Pathology

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4952

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The Endocytic Protein FCHO-1 is Essential in Preserving the Structural and Functional Integrity of Neurons
(2024-02-12) Jimenez, Monet Andrea; Bai, Jihong
Neurons are vital for animal physiology and behavior, demanding lifelong structural and functional integrity. This integrity is upheld by intricate endocytic pathways that sustain a multitude of critical neuronal features, such as cell polarity, circuit formation and maintenance, and rapid neurotransmission between neurons and their target cells. However, the precise convergence of these diverse pathways, comprising both unique and shared endocytic proteins, to collectively support the overall health of neurons remains elusive. In this thesis, I investigated the role of FCHO-1, a key endocytic protein, in C. elegans neurons. My studies revealed a remarkable and unexpected facet of FCHO-1, involving the preservation of neuronal structure and function. I showed that fcho-1 null mutant worms exhibited only moderate reductions in synaptic transmission strength and slightly diminished synaptic vesicle abundance — a surprising revelation. However, using electron microscope analysis, I found a distinct phenomenon: numerous abnormal “breaks” on the plasma membranes between neurons, suggestive of compromised neuronal boundaries. To delve deeper into this finding, I introduced a novel PAGEN assay, a fluorescence imaging method enabling quantification of cytoplasmic content exchange among live neurons. Using this method, I demonstrated that compromised neuronal membrane contacts in fcho-1 mutant worms allow for an elevated occurrence of abnormal cytoplasmic content exchange among neurons. Furthermore, I found that accumulation of adhesion molecule SYG-1 is increased in fcho-1 mutant neurons, comparing to wild type neurons. Collectively, these findings reveal FCHO-1’s critical and previously recognized role in maintaining neuron boundaries. This likely occurs through influencing the abundance of adhesion molecules on neuronal membranes, thereby potentially impacting the rigidity and stability of neuron-neuron contacts. In summary, my research uncovers an unexpected facet of the FCHO-1-dependent endocytic pathway — its pivotal role in preserving neuronal structural integrity.
Impact of proteasomal processing on immunopeptidome repertoires and therapeutic immune recognition.
(2023-01-21) Lahman, Miranda Claire; Chapuis, Aude G.
Acute myeloid leukemia (AML) is the most common acute leukemia in adults. AML often responds to initial chemotherapy, but a majority of patients will relapse with resistant disease leaving a critical need for more effective therapies. Hematopoietic stem cell transplantation can prevent relapse long term through donor T cell-mediated elimination of leukemic cells. However, this anti-leukemic effect is unpredictable, and transplant can have detrimental side effects, including graft versus host disease (GVHD). New therapies focus the anti-leukemic effect and bypass potential GVHD by using engineered T cells with a defined T cell receptor (TCRs) that recognize proteasome-generated peptide fragments from tumor-associated proteins. Such ‘TCR-T’ cells rely on the targeted peptide to be processed and then presented by the restricting human leukocyte antigen (HLA). Cells can express standard- and immuno-proteasome isoforms, which can generate ‘distinct’ (isoform-dependent) or ‘mutual’ (isoform-independent) peptides. We investigated a clinical scenario in which the targeted AML modulated proteasome composition to eliminate processing of the targeted ‘distinct’ peptide, leading to loss of recognition by the adoptively transferred TCR-T cells and AML progression. Instead, an alternative TCR recognizing a ‘mutual’ peptide could respond to the progressive AML. Our data point to a mechanism whereby AML can evade immune recognition through modulation of proteasome isoform expression. This further implies that TCR-T cell therapy could be enhanced if tailored to target ‘mutual’ peptides. To determine the relative proportions of HLA-restricted ‘distinct’ and ‘mutual’ peptides and to identify promising TCR-T cell targetable peptides less likely to evade recognition, we utilized an emerging peptide discovery platform, Artemis, that employs soluble HLAs to bypasses immunoprecipitation of endogenous HLAs. Artemis-identified peptides revealed that the proportions of ‘mutual’ and ‘distinct’ peptides were specific to each HLA allele, with HLA-A*02:01 presenting the fewest ‘mutual’ peptides and HLA-A*11:01 presenting the most. We recovered 37 peptides from three AML-associated proteins of interest, 19 (51%) were ‘mutual’ peptides and some peptides (11%) were previously identified. Our findings demonstrate that defining isoform-specific proteasomal processing is critical to optimal peptide selection for TCR-T cell therapy and Artemis is an effective platform to identify ‘mutual’ peptides.
An Automated System For High-Throughput Longevity and Healthspan Discovery in Caenorhabditis elegans
(2023-01-21) Blue, Benjamin; Kaeberlein, Matt
Over the last century, the study of aging biology has primarily been advanced through the development and use of animal models that share common molecular hallmarks with human aging. One major model system that has been widely used during the last several decades and through the present day is the roundworm Ceanorhabditis elegans. Its short lifespan, easy husbandry, and genetic tractability have allowed it to be easily adapted for studying the molecular biology of aging. For instance, research using C. elegans was used to discover the interplay between insulin signaling and rate of aging. However, while C. elegans research proved orders of magnitude more efficient (at the cost of a larger evolutionary gap) than using vertebrate models, such as mice or non-human primates, it’s use was still hamstrung by the necessity for manually collected lifespans. During the last decade, the lowering cost and increasing capabilities of digital microscopy and computer vision have led to researchers attempting to automate the most basic aging related experiment in C. elegans: the measurement of an individual’s lifespan relative to the population within which it resides. Some examples of this include the Automated Lifespan Machine, The WormMotel, as well as several different microfluidic platforms such as the NemaLife chip. These platforms sought to semi-automate or even fully-automate the collection and recording of lifespan measurements but often had large bottlenecks in their pipeline that precluded them from achieving the scale necessary to robustly advance the field of aging biology. This thesis will present the development and use of a novel robotics platform that, when paired with a suite of AI-powered computer vision, fully automates lifespan analysis of C. elegans.
Utility of kidney organoids for disease modeling and therapeutic development
(2022-04-19) Helms, Louisa; Freedman, Benjamin S
Chronic kidney disease (CKD) affects 1 in 7 adults and is the 10th leading cause of death in the United States in 2022. Kidney transplant and dialysis remain the leading treatment strategies for kidney failure despite their expensive and outdated technological innovation. Stem cell derived human kidney organoids aim to provide a vital tool to study complex diseases ranging from infectious to genetic, and a translationally relevant system to discover and probe novel therapeutic pathways to improve our ability to treat kidney disease. CKD is the greatest risk factor for developing severe COVID-19, the 3rd leading cause of death in the US in 2022, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported to cause acute kidney injury in 1 in 4 hospitalized COVID-19 patients. Utilizing genome-edited kidney organoids, SARS-CoV-2 variants, and clinical data, we investigated viral tropism, mechanism, and therapeutic approaches in the context of the kidney. SARS-CoV-2 infected proximal tubules in kidney organoids via angiotensin converting enzyme 2 (ACE2). Infected organoids produced replication competent virus and displayed apoptotic responses in the context of polycystic kidney disease (PKD), a genetic cause of CKD. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in kidney organoids. Replication is reduced by remdesivir treated and infection blocked by treatment with de novo–designed spike binder peptides. This work clarifies the impact SARS-CoV-2 infection has on the kidney and enables the assessment of viral fitness and emerging therapies in the context of infectious disease. Autosomal dominant PKD is a genetic kidney disease affecting 1 in every 400-1000 people worldwide, causing progressive fluid-filled cyst production followed by fibrosis in the kidneys, liver, and other organs, resulting in organ failure. PKD is caused by mutations in the polycystin proteins polycystin-1 (PC1) or polycystin-2 (PC2), but the molecular pathway causing cystogenesis remains elusive. Genome-edited PKD organoids phenocopy cystogenesis and previously identified that the myosin inhibitor blebbistatin resulted in cyst enlargement. I discovered that treatment with the myosin activator, EMD 57033 (EMD), prevented cyst growth and that treatment when cysts were already established was able to slow cyst expansion. Live-imaging of EMD-treated organoids expressing fluorescently-tagged non-muscle myosin II B (NMIIB-GFP) revealed increased apical-basal tubule contractility of PKD organoid tubules compared to controls, indicating that the PKD organoid tubules were poised to contract and may have intrinsic contractile dysfunction. Analysis of the slowly progressing Pkd1RC/RC mouse model reveals a concomitant expansion of phosphorylated myosin light chain 2 (pMLC2) expressing stromal pericytes and cyst growth, suggesting that a therapeutic reducing pMLC2 expression may rescue kidney fibrosis later in disease. Together, this work suggests both a tubular and stromal myosin contribution to PKD pathogenesis that can be therapeutically targeted using myosin activators early in disease and pMLC2 inhibitors in later stage disease progression. In conclusion, our studies of PKD and COVID-19 reveal great utility of kidney organoids for disease modeling and therapeutic development, advancing the translational applications of organoid technology.
Molecular and Spatial Regulators of Cardiac Fibroblast Cell State and Plasticity in Myocardial Infarction Injury
(2022-01-26) Bugg, Darrian; Davis, Jennifer M
A central obstacle in heart disease is the replacement of healthy kinetic muscle with stiff fibrotic scarring. Fibrotic deposition represents a major clinical burden because it rapidly progresses the heart towards failure and current antifibrotic therapies are ineffective. It has been proposed that myofibroblasts are the major cellular source of the fibrotic response, yet the molecular mechanisms governing the formation and maintenance of the myofibroblast cell state are poorly understood. Here it was found that infarct scar alignment acts as a positive regulator of the myofibroblast cell state through focal adhesion sensation and downstream p38-YAP-TEAD signaling. Furthermore, is has been suggested that the RNA binding protein Muscleblind-Like1 (MBNL1) binds to and post-transcriptionally regulates many targets in the p38 signaling axes, suggesting a post-transcriptional control point for mediating fibrosis. Using fibroblast specific gain and loss of function models it was shown that MBNL1 is not only necessary for mediating the fibrotic response, but that MBNL1 acts as a post-transcriptional switch required for cells to transit between mesenchymal, quiescent, and activated cell states. Collectively, these data deepen our understanding of fibroblast to myofibroblast cell state transitions, where MBNL1 is upregulated after the early fibroproliferative window following myocardial infarction (MI) to stabilize pro-differentiation transcripts providing sufficient activation energy for myofibroblast formation. Although MBNL1 expression remains high through the first 2 weeks of MI, it is suggested that ECM alignment helps to maintain the activation energy necessary for maintaining the myofibroblast cell state until remodeling has resolved. Finally, these data suggest that antifibrotic therapies must not only consider the reduction in overall fibrotic quantity but also the topography and content of the matrix being laid down by fibroblasts with altered function.
Enhancing Local delivery of Macrophage Checkpoint Inhibitors with Chemokine Gradients to Lure and Destroy Pediatric Brain Tumor Cells
(2021-07-07) Nealy, Eric Scott; Olson, James
Pediatric brain tumors (PBTs) are the leading cause of cancer-related death in children. These malignancies tend to occur in locations of the brain where complete resection and adjuvant therapy could lead to an impaired quality of life. Residual cells from incompletely resected tumors may invade nearby areas of the brain where they can cause recurrence and ultimately lead to patient death. This thesis explores whether hydrogel-based delivery chemokine gradients and macrophage checkpoint inhibitors (MCIs) into orthotopic xenograft PBTs can result in enhanced elimination of tumor cells. Tumor cells lured to an immunotherapy “trap” by chemokines would then become exposed to MCIs they would have otherwise avoided, and targeted for elimination by phagocytic cells in the brain. Our data confirms that gradients of CXCL12 are effective at eliciting chemotaxis of patient-derived, pediatric high grade glioma (pHGG) tumor cells in vitro. Use of CD47 mAb as a single agent was effective at promoting pHGG tumor cell phagocytosis in co-cultures with murine macrophages. Therefore, we engineered slow-release hydrogels to locally deliver CXCL12 and CD47mAb to mice bearing orthotopic xenograft pHGGs for timescales up to one month. Mice receiving intratumorally injected gels containing this combination demonstrated attenuated tumor growth compared to mice receiving gels containing CD47mAb alone. These pre-clinical results suggest a combination of chemokines and MCIs could be a safe and effective means to promote recruitment and immunological clearance of remnant PBT cells in patients.
Genomic Instability at Single Cell Resolution
(2021-07-07) Dowsett, Ian; Herr, Alan
All organisms maintain the integrity of their genome through highly precise DNA replication and repair. Errors in these mechanisms can lead to genetic instability that results in cellular dysfunction or malignancy. Modern sequencing technology has enabled the development of methods to interrogate these processes during individual cell divisions. Our lab previously devised a single cell resolution approach that suggested that Saccharomyces cerevisiae cells with abrogated replicative fidelity exhibit multiple mutator states, a phenomenon termed ‘mutator volatility’, but the data was also consistent with a hypothesis in which replication errors segregated unequally during cell division. The research reported in my dissertation uses an expanded approach in chapter two to confirm that mutator volatility exists in strong mutators. It also shows that unequal inheritance of replication errors occurs due to the sequential processes of semiconservative DNA replication and chromosome segregation. Using computational modeling, I show that unequal segregation may dramatically expand heterogeneity in the mutation burden in human tumors. In chapter three, I model the strongest human mutator allele in cancer (POLE-P286R) and find an even greater volatility. Although this mutator phenotype depends in yeast on the S-phase checkpoint, the volatility does not. In the fourth chapter, I present a novel single cell resolution method to detect loss of heterozygosity (LOH) in yeast and then use this method to investigate if LOH increases with replicative age. These findings highlight the numerous insights that may be gleaned by studying the processes that give rise to genome instability at the level of single cells.
The regulation and function of C. elgans flavin-containing monooxygenase-2
(2021-07-07) Rossner, Ryan; Kaeberlein, Matt; Mendenhall, Alex
In the last one hundred years, aging has become an increasingly tractable problem. The pioneering dietary restriction experiments of the 1920s and '30s, the robust evolutionary and molecular theories of the 1950s -'70s, and the identification of conserved longevity genes in the 1980s – 2000s all paved the way for the aging research field's current rapid expansion and mainstream traction. Along with metformin, senolytics, and numerous other promising avenues, the field is still characterizing the molecular mechanisms through which major interventions like dietary restriction and the inhibition of insulin and mTOR signaling promote longevity. In this dissertation, I use the nematode roundworm model species Caenorhabditis elegans to define the regulation and function of the conserved pro-longevity target gene flavin-containing monooxygenase-2 (fmo-2). I find that both the regulation and function of fmo-2 is dependent on endogenous sulfur amino acid metabolism, placing fmo-2 at a nexus of redox, cellular energetics, and other processes central to aging.
The Microbial Etiology of Colorectal Cancer
(2020-10-26) Kordahi, Melissa; DePaolo, Randy W
The microbiome of the Gastro-Intestinal tract is estimated at 100 trillion organisms which act in a symbiotic relationship with the surrounding tissue cells to maintain homeostasis. However, alterations in the gut microbiota caused by genetics or environmental factors can disturb this relationship and promote diseases such as colorectal cancer (CRC). CRC is the third most common form of cancer in both men and women and the second leading cause of cancer-related death worldwide, presenting a considerable disease burden. The intimate association between the microbiota and the cells of the colon sets the stage for a number of interactions that may contribute to carcinogenesis. While only a few specific commensal species may play a direct causal role in CRC, more general shifts in the composition may promote local inflammation through engagement of innate immune receptors encoded within the colonic tissue. Changes in gene expression within the microbiota may also be important by altering virulence factors and producing metabolites that may have detrimental effects on the tissue. In the first chapter of this Thesis, we explore the conceptual frameworks through which certain members of the microbiota are believed to cause CRC, the sensing of microbiota associated molecular patterns by innate immune receptors known as Toll-Like-receptors (TLRs) and the various strategies aimed at manipulating the microbiota and targeting the TLRs, in the hopes of developing new treatment approaches. In the second chapter of this body of Research, we focus on Bacteroides fragilis, a particular bacterial commensal species that has been correlated to CRC development in human studies. Mouse models have also shown that B. fragilis is capable of remodeling the mucosal immune response and colonic bacterial community to promote oncogenic changes in the epithelium. In our study, we analyzed the mucosal microbiome of patients undergoing CRC screening and noted a high prevalence of B. fragilis in patients with early CRC lesions. We isolated B. fragilis from mucosal biopsy samples for deeper characterization and showed that they phenotypically differed according to their geographical location in the colon and the presence of pre-cancerous lesions in the microenvironment they were isolated from. The results we gathered explore the relationship between B. fragilis and early-stage CRC and provide biological framework for microbiota-based biomarkers and therapeutic targets. This study shows that the pre-cancerous mucosal environment alters the immunogenicity of a gut commensal. Environmental factors that influence the gut microbiota are then further explored in the context of individual species in Chapter 3 using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF MS) technology. Another factor influencing the gut microbiota and, therefore the host immune response, is genetics. Development of the human immune system depends on various receptors capable of recognizing and responding to pathogens and commensals. As mentioned in Chapter 1, these receptors include toll-like receptors (TLRs) found on macrophages, dendritic cells, and intestinal epithelial cells. Using TLR6 knockout mice in chapter 4, our study aimed to understand how disruption in host recognition of the microbiota can exacerbate disease. Defective TLR6 signaling was shown to worsen the host susceptibility to inflammation associated colorectal cancer. Within the same study, analysis of the microbiota revealed a therapeutic potential by restoring microbial ecology. By better understanding how the gut microbiome influences the development of colorectal cancer, we can begin to think in terms of innovative therapies to approach the treatment of this disease that continues to be challenge for healthcare professionals and patients in the clinic.
Natural genetic and phenotypic variation for aging
(2020-10-26) Jin, Kelly; Promislow, Daniel
Aging is a complex, highly variable trait influenced by genes, environment, and the interaction between the two. While aging is universal across nearly all species in the animal kingdom, there are many different ways an organism declines in health. As such, in order to fully understand why and how we age, we must explore the variation of aging in populations, both genetically and phenotypically. Historically, two main approaches have been used to try and understand how and why we age, including evolutionary and mechanistic approaches. Evolutionary approaches to studying aging have largely utilized demography, population and quantitative genetics, and mathematical modeling methods to investigate aging from a population perspective. Mechanistic approaches, in contrast, use cellular and molecular methods to drill down to mechanism, usually in the context of highly specific genetic or cellular backgrounds. While these two approaches have each revealed a great deal about both the evolutionary theory underlying why we age, as well as specific cellular pathways that may explain how we age, there is room for integration of these two complimentary approaches in the current landscape of aging research. An integrated approach to studying aging would incorporate the advantages of population and quantitative genetics, mathematical modeling, and molecular techniques to learn more about aging in individuals within a genetically variable population. Recent advancements in high-throughput genetic and molecular tools, as well as the growing popularity of data sharing, have made such approaches more feasible. In this dissertation, I present three unique but complementary perspectives on how we can use systems biology methods and animal models of genetic variation to learn more about the aging process. In the first study, I investigate the genetic and metabolomic architecture underlying lifespan extension using a collection of 178 genetically variable Drosophila lines. In the second study, I introduce a novel model system for human aging and disease research, the companion dog, and demonstrate that dogs, like humans, show increased levels of comorbidity with age. In my final research chapter, I expand on the versatility of the companion dog as a model for aging by using canine epigenomic profiles to build predictive models of chronological age, and interrogate whether or not those models might also predict the overall health of the animals. Taken together, this collection of highly interdisciplinary studies paves the way for future studies of aging that integrate the advantages of both classical quantitative genetics and cellular approaches to gain a more complete understanding of the biology of aging.
Characterizing the role of DDX3X in DNA double strand break repair; Implications for lymphocyte biology
(2020-10-26) Cargill, Michael James; Warren, Edus
DDX3X is a human, ATP-dependent RNA helicase with roles throughout RNA metabolism. A number of human diseases are associated with alterations in this gene, particularly in hematological malignancies. We aimed to investigate a novel function for DDX3X in DNA double-strand break (DSB) repair and its role in lymphocyte biology. We confirmed a traditional role for DDX3X in regulating DNA DSB repair protein levels, and herein provide evidence for a novel role as an active participant in DSB repair. We propose that DDX3X is recruited to DSBs in actively transcribed regions to regulate DSB-induced RNA metabolism. Finally, we developed CRISPR/Cas9 techniques to interrogate the function of DDX3X, and its Y-homologue, in lymphocyte biology. Thus, DDX3X plays an important role in DSB repair likely consequential to lymphocyte health and disease.
Identification of hypothalamic neural circuits regulating glucose and energy homeostasis
(2020-08-14) Faber, Chelsea Leigh; Morton, Gregory J
Diabetes and obesity are among the most common and costly health issues facing modern humans. The World Health Organization (WHO) estimates that obesity rates have tripled in the last 50 years: as of 2016, nearly 40% of adults are overweight, and 13% are obese. Given the strong association between obesity and type 2 diabetes (T2D), a compelling need exists for improved understanding of their pathophysiology. Accumulating evidence shows that the brain plays a critical role in the regulation of energy and glucose homeostasis. However, the underlying neurocircuitry by which the brain regulates energy balance and glycemia is narrowly understood, due to the exceedingly complex and highly interconnected nature of mammalian neurocircuitry. In this dissertation, I will discuss how, using a combination of engineered mouse models and genetically encoded tools, along with sophisticated metabolic phenotyping, we advanced our understanding of brain neurocircuits regulating energy homeostasis.
Identifying and Characterizing Novel Targets against Rhabdomyosarcoma Disease Relapse
(2020-08-14) Pham, Thao; Chen, Eleanor Y
Rhabdomyosarcoma (RMS) is the most common soft tissue pediatric sarcoma. Patients with relapsed or metastatic disease are faced with a poor survival outlook. Self-renewal of tumor propagating cells (TPCs) is believed to be responsible for driving RMS disease relapse through resistance to conventional therapies and recapitulation of RMS tumor heterogeneity. Identifying novel regulators of TPC activity may provide insight into potential therapeutic targets. In this dissertation, I identify and characterize GRK5 and HDAC6 as novel regulators of RMS cell growth and self-renewal. GRK5, a G-protein receptor kinase, regulates cell cycle in a kinase-independent manner to promote RMS tumor cell growth. Loss of GRK5 results in significant reduction of RMS self-renewal capacity due to increased cell death. HDAC6, a histone deacetylase, promotes RMS tumor growth by modulating cell cycle progression and tumor cell differentiation. Depletion of HDAC6 reduces RMS self-renewal capacity by limiting dilution assays and expression of stem-cell markers SOX2, NANOG and OCT4. In vivo inhibition of GRK5 and HDAC6 with small molecule inhibitors results in reduced RMS tumor cell growth, demonstrating their potential as therapeutic targets. Current in vitro approaches for studying RMS TPCs are unspecific, with in vivo methods being time intensive and costly. I will present preliminary data identifying CD133 as a potential marker for embryonal rhabdomyosarcoma (ERMS) TPCs for rapid in vitro characterization of TPC behavior. Collectively, my findings demonstrate the potential of RMS TPCs as therapeutic targets against RMS disease relapse and metastasis.
Mechanism of Diabetes Remission Induced by the Central Action of Fibroblast Growth Factor 1
(2020-08-14) Brown, Jenny M; Schwartz, Michael W
The rising prevalence of type 2 diabetes mellitus (T2D) is a major health concern worldwide. Growing evidence of a role for the brain in glucose homeostasis has stimulated interest in therapeutic approaches that target the brain for the treatment of T2D. Our recent finding that a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) elicits sustained diabetes remission in rodent models of T2D supports a growing consensus that the brain is a key target for diabetes drug development. The goal of the research reported in this dissertation is to identify the brain area, signal transduction pathway, and peripheral mechanisms responsible for mediating sustained diabetes remission induced by central FGF1. We identified the hypothalamic arcuate nucleus (ARC) median eminence (ME) (ARC-ME), a brain area known to participate in glucose homeostasis, as being one of only two brain areas that show robust induction of MAP Kinase / ERK (MAPK/ERK) signaling (a marker of FGF receptor activation) following icv injection of FGF1, and that FGF1 microinjection localized to this brain area is capable of inducing sustained glucose lowering. Additionally, activation of the MAPK/ERK signaling pathway occurs in the ARC-ME for at least 24 hours post icv injection, and diabetes remission induced by the central action of FGF1 depends upon this prolonged hypothalamic MAPK/ERK signaling. Finally, in the Zucker diabetic fatty rat model of T2D, sustained glucose lowering induced by the central action of FGF1 involves both preservation of β-cell function and stimulation of hepatic glucose utilization through increased hepatic glucokinase activity. This work provides fundamental insight into mechanisms underlying the brain’s capacity to induce sustained diabetes remission.
Molecular and Cellular Mechanisms of Mycobacterial Glycolipid Recognition by Human T Cells
(2020-08-14) James, Charlotte A; Seshadri, Chetan
Tuberculosis (TB) is of high global health importance and disproportionately affects individuals in resource-limited settings. A major challenge to reducing the global burden of this disease is the lack of effective vaccines and diagnostics. At present, the intricacies of the immune response to this disease are not understood well enough to rationally develop efficacious vaccines. Peptide-specific T cells have been implicated as a critical component of the immune response to TB. However, there are few studies that investigate the role of T cells that recognize non-peptide antigens in the immune response to TB. T cells can recognize lipid antigens presented by CD1 molecules, but how these antigens are recognized and the impact that antigen recognition has on lipid-specific T cell activation and functional differentiation is not understood. Here, we elucidate the molecular and cellular factors that affect lipid antigen recognition by human T cells, and what impact these factors have on T cell activation and function. This work focused on a family of mycobacterial lipids, diacylated sulfoglycolipids (Ac2SGL), which are only expressed by virulent strains of Mycobacterium tuberculosis, the causative agent of TB. The first aim of this work utilized synthetic Ac2SGL analogs, a panel of T cell clones, and antigen presenting cells that express mutated CD1 molecules to probe the specificity with which these antigens are recognized by T cells to inform which molecular moieties are essential for Ac2SGL recognition by T cells. The second aim investigated the impact of T cell receptor co-receptors on antigen recognition at the cellular level, as this influences the magnitude of activation and functional differentiation of T cells. We found that co-receptors augmented T cell affinity for Ac2SGL and these molecules impact T cell function in vitro and ex vivo. Together, our data support an emerging model that related but chemically distinct antigens and T cell subpopulations should be studied independently to fully understand the T cell response to mycobacterial lipid antigens. As Ac2SGL holds promise as a target for novel vaccination and diagnostic strategies for TB, these studies will inform the development of tools to address these two major needs.
Deciphering the relationship among nutrition, host and microbe
(2020-02-04) Chac, Denise; DePaolo, Randy W
Within the human gastrointestinal tract, there are trillions of resident microbes collectively known as the gut microbiota. These organisms have a profound impact on host physiology, particularly the immune system. Upon birth, the gut microbiota begins to take shape with input from various influences including genetics and environmental factors such as dietary habits, antibiotic use, and stress. While the gut microbiota has been shown to be necessary for proper development and immunity, it has also been implicated in the development of several environment linked diseases including non-alcoholic fatty liver disease (NAFLD), inflammatory bowel disease (IBD) and colorectal cancer (CRC). The consequences of the changing microbiota in these disease states and how the microbiota can be used therapeutically has yet to be fully explored. In this body of research, alterations to the microbiota due to diet (Chapter 2) and host genetics (Chapter 5 and 6) in the context of disease are analyzed. Environmental factors that influence the gut microbiota are also explored in the context of individual species (Chapter 3 and 4). One of the key players in shaping the gut microbiota is diet. To study how dietary fats could alter host microbiota and liver pathology, various diets were used in a model of NAFLD. A NAFLD-inducing diet high in cholesterol and sucrose was used to induced steatosis and liver inflammation while two intervention diets of either low fat and low fiber or a high fish oil diet were developed. While switching from the NAFLD-inducing diet to either of the intervention diets drastically reduced the steatosis and improved liver pathology, the corresponding microbiotas from the intervention diets were not sufficient to resolve hepatic steatosis and may even exacerbate the liver inflammation in the absence of dietary change (Chapter 2). To study the effects of dietary factors directly on the microbes, dietary fatty acids were applied directly to the enteric pathogen, Yersinia enterocolitica. Arachidonic acid is an omega-6 fatty acid that is found in high concentrations in the Western diet and has been associated with inflammation. Therefore, this study analyzed how arachidonic acid altered the protein signature using a technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Chapter 3) and virulence through both in vitro and in vivo assays. Following exposure to physiological levels of arachidonic acid, Y. enterocolitica became highly virulent with increased invasion into colonic epithelial cells and rapid systemic infection of mice (Chapter 4). Another factor influencing the gut microbiota and, therefore the host immune response, is genetics. Development of the human immune system depends on various receptors capable of recognizing and responding to pathogens and commensals. These receptors include toll-like receptors (TLRs) found on macrophages, dendritic cells, and intestinal epithelial cells. Using TLR1 and TLR6 knockout mice, these studies aim to understand how disruption in host recognition of the microbiota can exacerbate disease. In Chapter 5, aberrant TLR1 signaling led to increased mucosal-adherent microbes and defective mucosal immunity. These changes consequently exacerbated the host response to a model of colonic injury and recovery. On the other hand, defective TLR6 signaling worsens the host susceptibility to inflammation associated colorectal cancer (Chapter 6). Within the same study, analyze of the microbiota revealed a potential therapeutic by restoring microbial ecology. By investigating the various influences on the microbiota and the host in the context of nutrition and disease we can begin to understand the complexity of the microbiome and develop therapeutics. The diverse studies in this body of research ultimately reveal how environmental stimuli and disrupted sensing of the microbiota can have prolonged immunological impact.
Modeling autoimmune associated genetics in primary human T cells using CRISPR/Cas9 gene editing
(2019-10-15) Anderson, Warren Robert; Rawlings, David J
Genome wide association studies have identified genetic risk variants associated with multiple autoimmune diseases, thereby impacting large numbers of patients. Prominent examples are found within the phosphatase encoding genes PTPN22 and PTPN2. Studies have shown that risk variants in these genes impact a variety of cell types, but their expression or ablation in lymphocytes alone can be sufficient to drive autoimmunity in certain mouse models, due in part, to hyper-active lymphocyte signaling. Notably, cross-sectional studies of human carriers of the PTPN22 or PTPN2 risk variants have shown these donors to possess T cells that are hypo-responsive to TCR or cytokine stimuli. To investigate this discrepancy and understand the functional impact of these variants, new methods of studying these genes are required. The purpose of this study is to establish methods of using gene editing approaches in primary human T cells to modify expression the genes PTPN22 and PTPN2 and assess the impact of altered gene expression. Using this approach we found that ablation of PTPN22 and PTPN2 in primary human CD4+ T cells results in T cell responses which mirror current mouse models, with increased responses to TCR stimulation upon disruption of either gene, and enhanced responsiveness to IFNγ and IL-2 in PTPN2 disrupted cells. Interestingly, in the case of PTPN2 disruption, we found responses to IL-2 to be dynamic, eventually resulting in loss of responsiveness to IL-2, mirroring current human data from carriers of a reduced expression variant. Furthermore, we have explored several efficient methods to alter the coding sequence of PTPN22 to reflect its risk variant in non-risk donor T cells. Collectively our data shows that gene editing is a powerful tool for investigating how gene variants can contribute to disease, and that the effects of genetic risk variants may impart contextual and dynamic phenotypes on human lymphocytes. Finally, the methods we have established in this study are applicable to many other gene variants, and potentially could be utilized in multiple primary cell types.
miR-155 expression modulates microglia functions in vitro and in the APP/PS1 mouse model of Alzheimer’s disease
(2019-10-15) Aloi, Macarena Sofia; Garden, Gwenn A
Alzheimer’s Disease (AD) is characterized by the accumulation of extracellular Amyloid-β (Aβ) as well as both CNS and systemic inflammation. Microglia, myeloid cells resident to the CNS, use microRNAs to rapidly respond to inflammatory signals. MicroRNA (miRNA) profiles are altered in the tissue, circulating monocytes, and serum of AD patients. MiR-155 is a specific miRNA that modulates the phasic inflammatory responses of innate immune cells, however its precise role in AD pathogenesis remains unknown. We hypothesized that miR-155 participates in AD pathophysiology by regulating microglia responses to Aβ in vitro and in vivo. In cultured neonatal microglia, we observed that modulation of miR-155 expression impacts the internalization of fibrillar Aβ at the plasma membrane and to low-pH compartments. In mouse models of AD, microglia specific knock-out of miR-155 decreased accumulation of Aβ. In addition, we also observed that microglia specific deletion of miR-155 acutely increased seizures and seizure-related mortality in two mouse models of AD. Reduced Aβ plaques after miR-155 deletion in microglia suggests increased clearance and the hypothesis that in AD models, microglia facilitate epileptogenesis by increased internalization of synaptic material along with removal of Aβ. Together, these findings identify miR-155 expression in microglia as a potential regulator of synaptic homeostasis and microglia responses to Aβ in mouse models of AD.
Molecular genetics of myeloid malignancy predisposition: Insights into pathogenesis and therapeutic translation
(2019-10-15) Krutein, Michelle; Horwitz, Marshall S
Preleukemic diseases are highly informed by genetic predisposition and require appropriate models for studying pathogenesis and the progression to hematological malignancy. Although rare, familial platelet disorder (FPD) and severe congenital neutropenia (SCN) have few or no therapies available to patients and also possess high rates of leukemic transformation to myeloid malignancy. With these points in mind, my graduate work aimed to elucidate the molecular mechanisms of ELANE-associated severe congenital neutropenia, identify new genetic mutations causing preleukemic diseases, and evaluate novel therapies for platelet disorder with predisposition to myeloid malignancy. Familial platelet disorder with predisposition to acute myelogenous leukemia is an autosomal dominant disorder caused by monoallelic mutation of RUNX1, initially resulting in half-normal levels of RUNX1 protein. Patients develop leukemia only after a protracted prodrome consisting of thrombocytopenia and bleeding diathesis relating to functional platelet granule deficiency, suggesting that early intervention affords an opportunity for preventing malignant transformation. We hypothesize that pharmacological inhibition of RUNX1 protein degradation may normalize RUNX1 protein levels and restore platelet numbers and function. RUNX1 is rapidly degraded through the ubiquitin-proteasome pathway. Moreover, RUNX1 auto-regulates its own expression. A predicted kinetic property of auto-regulatory circuits is that transient perturbations of steady-state levels result in continued maintenance of expression at adjusted levels, even after inhibitors of degradation or inducers of transcription are withdrawn, suggesting that transient inhibition of RUNX1 degradation may have lasting effects. Here we evaluate cell lines, FPD/AML patient derived induced pluripotent stem cells (iPSC), and FPD/AML primary bone marrow cells and show that, in some circumstances, transient expression of exogenous RUNX1 or inhibition of steps leading to RUNX1 ubiquitylation and proteasomal degradation restore RUNX1 levels, thereby advancing megakaryocytic differentiation in vitro. Thus, drugs retarding RUNX1 proteolytic degradation may represent a therapeutic avenue for treating bleeding complications and preventing leukemia in FPD/AML. Heterozygous mutations in ELANE, encoding the potent serine protease, neutrophil elastase (NE), cause cyclic neutropenia (CyN) and are the most common cause of severe congenital neutropenia (SCN). Patient presentation is marked by profoundly low neutrophil counts accompanied by predisposition to myelodysplasia (MDS) and acute myeloid leukemia (AML). There is no unified theory of SCN or CyN pathogenesis. However, out of the >100 mutations recorded in SCN and CyN, none of these mutations have been found to encompass the three catalytic residues necessary for NE proteolysis, which may suggest retention of these residues is important for SCN and CyN pathology. To address this question, I developed novel iPSC models of EA-associated severe congenital neutropenia via genome editing strategies using CRISPR-Cas9 targeting with homology directed integration of synthetically designed non-viral vectors. These vectors either harbored an aggressive SCN mutation or a single residue substitution of the catalytic serine of neutrophil elastase. Additional work must be performed to generate iPSC lines possessing both the SCN and catalytic inactivation mutation in cis as well as a wild type ELANE control iPSC line. All vectors contained a green fluorescent protein (GFP) gene trap whereby mutant NE expression is announced through GFP reporting. These cell lines allow us the opportunity to investigate how catalytic activity of neutrophil elastase influences neutrophil development and SCN pathology, both questions that are unanswered or under scrutiny in the field. It is also critical to mention that our method of genome editing creates models whereby expression of the mutant protein is detectable through a reporter. This characteristic makes our models superior to other existing iPSC models that have not been able to achieve mutant protein reporting due to direct reprogramming of primary SCN samples. Additionally, these integration vectors can be easily adapted to harbor any desired changes in exon 4 or 5 of ELANE. Lastly, although neither homozygous or S202A mutations in ELANE have been observed in normal, SCN, or CyN individuals, these novel cell models will provide us with the tools to determine the interaction between NE proteolysis and granulopoiesis in both normal and diseased states. Congenital neutropenia is a genetically heterogeneous disease whereby our lab has contributed to this growing list of genetic factors found causative of neutropenia. In order to expand the current knowledge of neutrophil development and biology it is critical for us to continue the search for novel genes or mutations that produce a neutropenic phenotype. Genetic screening of neutropenic children in two unrelated families revealed the same T679I variant of unknown significance in the gene SUZ12. A critical transcription factor governing stem cell differentiation, SUZ12 protein normally facilitates epigenetic remodeling through global H3K27me3 yet has not been reported as having a role in neutrophil development specifically. I reprogrammed primary patient samples to SUZ12-iPSCs and subsequently subjected them to hematopoietic stem cell (HSC) and neutrophil differentiation which recapitulated phenotypes observed in patients. Epigenetic landscape evaluation of SUZ12-iPSCs via western blot and chromatin immunoprecipitation sequencing (ChIPseq) revealed reduced H3K27me3 repressive genome markers, elevated H3K4me3 activation markers, and some differences in SUZ12 binding. These studies reflect the first report of mutations in epigenetic proteins, more specifically SUZ12, as being causative of SCN.
The Role of Early Autocrine IL-2 Signals in Programming Antigen-Specific CD8 T cell Responses
(2019-10-15) Yuzefpolskiy, Yevgeniy; Sarkar, Surojit
IL-2 is a potent cytokine in mediating antigen-specific CD8 T cell responses. Following antigen priming, IL-2 signals drive the differentiation of resting cytotoxic T cells into potent effector cells to elicit rapid expansion, and the expression of effector molecules critical for eradicating intracellular infections. Antigen-specific CD8 T cells more sensitive to early IL-2 signals undergo pronounced effector expansion as they differentiate into terminal effector cells that subsequently undergo substantial contraction following antigen clearance. Conversely, cells expressing lower levels of the high affinity IL-2 receptor, CD25, exhibit lesser expansion and preferentially form long-lived memory CD8 T cells, which provide protection from future infections. Interestingly, one of the hallmark features of these memory CD8 T cells is their retained function of autocrine IL-2 production. This suggests that autocrine IL-2 is intrinsically linked to the differentiation and maintenance of effector and memory cells. In our studies we have utilized a murine model of acute infection in which we studied the effects of autocrine IL-2 ablation on antigen-specific CD8 T cells. We were able to show that autocrine IL-2 signals were dispensable to antigen-specific CD8 T cells for mounting a primary immune response; the T cells showed no defect in effector expansion or function, and were capable of forming long-lived, polyfunctional memory CD8 T cells. However, upon antigenic rechallenge memory cells deficient in autocrine IL-2 signals underwent increased cell-death resulting in a compromised secondary response. We then used temporal ablation of autocrine IL-2 to determine that these signals were critical during the initial priming of antigen-specific CD8 T cells. Unlike acute infections, where antigen is cleared by the effector response, during chronic infections antigen persists in the host exhausting the antigen-specific T cells via constant stimulation. However, the initial priming events are similar in both acute and chronic IL-2 infections. Thus, we wanted to determine if autocrine IL-2 signals also program survival in CD8 T cells in a system where the cells are continuously stimulated with antigen. We discovered that in the absence of autocrine IL-2, antigen-specific CD8 T cells undergo lower expansion but are not affected in their ability to elicit effector function. Following establishment of exhaustion, CD8 T cells lacking autocrine IL-2 over-expressed inhibitory receptor PD-1, and had a pronounced defect in maintenance. Surprisingly, T cells lacking autocrine IL-2 underwent significantly better expansion following PDL1 checkpoint blockade therapy, expanding to levels similar to their WT counterparts. These data show for the first time a critical role for autocrine IL-2 during chronic infections. Furthermore, this research suggests that it is possible to tune exhausted CD8 T cells using IL-2 signals, to boost their responsiveness to checkpoint blockade therapy.

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