Browsing by Subject "inflammation"
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Item Contributions Of Signal Transducer And Activator Of Transcription 3 To Tumor And Immune Cell Functions In Breast Cancer(2016-10) Chuntova, PollyThe studies presented in this thesis demonstrate a novel link between activation of the FGFR pathway, alterations of the tumor microenvironment and tumor immune response in mammary tumorigenesis. These studies are the first to demonstrate that FGFR signaling in epithelial cells leads to accumulation of the ECM component hyaluronan (HA) through increased production of pro-inflammatory cytokines and activation of the STAT3 pathway. Therapeutic inhibition of STAT3 in vivo reduced HA accumulation, which correlated with reduced tumor burden. Nonetheless, STAT3 inhibition did not result in tumor regression, suggesting that additional pro-tumorigenic mechanisms are able to sustain tumor growth. Previous work has shown that FGFR1 activation leads to rapid recruitment of macrophages with pro-tumorigenic functions. We hypothesized that as TAMs differentiate in the presence of FGFR1-driven IL-6 family of cytokines, the STAT3 signaling pathway would be activated and would influence TAM differentiation. Therefore, further studies focused on delineating the STAT3-dependent phenotype and function of mammary TAMs. Utilizing a mouse model of genetic STAT3 ablation within myeloid cells demonstrated decreased tumor latency and increased tumor growth rate in conditional-STAT3∆/∆ mice compared to control animals. These results provide evidence to the hypothesis that STAT3 activation in different tumor and immune cell populations can result in both pro- and anti-tumor phenotypes, and detailed understanding of these mechanisms is necessary for developing effective therapeutic approaches.Item Defining Mechanisms of Inflammation and Fibrosis In Cardiac Valve Disease(2019-05) Meier, LeeCardiovascular disease (CVD) has been the leading cause of death for over a century and it will continue to be for the foreseeable future. While it has long been hypothesized that inflammation and the immune system play contributory roles in CVD initiation and progression, only recently was confirmatory evidence acquired from a large-scale clinical trial in humans. Thus, despite the existence of significant scientific and clinical efforts invested in understanding immunopathology in CVD, only recently has definitive evidence in support of this line of reasoning been acquired. Despite numerous scientific advances in recent years, our understanding of CVD and the role of inflammation therein remains limited. To date, no FDA approved drugs exist that specifically target aspects of inflammation in CVD despite the breadth of scientific data that underlies its importance. As such, continued investigation of the cellular and molecular mechanisms that govern CVD initiation and progression are needed. In this dissertation I seek to expand the scientific and clinical community’s understanding of the role for inflammation in the development of cardiac valve disease, a subset of CVD and a significant contributor to morbidity and mortality worldwide. Firstly, Chapter 1 provides an overview of cardiovascular structure and normal physiology. Secondly, Chapter 2 provides a brief overview of various forms of acquired CVD including atherosclerotic CVD, with emphasis on valvular heart disease (VHD). Therein, I include an introductory discussion of the role of inflammation in acquired CVD development. Thirdly, Chapter 3 provides a discussion of the current understanding of antibodies with specificity to self-epitopes (i.e. autoantibodies) during the initiation and progression of CV inflammation and its downstream chronic consequences on CV function. Therein, I provide motivation for studying the role of autoantibodies in CVD initiation and progression and provide insight into the current understanding of how autoantibodies interface with elements of the circulatory system to further CVD progression. As our animal model of systemic inflammation and cardiac valve disease is driven by autoantibodies, the discussion in this review provides a framework for the experimental studies that follow in later chapters of this dissertation. Chapter 4 demonstrates that discrete population of mononuclear phagocytes (MNP) are critical for MV inflammation and fibrosis. Therein, I provide evidence that MNPs are necessary for disease initiation and progression and are critical orchestrators of MVD through cytokine secretion in response to activating FcγR signaling. Downstream of MNP cytokine production, activation of the MV interstitium drives recruitment of additional inflammatory cells. On a chronic timescale, MV fibrosis results. Finally, I provide evidence for upregulation of similar pathways in samples acquired from human inflammatory MVD. Chapter 5 expands on the observations set forth in Chapter 4, and provides additional mechanistic clarification of the role for MNPs during the initiation and progression of MV inflammation and fibrosis in K/B.g7 mice and in humans. Firstly, I demonstrate that type-2 inflammation is required for disease in K/B.g7 mice and acts through multiple levels to orchestrate both systemic inflammation and cardiac valve-localized inflammation and fibrosis. Within the context of MV disease, I provide evidence that IL-13 (and not IL-4) is an important aspect of valve fibrosis. Next, I demonstrate a role for apoptotic cell accumulation and expression of the ‘don’t eat me’ signal, CD47, during the initiation and progression MVD. I show that blockade of this immune checkpoint enhances phagocytic cell clearance, and decreases MNP production of TNF, IL-6, and IL-13. The MVD dampening effect is seen when CD47 blockade is conducted both preventatively and therapeutically, thereby underscoring the central role of this pathway in disease progression. Lastly, I provide evidence for upregulation of these pathways in human RHD samples, again providing evidence for the translational potential these results hold. Finally, Chapter 6 provides preliminary evidence that a hallmark of MV inflammation in K/B.g7 mice is induction of endothelial-mesenchymal transition (EndoMT). Therein, I show that MV endothelial cells upregulate CD47 during pathological EndoMT and that this process is attenuated in the setting of CD47 checkpoint blockade. Additional work is needed to better clarify the role for EndoMT in development of MV inflammation and fibrosis, however, it is tempting to speculate about a putative pro-fibrogenic role for endothelial-derived mesenchymal cells during the course of chronic MV inflammation and fibrosis. Collectively, the results presented here provide substantial mechanistic insight into the underlying cellular and molecular pathways that contribute to cardiac valve fibrosis in the setting of chronic, autoimmune inflammation. These studies were the first to definitively implicate myeloid cells in the fibrotic remodeling of the MV and, in doing so, identified nearly ten putative therapeutic targets for exploration in human disease. Future investigation will involve more definitive identification of specific cell population(s) that drive disease progression and their individual developmental origins.Item Epigenetic Mechanisms in Lung Cancer(2018-09) Seiler, ChristopherEpigenetic control of gene expression involves covalent reversible modifications of DNA, RNA, and histones which lead to changes in chromatin structure and accessibility. The ability to maintain precise control over gene expression in cells and tissues is critical for ensuring normal cellular development and homeostasis. The most important epigenetic mark of DNA is methylation of cytosine at the C5 position (MeC). This stable epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained through cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases oxidize 5-methylcytosine (MeC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. A precise balance of DNA methylation and demethylation is important for establishing tissue specific gene expression patterns, maintaining cell identity, and guiding development. However, inflammation and exposure to exogenous agents can lead to changes in DNA methylation patterns or “epimutations” which together with genetic mutations can lead to the development of cancer. Chapter I of this Thesis provides an overview of the major mechanisms of epigenetic regulation including epigenetic marks of DNA, non-coding RNAs, and histone modifications. Chapter I then describes epigenetic dysregulation in cancer and other human diseases. We then go on to describe how epigenetic changes in DNA can be detected and quantified using antibodies and mass spectrometry-based approaches. After considering global quantitation of epigenetic DNA modifications, we discuss the methods available for mapping epigenetic modifications along the genome. In Chapter II of this thesis, the effects of C5-cytosine substituents with increased steric bulk on catalytic activity of maintenance DNA methyltransferase (DNMT1) were examined. This protein specifically recognizes 5-methylcytosine (MeC) bases at hemimethylated CG sites in DNA and conducts maintenance methylation. Maintenance methyltransferase activity was the highest towards DNA containing the natural DNMMT1 substrate, MeC. The enzyme was capable of performing maintenance methylation when 5-ethyl-dC was the substrate, while the more rigid and bulky C5-alkyl substituents such as 5-vinyl- dC, and 5-propyl-dC could not direct maintenance methylation. In Chapter III, we investigated the kinetics of maintenance DNA methylation towards DNA duplexes containing oxidized forms of MeC (hmC, fC, and caC). We also employed a molecular dynamics simulation of the enzyme with the DNA to understand the interactions of oxidized forms of MeC with the DNMT1 enzyme. We found that methyl transfer rates were reduced when MeC was oxidized to hmC, fC, and caC, consistent with the model that Tet mediated oxidation contributes to passive DNA demethylation. In Chapters IV and V, we investigated inflammation-mediated epigenetic changes in the lung using A/J mouse model of smoking induced lung cancer. In collaboration with NuGEN (Santa Carlos, CA), we developed a novel reduced representation bisulfite sequencing (RRBS) methodology to map both MeC and hmC genome-wide. Our studies provide evidence that inflammation of the lung induces both global and site-specific epigenetic changes in DNA methylation and hydroxymethylation, alters global histone acetylation, and deregulates gene expression. These studies also provide evidence that exposure to cigarette smoke can alter site-specific DNA methylation and hydroxymethylation of genes that are associated with the cancer phenotype. The final chapter of this dissertation (Chapter VI) employs affinity proteomics to identify protein readers of epigenetic marks of DNA in the lung. DNA duplexes functionalized with C, MeC, hmC, fC, and caC were attached to solid support and incubated with nuclear protein extracts from human bronchial epithelial cells (HBEC). Proteins specifically recognizing DNA epigenetic marks were identified using Orbitrap Velos mass spectrometer and quantified using 8-plex TMT tags. This chapter details the development of a method for carrying out the affinity proteomics experiments, including solid phase synthesis of DNA targets, peptide tagging, sample clean-up, fractionation, and nanoHPLC-ESI+-MS2 based methodology for protein identification and quantification. Overall, during the course of the studies described in this thesis, we have investigated the specificity and kinetics of human maintenance DNA methyltransferase (DNMT1), employed animal models to characterize epigenetic changes in the lung caused by inflammation and exposure to cigarette smoke, and examined novel mechanisms of epigenetic regulation at oxidized forms of MeC. Overall, this work contributes to current understanding of epigenetic regulation in normal cells and epigenetic deregulation in cancer.Item Exploration of Platelet and Mast Cell Communication: A Study of Membrane Lipids, Asthma, and Inflammation(2016-05) Gruba, SarahThis work examines how environmental factors such as lipid membrane concentration, opioid agonist exposure, and inflammatory diseases impact cell communication. It explores the use of different cell models, specifically platelets and mast cells, to understand how disease states can impact cellular function. Throughout the thesis, a variety of analytical techniques including electrochemistry, mass spectrometry, dark field imaging, and microfluidics, are used to understand exocytosis, lipid concentration, manufactured inflammatory mediators, adhesion, and shape change in platelets and mast cells. Platelets are cell-like bodies that travel through the bloodstream and are known for their role in hemostasis and diseases like stroke and myocardial infarction. They have also been implicated in inflammatory diseases such as asthma. In addition, their anucleate nature and easy isolation make them an ideal model for studying variations in cell communication upon the modification of their lipid content. Platelets communicate through the exocytosis of their three distinct granule types (δ, α, and lysosome). These granules contain molecules that assist in the transmigration of immune cells to the site of activation and help with additional platelet aggregation and adhesion. In contrast, mast cells are found throughout the body in connective tissue and are one of the immune system’s first lines of defense. They are primarily known for their role in allergies and asthma. Upon detection of antigens that they are sensitized to, the mast cell secretes manufactured chemokines and pre-formed granule mediators, including histamine and serotonin, calling other inflammatory cells to the site of infection. Chapter One reviews single cell analysis techniques with a particular emphasis on the techniques used in this thesis, including electrochemistry and mass spectrometry. Chapter Two through four are focused on understanding how variations in membrane lipids and structure affect platelet function and exocytosis in general. Chapter Two focuses on understanding the variations that the fusion pore undergoes when granules are being exocytosed. Traditionally, a granule release event, monitored using carbon-fiber microelectrode amperometry, has a quick rise in current (spike) and gradual decay. The variations to this spike are classified as different forms of pre- and post-spike features and non-traditional granule secretion events. The role of cholesterol in changing the frequency and duration of these features is also discussed. Chapter Three discusses the role of phosphatidylserine (PS) in cellular communication using a platelet model. In this chapter, we explore how the stereochemistry of the head group and concentration of PS affects various platelet functions including granular content secretion, manufactured lipid release, and adhesion. The cholesterol level change upon addition of PS is also monitored. Finally, Chapter Four aims to understand how natural lipid variations affect cell function by comparing platelets from different species. This chapter highlights the importance of understanding your cell model relative to the actual cells involved in the disease or function being studied. Chapter Five and Six progresses from lipid function into developing a better understanding of how platelets respond to their environment, particularly in the context of inflammatory diseases. Chapter Five’s focus is on platelet response to opioids like those that are used in the treatment of pain due to inflammatory diseases, cancer, or surgery. Specifically, the effects on cell exocytosis as well as the presence of and role that opioid receptors play in platelets are characterized. Chapter Six focuses on studying how platelets respond to allergic asthma, including response to allergens and the chemoattractants (CXCL10 and CCL5) released during an asthma attack. Using bulk and single cell methods in conjunction allows us to obtain in-depth information on both the overall response and the granule fusion pore during exocytosis. Chapter Seven and Eight focus on mouse peritoneal mast cell (MPMC) function in the context of inflammatory diseases including allergic asthma and neurogenic inflammation, respectively. Chapter Seven aims to state the importance of understanding the cell line you are using since variations in response to allergens are noted between commonly used mast cell models (rat basophilic leukocytes cell line and primary culture MPMC). In addition, MPMC response to CXCL10 and CCL5 was monitored. Finally, Chapter Eight explores the role of MPMC in neurogenic inflammation, a process wherein neurons release the neuropeptides substance P and calcitonin gene-related peptide. Mast cell response to these neuropeptides has been highly disputed, and this chapter focuses on the impact of IgE on MPMC bulk granular content secretion. It also aims to understand how these neuropeptides affect the fusion pore opening and closing during exocytosis.Item Ffar4 regulates cardiac oxylipin balance to promote inflammation resolution in HFpEF secondary to metabolic syndrome(2023-02) Zhang, NaixinHeart failure with preserved ejection fraction (HFpEF) is a complex clinicalsyndrome, but a predominant subset of HFpEF patients has metabolic syndrome (MetS). Mechanistically, systemic, non-resolving inflammation associated with MetS might drive HFpEF remodeling. Free fatty acid receptor 4 (FFAR4) is a GPCR for long-chain fatty acids that attenuates metabolic dysfunction and resolves inflammation. Therefore, we hypothesized that FFAR4 would attenuate remodeling in HFpEF secondary to MetS (HFpEF-MetS). To test this hypothesis, mice with systemic deletion of FFAR4 (FFAR4KO) were fed a high-fat/high-sucrose diet with LNAME in their water to induce HFpEF-MetS. In male FFAR4KO mice, this HFpEFMetS diet induced similar metabolic deficits, but worsened diastolic function and microvascular rarefaction relative to wild-type (WT) mice. Conversely, in female FFAR4KO mice, the diet produced greater obesity but no worsened ventricular remodeling relative to WT mice. In FFAR4KO males, MetS altered the balance of inflammatory oxylipins systemically in HDL and in the heart, decreasing the eicosapentaenoic acid-derived, pro-resolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE), while increasing the arachadonic acid-derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE). This increased 12-HETE/18- HEPE ratio reflected a more proinflammatory state both systemically and in the heart in male FFAR4KO mice, and was associated with increased macrophage numbers in the heart, which in turn correlated with worsened ventricular remodeling. In summary, our data suggest that FFAR4 controls the proinflammatory/pro-resolving oxylipin balance systemically and in the heart to resolve inflammation and attenuate HFpEF remodeling.Item Lipocalin 2 Deficiency Influences Transforming Growth Factor-Beta Effect On Inflammation And Extracellular Matrix Remodeling In Inguinal Adipocytes(2015-09) Pfarr, AmbriaChronic low-grade inflammation present in hypertrophic obesity has the ability to cause remodeling of the adipose tissue due to the increased presence of macrophages and pro-inflammatory cytokines. Transforming Growth Factor-β (TGF- β) is a cytokine released from macrophages that is increased in obesity and plays a major role in extracellular matrix (ECM) remodeling of tissues. Lipocalin 2 (Lcn2), a cytokine expressed in adipose tissue, is related to inflammation and ECM remodeling. Due to the qualities that both adipokines possess the role in extracellular matrix remodeling and inflammation, we looked into the TGF- β effect on the regulation of ECM and inflammatory cytokines in Lcn2 deficient adipocytes to acquire more information about the function of Lcn2 under inflammatory stimuli and the association with metabolic diseases. Therefore, we performed experiments to address how Lcn2 knock out (KO) influences the response of adipocytes to TGF- β in the production of proinflammatory and anti-inflammatory cytokines and ECM proteins. Our results demonstrate that TGF- β down-regulates Lcn2 expression at both the mRNA and protein levels in inguinal adipocytes. Lcn2 KO adipocytes have lower levels of TGF- β expression, but normal levels of p70S6K phosphorylation and normal response to TGF- β stimulation in mammalian target of rapamycin complex 1 (mTORC1) signaling activation. However, the inhibitory effect of rapamycin on TGF- β expression is attenuated in Lcn2 KO adipocytes. Moreover, Lcn2 KO impairs the rapamycin potentiation of TGF- β effect on the expression of ECM proteins, but shows little effect on dysregulation of cytokines.Item Understanding The Regulation Of Adipogenesis And Adipocyte Metabolism In Obesity(2015-08) Zhao, MingInflammation in hypertrophic adipose tissue is a critical inducer of adipose tissue dysfunction, which ultimately leads to systemic insulin resistance and type 2 diabetes. Elevation of lipopolysaccharide (LPS) induced by high fat diet has been recently proposed to be one of the potential mechanisms contributing to the development of inflammation and metabolic dysfunction of adipose tissue in obesity. Therefore, it is of interest to expand our understanding of LPS effect on adipocyte metabolism and to identify the molecular mechanism by which LPS deteriorates adipose tissue metabolism. In the first study, we investigated the effect of LPS on the adipogenic capacity and cellular senescence of adipocyte progenitors by using stromal-vascular (SV) cells isolated from inguinal adipose tissue of C57BL/6 mice. We found that LPS treatment for 24-hour prior to the induction of differentiation led to the inhibition of adipogenesis. In addition, LPS induced features of premature senescence of SV cells. Further studies showed that LPS treatment caused a reduction in Zfp423 and PPARγ expression in SV cells, suggesting that LPS impairs pre-adipocyte differentiation. In the second study, we explored the role of endosomal/lysosomal protein NPC2 (Niemann-Pick disease, Type C2) in regulating lysosomal activity and in mediating LPS effect on adipocyte inflammation and function. NPC2 knockdown reduced lysosomal protease cathepsin B levels and impaired autophagy-lysosomal activity in 3T3-L1 adipocytes. Interestingly, NPC2 knockdown diminished LPS effect on inflammatory response and blunted LPS-induced glucose uptake in adipocytes. In the third study, we determined the effect of eicosapentaenoic acid (EPA) on promoting metabolic health of adipocytes, specifically the browning of subcutaneous white adipocytes. When added to SV cell cultures during 8-day adipocyte differentiation, EPA significantly increased the expression of thermogenic genes as well as mitochondrial DNA content. These results indicate that EPA enhances energy expenditure capacity by recruiting beige adipocytes. In summary, we have demonstrated a new mechanism by which LPS disrupts adipogenesis and adipocyte metabolism. We have also characterized the role of NPC2 as an important molecular mediator of LPS-induced adipocyte inflammation and the effect of EPA on promoting the browning of subcutaneous adipocytes.Item Understanding The Role Of Pentraxin 3 In Adipose Tissue Inflammation And Aging(2022-01) Lin, Te-YuehObesity and aging are often accompanied by chronic low-grade inflammation in adipose tissue. Metabolic endotoxemia (elevated circulating lipopolysaccharide [LPS] level) is the inducer of systemic and adipose tissue inflammation and dysfunction, which is developed during obesity and aging. As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays a vital role in innate immunity and can be induced by inflammatory stimuli in adipose tissue and adipocyte. Altered PTX3 levels have been observed in adipose tissue and blood during obesity and aging, while the role of PTX3 in adipose tissue during inflammation and aging is not fully understood. My Ph.D. research attempts to understand the role of PTX3 in adipose tissue inflammation and aging. In the first project, we investigated how PTX3 regulates inflammation in adipose tissue, and we found that PTX3 plays an anti-inflammatory role partially by regulating miR-21 expression and secretion in brown adipocytes. In the second project, we explored PTX3 secretion from adipose tissue and adipocyte and found that PTX3 is secreted mainly through conventional protein secretion, while a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes. In the third project, we examined the physiological role of PTX3 in adipose tissue during aging, and we showed that PTX3 deficiency induced senescence and inflammation in adipose tissue and promoted lipid transport and oxidation in white adipose tissue of old female mice. In summary, my doctoral research reveals that adipose-derived PTX3 plays an essential role in regulating LPS-stimulated inflammation and cellular senescence during aging, and adipocyte-derived PTX3 is secreted via conventional protein secretion and exosomes.