Browsing by Subject "macrophage"

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    Defining Mechanisms of Inflammation and Fibrosis In Cardiac Valve Disease
    (2019-05) Meier, Lee
    Cardiovascular 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.
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    The functional impact of Retinoic acid and RIP140 in macrophages: from chromatin to physiology
    (2016-08) Lee, Bomi
    Innate immunity consists of two systems; humoral and cellular systems. The humoral system includes anti-microbial peptides and opsonins while the cellular system involves specialized cells including phagocytes. The major functions of phagocytes are scavenging toxic compounds and producing inflammatory mediators to destroy infectious organisms and to transfer signals to other immune cells. Dysregulation of the phagocytic system by various conditions including the defect of immune cells and insufficient nutrition can lead to inflammatory diseases. Therefore, understanding the characteristics of phagocytes and the molecular mechanism in response to extracellular stimulations is critical for the development of therapeutics for inflammatory diseases. The focus of my thesis was to evaluate cellular signaling and its pathophysiological relationship in one type of phagocytes, macrophages. All-trans retinoic acid (RA) and its derivatives have been proved as potent therapeutics for inflammatory diseases, but the molecular mechanism of RA action in macrophages was not well established. In order to shed light on the functional role of RA in macrophages, I first found that topical application of RA significantly improved wound healing and the co-stimulation with RA and IL-4 synergistically activated Arginase-1 (Arg1), a critical gene for tissue repair, in macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to remodel the +1 nucleosome of Arg1 for transcription initiation and to facilitate transcriptional initiation-elongation coupling by recruiting elongation factor TFIIS. This study demonstrated RA’s modulatory activity in IL-4-induced anti-inflammatory macrophages, which involves synergistic activation of Arg1 by RA and IL-4 and a functional role of Med25 in chromatin remodeling of this gene promoter. In macrophage activation, there are two well-established phenotypes; classically activated (M1, pro-inflammatory) and alternatively activated (M2, anti-inflammatory) macrophages. The switch from M1 to M2 is critical for the control of inflammatory responses including wound repair. Previously, it has been found that Receptor-interacting Protein 140 (RIP140) is an enhancer of M1 by acting as a co-activator for NF-κB. Related to this, my research has uncovered that RIP140 delays the wound healing process by suppressing the macrophage phenotype switch from M1 to M2. With regards to mechanism, IL-4 treatment stimulates RIP140 export from the nucleus to the cytoplasm to form a complex with calpain regulatory subunit (CAPNS1) to activate calpain1/2 that enhances the activity of PTP1B, a negative regulator for STAT6 in M2 macrophages. Together, these results have established a new modulatory role of RIP140 in macrophage phenotype switch during wound healing by regulating both M1 and M2 activations (enhancing M1 and suppressing M2 activation). Another new finding I discovered about RIP140 was its repressive effect on osteoclast (OC) differentiation. OCs are derived from monocyte/macrophage lineage of hematopoietic cells and are responsible for bone resorptive activity. OCs maintain a balance in bone remodeling with osteoblasts (OB) that are involved in bone formation. RIP140 forms a complex with orphan nuclear receptor TR4 in pre-osteoclastic cells to suppress OC differentiation. Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) induces RIP140 protein degradation and represses TR4 mRNA level, which terminates the repressive activity of TR4/RIP140 complex in OC differentiation. In vivo micro CT analysis of macrophage/monocyte-specific RIP140 KD (mϕRIP140KD) mice showed an osteopenia phenotype with reduced OB function and increased OC activity, indicating uncoupling between OC and OB. This study demonstrated RIP140’s additional role in OC differentiation and bone diseases such as osteoporosis. Taken all together, these studies have established fine-tuning molecular mechanisms in macrophages, including their phenotypic switch and polarization/maturation. Specifically, we uncovered additional pathways of signal inputs and stimuli that regulate these processes such as RA, IL-4 and RANKL, and determined their physiological relevance in wound healing, inflammation and osteoclastogenesis. Differential activation of macrophages by these biological cues further confirms the plastic nature of macrophages. These findings contribute to our understanding of signaling mechanisms in macrophage polarization and their impact on diseases.
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    Melanoma displays an evolutionarily conserved resistance to modulation of phagocytic signals
    (2017-08) Anderson, Katie
    Therapeutic activation of macrophage phagocytosis has the ability to restrain tumor growth through phagocytic clearance of tumor cells and activation of the adaptive immune response. The objective of this thesis was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. We observed that melanoma cells from mice, humans, and dogs displayed an unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the “don’t eat me” signal CD47 or by chemotherapeutic enhancement of known “eat me” signals. In addition, combination doxorubicin chemotherapy and CD47 blockade did not consistently promote an anti-tumor adaptive immune response. Phagocytosis of melanoma cells was not enhanced by inhibition of secretory pathways, and phagocytosis of sensitive lymphoma tumor cells was not impaired in the presence of melanoma culture supernatants, indicating that soluble factors did not mediate phagocytosis resistance. siRNA mediated knockdown of 47 candidate “don’t eat me” signals similarly did not enhance melanoma cell phagocytosis, suggesting that these proteins do not disable macrophage phagocytosis. We conclude melanoma cells possess a mechanism of resistance to phagocytosis. Further investigation will be needed to define this mechanism and to develop strategies to overcome melanoma cell resistance to the innate immune response.
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    Regulation of the kinases LynA and LynB and function in autoimmune disease
    (2021-04) Brian, Ben
    Cell-surface receptors on immune cells direct immune-cell function by sensing and responding to signs of pathogens and tissue damage. Signaling initiated by immunoreceptors is responsible for essential aspects of the immune response, including phagocytosis by myeloid cells, and antigen sensing by B and T cells. Efficient regulation of immunoreceptor signaling ensures that inflammation arising from pathogen clearance is limited in order to prevent tissue damage. Autoimmune diseases, such as systemic lupus erythematosus, can occur when these signaling pathways are improperly regulated. The tyrosine kinase Lyn is an important regulator of immune function due to its unique ability to both initiate signaling that can generate inflammation, and also recruit and activate proteins that dampen cellular activation. Alterations to Lyn expression and signaling in both human and mice can worsen or cause autoimmune disease. Understanding how Lyn is regulated and balances these roles is necessary to develop therapies that selectively limit autoinflammation but preserve pathogen clearance. Alternative splicing of the lyn gene produces two proteins, LynA and LynB, that differ by the presence of a 21-amino-acid insert present in LynA and absent in LynB. Here, we demonstrate the LynA and LynB are differentially regulated in immune cells. Phosphorylation of LynA at Tyrosine 32 in its unique region causes LynA to be rapidly, and selectively, poly-ubiquitinated by an E3 ligase, c-Cbl, and degraded. We show that differential expression of c-Cbl in macrophages and mast cells controls LynA protein levels, degradation, and signaling responses following Src-family kinase activation. Furthermore, we created novel knockout mice to study the roles LynA and LynB play in regulating autoimmune disease. Using these novel mice, we show that LynB prevents the development of splenomegaly and autoimmunity by limiting myeloid cell expansion and B cell activation. We also demonstrate that LynB-deficient mice have elevated responses to Toll-like receptor activation. Together, these results indicate that LynA and LynB are differentially regulated and have unique roles in the immune response. Therefore, understanding LynA and LynB signaling and regulation could yield targets that limit inflammation but preserve normal immune function.