Below are summaries of the projects of NIH intramural scholars funded as part of the Maximizing Opportunities for Scientific and Academic Independent Careers (MOSAIC) Program.
Project Title: Delineation of Macrophage-Derived Transglutaminases Role in Adipose Tissue Health and Inflammation in ObesityInstitution: Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentGrant ID: DK136921
Abstract Text: Obesity is classified into either healthy or unhealthy metabolic states. Unhealthy obesity is characterized by decreased preadipocyte proliferation, increased adipocyte enlargement (i.e. hypertrophy), altered inflammatory states and increased infiltration of macrophages. Invariably, this condition promotes the development of cardiovascular disorders and type 2 diabetes. Although the pathological burden is well established, the underpinning molecular mechanics that drive deregulation of immunometabolic activities remain unclear. Preliminary data shows transglutaminases (TGases), such as TGM2, TGM4 and F13A1, are novel soluble modulators of immunometabolism that can shift obesity into a metabolically unhealthy state. High levels of TGases were associated with adipocyte hypertrophy, macrophage infiltration, increased IL-10, and deregulation of collagen deposition into the extracellular matrix. Blocking TGase expression reversed and/or normalized several of these indices. Interestingly, macrophages were identified to be a major source of TGases. Reports have shown M2 macrophages are key producers of TGases, but no data has linked this to immunometabolic regulation. Preliminary data herein now supports those findings and extend the importance of the TGaseproducing M2 macrophage subsets as pivotal players governing the shift towards unhealthy obesity states. Taken together, these studies will evaluate the hypotheses that TGases serve as a pivotal modulator in balancing the shift between healthy vs. unhealthy obesity, particularly by regulating adipocyte hypertrophy, lipotoxicity, and inflammation. To directly address this hypothesis, studies will evaluate the mechanisms by which TGases regulate adipose tissue functioning in obesity (Aim 1). Lentiviral vector-based delivery of shRNA silencing of varying TGases in HFD-treated mice in vivo will be employed to resolve the direct role on adipocyte growth, differentiation and metabolic activities, as well as accompanying changes in extracellular matrix composition/assembly and vascularization. In tandem, investigations will evaluate the role of TGases in regulation of inflammation and immunity (Aim 2). Using similar shRNA in vivo silencing approaches, studies will delineate the key role of TGase in regulating macrophage functions, immune cell infiltration and the cytokine inflammatory (cytokine) environment in adipose tissues during obesity. Lastly, studies will develop a novel lentiviral-based conditional knockout mouse model as a tool to concretely evaluate the role of TGase-producing immune cells in modulating the immunometabolic environment of obesity (Aim 3). The lentiviral vector will be engineered to carry a cell-specific promoter upstream of the cre recombinase transgene for in vivo delivery into floxed TGase mouse models. This will unravel the regulatory role of specific TGase-producing immune cell subsets in modulating the adipose tissue microenvironment during obesity. Successful completion of these aims will advance our understanding of the immunometabolic regulatory network orchestrated by TGases and M2 macrophages and define its effect on shifting the balance between healthy vs unhealthy obese states.
Public Health Relevance Statement: Incidence of obesity and type 2 diabetes has dramatically increased in the US and although thoroughly investigated, it isn’t until recently that the immunological and inflammatory components of the diseases have been appreciated, as playing a chief role in disease etiology and progression. Building on this insight, these investigations will advance our understanding of how novel inflammatory mediators contribute to metabolic unhealthy obese states. Knowledge from the studies will fill major gaps in our understanding of the disease pathology and present opportune avenues for development of innovative therapies to alleviate the burden.
Project Title: The role of host mRNA cleavage by RNase L in viral infectionsInstitution: National Institute of Diabetes and Digestive and Kidney DiseasesGrant ID: K99GM143484
Abstract Text: Viral infections remain a challenging public health issue worldwide. The presence of many viruses, such as Influenza A and Hepatitis C, activate a latent Ribonuclease (RNase L) in human cells. Activated RNase L cleaves the single stranded regions of viral and host mRNAs. Cleavage of these RNAs leads to physiological changes in the cell, such as autophagy, senescence, decreased cell motility, interferon production and cell death. However, many aspects of RNase L activation in cells are not yet understood. Recent in vivo and in vitro kinetic studies suggested that RNA cleavage by RNase L is modulated in the cell by an unidentified factor. RNase L can directly interact with the ribosome and with several translation factors involved in different steps of protein synthesis. Due to these interactions it was proposed that RNase L’s cleavage activity is modulated by the translation of the host messenger RNAs (mRNA). Therefore, in specific aim 1, I will investigate this relationship between host mRNA cleavage by RNase L and translation at the global and individual gene level by combining two high-throughput sequencing methods, ribosome footprint profiling and RNA sequencing in RNase L activated human cells. In addition, ribosome-mediated RNase L cleavage activity will be also directly observed at the individual gene level by a novel technique, the real-time fluorescent single molecule detection of translating nascent peptides (SINAPS) in living cells. In aim 2, I will also explore the potential involvement of translation factors in translationmediated RNA cleavage by RNase L by first mapping the details of interactions of RNase L and translation factors by mutagenesis studies and Cryo-Electron Microscopy. Then these interactions will be disrupted in living cells to probe their potential involvement in mediating RNA cleavage by RNase L. Furthermore, RNase L activation can lead to many physiological changes in the cell, such as autophagy and apoptosis. It is plausible that the level of active RNase L is the determinant of which physiological processes will occur. To investigate the correlation between active RNase L levels and RNase L mediated changes in the cell, I will develop a fluorescent RNase L activation indicator that will allow us to sort cells into homogenously activated populations in aim 3. Subsequently, in these sorted cells I will detect changes in the transcriptome and translatome and assess signatures of physiological changes of autophagy and apoptosis in the context of RNase L activation levels. In addition, I will also study how RNase L cleaved host RNA fragments contribute to activation of innate immune response pathways in cells by using Cross-linking Co-Immunoprecipitation and sequencing (CLIP-seq). In summary, the proposed project will investigate unexplored aspects and outcomes of RNase L activation. Uncovering new aspects of the defense against viral infections will enable further studies and potentially contribute to the development of new therapeutic strategies.
Public Health Relevance Statement: The persistent presence and continuing emergence of viral infections is one of the major obstacles to improved human health. The majority of viruses, such as human immunodeficiency virus 1 and potentially SARS-CoV2, activate an endoribonuclease, RNase L, that consequently induces several physiological changes in the cell from autophagy to apoptosis. In the present proposal I will investigate how the activity of RNase L is modulated and its role in determining cell fate by using a combination of new innovative and classical molecular biology techniques.
Project Title: Multiplex Imaging in Therapy Refractory Tumors: Understanding the Spatiotemporal Facets of an Immunosuppressive EnvironmentInstitution: National Institute of Allergy and Infectious DiseasesGrant ID: GM147841
Abstract Text: Background/Significance. In recent years, it appeared that the cancer treatment revolution had arrived: immunomodulatory therapies. Although these breakthroughs unleash the immune system and can be deadly to tumors, there are a high percentage of cancers that fail to respond. One complicating aspect is tumor heterogeneity, both within the same cancer type and within a single patient. This aspect is especially prominent in pancreatic adenocarcinoma (PDA), which is notoriously resistant to many frontline immunotherapies and has a low 5-year survival rate. Although mouse models have made research into PDA more accessible, these models frequently fail to capture the spectrum of tumor heterogeneity in human PDA. Innovation. To address this limitation, I will use a unique collection of mouse pancreatic tumor clones with distinct and reproducible responses to treatment, mimicking the tumor heterogeneity seen in the clinic. This will provide me with unparalleled comparative and combinatorial power to interrogate the tumor microenvironment. Additionally, my proposed experimental approach will leverage emerging high-content imaging methods that are able to reveal the phenotype, activity, and spatial organization of the tumor microenvironment in situ.
Research Goals and Aims. The overall theme of my research is to understand the fine-grain cellular interactions that lead to an immunosuppressive tumor microenvironment, and how to then subvert and reprogram those interactions to improve synergistic combination therapies. Specifically, I propose two conceptually related aims that explore mechanisms to overcome the immunosuppressive tumor microenvironment. I aim to dissect how the myeloid compartment (Aim 1) and immunogenic cell death (Aim 2) can be manipulated to reinvigorate anticancer immune responses. Through these aims I will develop a platform for interrogating the underlying functional immunity of the tumor site, host-pathogen interactions, and the impact of therapeutic intervention.
Career Development Plan and Environment: My mentor, Dr. Ronald Germain, a renowned expert in immunology and microscopy, is an investigator in the NIH Intramural Research Program, one of the largest research centers in the world. In this unique environment, I will directly benefit from the numerous resources in place to support my research project and career development, including microscopy and immunology core facilities, frequent seminars and opportunities to engage colleagues/mentors, and regular workshops on grantwriting, mentoring, and laboratory management. Furthermore, I have facilitated multiple collaborations that will aid in expeditious attainment of my proposed research aims and my transition to independence. By the end of my training plan, I will be well-positioned to launch a productive independent career at the intersection of microbiology and cancer immunology.
Public Health Relevance Statement: Pancreatic cancer is an aggressive and therapeutically difficult cancer to treat. Mouse models of pancreatic cancer are therefore paramount to our mechanistic understanding of tumor microenvironment dynamics and immunomodulatory interventions. The overall goal of this research is to use cutting-edge microscopy techniques to mechanistically understand the fine grain cellular interactions that dictate tumor responsiveness and leverage them to improve synergistic therapeutic strategies.
Project Title: enomic Regulation of Immune Response by a Stat1 Gain of Function MutationInstitution: ational Institute of Arthritis and Musculoskeletal and Skin DiseasesGrant ID: AI177758
Abstract Text: The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1- GOF mouse model, we demonstrate that STAT1-GOF mice exhibit an impaired NK and CD8 T cell effector response and develop a cytokine storm with viral infection. This is due to an impaired tissue-specific and microbiome-dependent deficit in IFN-gamma production by liver NK cells, ILC1s, and iNKT cells early during infection. Continuing with these studies, the goal of this application is to understand how perturbations in cytokine signaling have downstream and long-term effects in immune response. This proposal tests the hypothesis that prior exposure to cytokine stimuli have long-term effects on gene expression and immune cell function. The approach is divided into three specific aims: first, to determine the epigenetic mechanisms underlying how a STAT1-GOF mutation alters transcriptional output; second, elucidate the mechanism underlying the tissue-specific reduction of IFN-gamma in STAT1-GOF mice with viral infection; and three, to determine the consequences of a cytokine storm on CD8 T cell memory. During the mentored phase of the K99, the candidate will be trained in various genomic sequencing technologies and bioinformatic analysis to investigate AIM 1. Alongside research training, the candidate will participate in career development courses and workshops about grant writing, laboratory management, and teaching to fulfill her goal to attain a tenure-track independent research position. The training phase will be in Dr. John O’Shea’s laboratory in NIAMS at the NIH, supported by excellent resources, expertise in the laboratory, and vibrant NIH research community. These studies and career development plan will launch the R00 phase of independent research to investigate AIM 2 and AIM 3. The broader implication of this work is it will uncover basic cytokine signaling mechanisms broadly applicable to the field of immunology and identify potential therapeutic options for patients with altered set points.
Public Health Relevance Statement: Whether it is from autoimmunity, chromosomal abnormalities, or monogenic diseases, patients with interferonopathies paradoxically struggle with viral infections. This research will identify how an unrestrained STAT1 and cytokine storm affects gene expression and immune cell response to viral infections.
Project Title: Investigating Molecular Mechanisms of Endocytosis of the Activated B Cell Receptor in Health and DiseaseInstitution: National Heart, Lung and Blood InstituteGrant ID: GM152952
Abstract Text: B cell activation and antibody production is central to the human adaptive immune response to pathogens. The B cell receptor (BCR), is the primary cell surface receptor that engages with foreign antigens and initiates activation of the cell. BCR stimulation induces receptor clustering and endocytosis. Details about the molecular mechanisms that drive internalization of large antigens in B cells are unclear. Clathrin mediated endocytosis (CME) is responsible for internalization of small BCR clusters, but studies have shown that many BCR clusters can grow far beyond the size of the average clathrin coated pit. In previous studies I identified novel plasma membrane structures that are highly co-localized with large BCR clusters. These structures are smooth raised membrane invaginations that also have small clathrin lattices associated with them (cSRM structures). The overall hypothesis investigated in this research is that cSRM structures represent a unique mechanism of hybrid endocytosis that allows B cells to internalize large antigens. Aim1 is to map the molecular interactions that are required for hybrid endocytosis using three methods: (1) super resolution correlative light and electron microscopy to determine the nanoscale localization of endocytic proteins around cSRMs (2) proximity labeling of the BCR in live cells to identify a core proteome of molecular interactions involved in hybrid endocytosis and (3) live super resolution imaging of stimulated B cells to examine the kinetics of actin and clathrin remodeling around large B cell clusters during hybrid endocytosis. These studies will generate a more clear picture of the molecular players and kinetics of protein interactions involved in hybrid endocytosis. In the independent phase of this award (Aim 2) I will determine how hybrid endocytosis plays a role in the pathogenesis in Activated B Cell like Diffuse Large B Cell Lymphoma (ABC DLBCL). BCR mutations associated with ABC DLBCL cause defects in receptor internalization via CME. Since the BCR is also endocytosed through clathrin-independent pathways, the goal of Aim 2.1 is to determine what affect BCR mutations have on internalization through hybrid endocytosis. BCR mutations that are associated with malignant B cells in ABC DLBCL will be introduced into the DG-75 B cell line and then cells will be evaluated to determine if they are able internalize large BCR clusters and generate cSRMs. Another feature of malignant B cells in ABC DLBCL is the persistence of spontaneous BCR clusters at the plasma membrane. These clusters likely contribute to the pathogenesis by perpetually amplifying positive signaling from the BCR. Sub Aim 2.2 will examine the plasma membrane features associated with spontaneous BCR clusters using electron microscopy and live cell imaging to determine if there is a defect in recruitment or function of endocytic proteins around these BCR clusters. In total these studies will elucidate the molecular mechanisms of a novel form of hybrid endocytosis in B cells, and then determine the role of hybrid endocytosis in contributing to disease pathogenesis in Lymphoma.
Public Health Relevance Statement: This study will investigate the molecular mechanisms of endocytosis of large B cell receptor clusters, and determine how receptor endocytosis play a role in the pathology of lymphoma. This work is of public health relevance because endocytosis is an essential function for all cells and B cells in particular are critical for a functioning human immune response. Furthermore, research into the molecular mechanism that facilitate pathology in lymphoma is instrumental to develop novel treatments for this disease.
Project Title: Studying the modulators and the physiological functions of RNA tailing in the C. elegans oocyteInstitution: National Institute of Diabetes and Digestive and Kidney DiseasesGrant ID: GM149822
Abstract Text: The decreasing quality of oocytes, associated with maternal age, is a major component of reduced fertility in older women. Many molecular markers are observed in deteriorating oocytes, including transcriptomic changes. Interestingly, in most animal models, the end of oogenesis and early embryogenesis proceed in the absence of transcription, relying on stored messenger RNAs (mRNAs). The developing oocyte carefully manages different populations of RNA during maturation, fertilization, and zygotic genome activation to successfully jumpstart embryogenesis. The accumulation of these maternal transcripts during oogenesis is therefore essential. Yet we know very little of how the transcriptome is sculpted after the transcriptional machinery is inactivated. What drives transcriptomic changes and how are they contributing to oocyte quality? RNA tailing, or the addition of untemplated nucleotides to the 3' end of RNA molecules, is a posttranscriptional process that has long been associated with the regulation of RNA stability and translation. Both nucleotide composition and length of tails can determine tail function. RNA tails are especially dynamic in the germline and the early embryo. The objective of this work is to identify the terminal nucleotidyl transferases and exonuclease that modulate RNA tails and to elucidate the mechanism that mediate the downstream effects on RNA stability and translation in the oocyte. First, I will examine the physiological function of TNTs and exonucleases in C. elegans fertility. Second, I will characterize changes in RNA tail length and composition during oogenesis and early embryogenesis to shed light on conserved pathways involved in generating viable and competent oocytes. Third, I will identify the co-factors that act upstream and downstream of TNTs and exonucleases, to coordinate their activity, and modulate tail-mediated regulation of RNA stability and translation. I am uniquely qualified to conduct this research, having studied different types of RNA and their biology throughout my graduate and postdoctoral training. In Katherine McJunkin’s laboratory, I have used the C. elegans model system and large-scale genomic screens to identify TNTs responsible for miRNA tailing and to assess its impact on microRNA turnover. In the proposed work, I will apply state-of-the-art techniques to dissect the machinery responsible for mRNA tailing in the context of reproduction and fertility. This work seeks to explore post-transcriptional mechanisms regulating gene expression during the oocyte-to-embryo transition and their contribution to oocyte quality.
Public Health Relevance Statement: The principal goal of this project is to dissect and study the machinery that modulates RNA tailing and its effects on RNA stability and translation. These mechanisms will be explored during C. elegans oogenesis and early embryogenesis, taking advantage of the window of transcriptional inactivity when post-transcriptional regulation of gene expression is critical. In the process, this project will also identify essential genes for oogenesis and shed light on how their regulation is critical to their physiological function in the oocyte.
Project Title: Quantitative Characterization of the Extracellular Matrix Components of Connective Tissue: Fingerprinting Macromolecular Components through Low-Field Magnetic ResonanceInstitution: Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentGrant ID: K99GM140338
Abstract Text: Fibrotic activity, the accumulation of macromolecules, alters the composition and microstructural organization musculoskeletal connective tissue. The ability to non-invasively quantify and characterize the significant extracellular matrix components such as proteoglycan content and collagen fibrils organization is important clinically since fibrosis is a common inflammatory response that plays a role in many pathologies. Recent advances in instrumentation for low field magnetic resonance (MR) has enabled its adoption in the field of macromolecular characterization, porous media, and recently biological tissues. LF MR is advantageous as an affordable non-cryogen alternative to high-field MR imaging with a greater detectable dynamic range of quantitative MR parameters. This adaptation for this imaging modality is limited by the lack of appropriate phantoms of connective tissue and the identification of biomarkers for healthy and diseased tissue. Composite gels that replicate the salient structural and compositional features of connective tissue will be developed and used to optimize low field MR methods and identify LF biomarkers. These methods will be applied to articular cartilage and lumbodorsal fascia connective tissues. The project is anticipated to have a significant positive impact on the clinical capability and utility of LF MR as an affordable point of care diagnostic application.
Public Health Relevance Statement: Musculoskeletal (MSK) conditions are the leading contributor to disability worldwide, between one and three, and one and five people live with a musculoskeletal pain condition. Regular assessment of the state of MSK connective tissue is needed to better identify the pathophysiological origin of the inflammatory and fibrotic response. This research will advance the adaptation of low field magnetic resonance imaging (MRI) as a novel, noninvasive, and affordable point of care diagnostic imaging method capable of assessing the state of MSK connective tissue and effective of regenerative medicine.
