc protein along with CpG adjuvant induced high levels of durable mucosal and systemic adaptive immune responses and, importantly, generation of lung-resident memory T cells. FcRn-dependent antigen delivery was corroborated when substantial protection characterized by significantly increased survival and reduced pulmonary pathology was observed in the HA-Fc-immunized wild-type (wt) mice. In contrast, control groups of wt and FcRn-deficient mice immunized with HA-Fc, a mutant version of HA-Fc that lacks FcRn binding capacity, HA alone, or PBS, experienced substantial morbidity, mortality, and lung damage. As the influenza nucleoprotein (NP) is highly conserved among strains, it is an attractive vaccine target. Thus I produced soluble NP-Fc fusion proteins as potential influenza vaccines. The preliminary study demonstrated that intranasal immunization of NP-Fc with CpG resulted in FcRn-mediated delivery of NP-Fc protein across the respiratory barrier and the induction of high levels of antibody titer compared to groups of control mice. Immunization with NP-Fc may be further explored for developing a universal mucosal influenza vaccine. Taken together, for the first time, my results prove that FcRn can effectively deliver an influenza antigen across the respiratory epithelial barrier, providing substantial protection against lethal respiratory infection. This study further suggests FcRn-mediated mucosal vaccination could be used to deliver a universal influenza vaccine antigen or protective antigens from other common respiratory pathogens.Item CHARACTERIZING THE INHIBITION OF INNATE IMMUNE SIGNALING BY MYCOBACTERIUM TUBERCULOSIS(2017) Ahlbrand, Sarah; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Numerous cytokines are induced as a consequence of infection by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that is responsible for millions of deaths each year. These cytokines play varied roles in eliciting and regulating the innate and adaptive immune responses against Mtb and often determine disease outcome. The pro-inflammatory cytokine interleukin 1β (IL-1β) is required for Mtb maintenance and clearance, as mice lacking the ability to produce IL-1β succumb very quickly to infection compared to infected wildtype mice. In contrast, interferon β (IFNβ) is largely thought to be detrimental to the host during Mtb infection based on collective studies in patients and mouse models. Both IL-1β and IFNβ are induced during Mtb infection, but here we demonstrate that Mtb has also evolved mechanisms to reduce their production and/or signaling. Previously, we’ve shown that Mtb can inhibit IL-1β production induced by the Absent in Melanoma 2 (AIM2) inflammasome. These findings were expanded upon by investigating the mechanisms by which Mtb inhibits AIM2 activation. A gain-of-function screen was also utilized in the non-pathogenic mycobacterial species Msmeg to identify Mtb genomic regions contributing to this phenomenon. In addition, we demonstrate that Mtb inhibits IFNβ signaling by inhibiting IFNβ-induced JAK1 and Tyk2 phosphorylation, leading to changes in the host type I interferon transcriptional profile. These studies provide insight into two previously undescribed mechanisms Mtb utilizes to manipulate the host immune response.Item Genome Wide Association Studies of Phagocytosis and the Cellular Immune Response in Drosophila melanogaster.(2016) Nazario-Toole, Ashley Elizabeth; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Phagocytosis of bacteria by specialized blood cells, known as hemocytes, is a vital component of Drosophila cellular immunity. To identify novel genes that mediate the cellular response to bacteria, we conducted three separate genetic screens using the Drosophila Genetic Reference Panel (DGRP). Adult DGRP lines were tested for the ability of their hemocytes to phagocytose the Gram-positive bacteria Staphylococcus aureus or the Gram-negative bacteria Escherichia coli. The DGRP lines were also screened for the ability of their hemocytes to clear S. aureus infection through the process of phagosome maturation. Genome-wide association analyses were performed to identify potentially relevant single nucleotide polymorphisms (SNPs) associated with the cellular immune phenotypes. The S. aureus phagosome maturation screen identified SNPs near or in 528 candidate genes, many of which have no known role in immunity. Three genes, dpr10, fred, and CG42673, were identified whose loss-of-function in blood cells significantly impaired the innate immune response to S. aureus. The DGRP S. aureus screens identified variants in the gene, Ataxin 2 Binding Protein-1 (A2bp1) as important for the cellular immune response to S. aureus. A2bp1 belongs to the highly conserved Fox-1 family of RNA-binding proteins. Genetic studies revealed that A2bp1 transcript levels must be tightly controlled for hemocytes to successfully phagocytose S. aureus. The transcriptome of infected and uninfected hemocytes from wild type and A2bp1 mutant flies was analyzed and it was found that A2bp1 negatively regulates the expression of the Immunoglobulin-superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of A2bp1 and Dscam4 in hemocytes rescues the fly’s immune response to S. aureus indicating that Dscam4 negatively regulates S. aureus phagocytosis. Overall, we present an examination of the cellular immune response to bacteria with the aim of identifying and characterizing roles for novel mediators of innate immunity in Drosophila. By screening panel of lines in which all genetic variants are known, we successfully identified a large set of candidate genes that could provide a basis for future studies of Drosophila cellular immunity. Finally, we describe a novel, immune-specific role for the highly conserved Fox-1 family member, A2bp1.