Indiana University-Purdue University Indianapolis (IUPUI) / Sensing, integrating, and relaying signals from the environment through proteins, metabolites, and lipids to the lung are critical for proper development. Moonlighting proteins, such as AIMP1, are a unique subset that serves at least two independent physiological functions. Encoded by gene AIMP1, AIMP1 has two known functions: (1) C-terminus EMAP II domain of full-length AIMP1 can be secreted out of the cell to chemoattract myeloid cells; (2) intracellular full-length protein interacts with tRNA synthetases in protein translation. However, despite the linkage of protein expression levels of with several lung pathologies such as bronchopulmonary dysplasia (BPD), effectively targeting the protein encoded by AIMP1 has been a challenge due to poorly understood mechanisms. This thesis explores physiological, signaling, and immunological moonlighting mechanisms of first, the extracellular EMAP II then the intracellular AIMP1. Experiments utilize both in vitro and in vivo models, including a murine model of BPD and Cre-mediated exon-deletion knockout. Experimental results provide evidence that in the BPD model, EMAP II levels are elevated and sustained – first in bronchial epithelial cells then in macrophages. Mice exposed to sustained and elevated EMAP II protein levels resemble the BPD phenotype while neutralization partially rescued the phenotype, implying EMAP II as a potential therapeutic target against BPD. Results from studies exploring EMAP II’s signaling mechanism identify transient stimulation of JAK-STAT3 phosphorylation, commonly found in inflammation-resolving macrophages. In contrast, it induces unique transcriptional changes that are reversible both by JAK-STAT inhibitor and siRNA-mediated knockdown of Stat3. Studies using AIMP1 knockout mouse reveal a novel function for the intracellular AIMP1. AIMP1 knockout mice exhibited neonatal lethality with a respiratory distress phenotype, decreased type I alveolar cell expression, and disorganized bronchial epithelium, suggesting a role in lung maturation. In vitro experiments suggest that a portion of AIMP1 residing in the cell’s membrane interacts with various phosphatidylinositols and contributes toward F-actin deposition and assembly. Data from these experimental studies provide insight into how the various functions of the promiscuous AIMP1 gene affect lung development. These studies exemplify not only characterize novel moonlighting mechanisms of AIMP1, but also highlight the importance of characterizing moonlighting proteins to promote therapeutic preventions. / 2020-02-21

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes is a morbid metabolic disorder that affects almost 500 million people worldwide. Although multiple factors contribute to diabetes pathogenesis, pancreatic islet inflammation and dysfunction are shared characteristics of its major forms. 12- lipoxygenase (12-LOX), an enzyme involved in lipid metabolism, has been implicated in islet inflammation. 12-LOX generates reactive oxygen species (ROS) that activate inflammation and serve as major contributors to islet dysfunction. Importantly, since ROS are transient moieties, they are challenging to study in vivo. Hence, establishing better animal models of ROS-mediated stress is critical to facilitate the discovery and preclinical testing of novel diabetes therapeutics. Here, I have adapted a zebrafish model of conditional β-cell injury, which is regulated by the administration of the prodrug metronidazole (MTZ), to study responses to ROS in vivo. I demonstrate that with MTZ treatment, ROS are generated within β-cells and subsequently exhibit recruitment of macrophages into the islet and induction of β-cell death. I utilized this model to uncover roles for macrophages and 12-LOX during islet injury. Excessive macrophage infiltration exacerbates islet inflammation and dysfunction. Interestingly, on the depletion of macrophages in zebrafish, I observed that β-cells recovered normal function upon cessation of prodrug treatment. This suggests that infiltrating macrophages promote maladaptive inflammation and premature removal of damaged β-cells. Thus, limiting the macrophage infiltration may be a therapeutic approach to restoring β-cell function. Based on the established roles of 12-LOX in other contexts, I hypothesized that its inhibition would prevent the localized infiltration of proinflammatory macrophages. To test this, I used both zebrafish and mouse models and observed a significant reduction in macrophage migration upon loss of 12- LOX activity. Furthermore, I found that expression of CXCR3, a crucial receptor regulating migration, was significantly reduced in 12-LOX loss-of-function macrophages. These data suggest a role for 12-LOX in macrophages, which is conserved across species. Collectively, my study reveals novel roles for 12-LOX in macrophage function and provides testable therapeutic targets for the resolution of inflammation-induced damage in the pancreatic islets. / 2020-11-19

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19629号 / 医科博第67号 / 新制||医科||5(附属図書館) / 32665 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 椛島 健治, 教授 岩井 一宏, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20799号 / 医博第4299号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 武藤 学, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21418号 / 医博第4408号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 髙橋 良輔, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of abnormal liver function in countries with western-style high fat, high cholesterol diets. Liver damage associated with NAFLD may lead to liver cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC). Additionally, recent data suggest that nonalcoholic steatohepatitis (NASH), the inflammatory phase of NAFLD, is linked to increased cardiovascular risk independent of the broad spectrum of risk factors of metabolic syndrome. Therefore, novel therapies are needed to inhibit the inflammatory liver damage that drives NAFLD. Hepatic macrophages (HMΦ’s), which include resident Kupffer cells and monocyte-derived macrophages, are the primary drivers of liver inflammation in both human and mouse models. In macrophages, chronic lipid exposure promotes pro-inflammatory polarization and the activation of pyroptosis via the NLRP3 inflammasome. While the role of the canonical pyroptosis pathway has been studied in NAFLD, the role of the newly discovered noncanonical (caspase-11/-Gasdermin-Ddependent) pathway has not been defined. Diet-induced NAFLD promoted hepatic steatosis and lobular inflammation in male WT mice. Caspase-11 deficiency decreases macrovesicular steatosis and total NAFLD Activity Score (NAS). High fat feeding promoted recruitment and activation of HMΦ in both Caspase-11 deficient (Casp11KO) and WT male mice, however, noncanonical pyroptosis (caspase-11 activity, surface Gasdermin-D, expression, liver IL-1β secretion) was ablated in HMΦs from Casp11KO mice. Bone marrow transplantation restored capacity for noncanonical pyroptosis in Casp11KO mice. RNAseq and microarray analysis revealed that lipid peroxidation and trained immunity mediate noncanonical pyroptosis in diet induced NALFD. / Biomedical Sciences / Accompanied by 1 PDF file: DrummerIV_temple_0225E_171/CED

Rationale. Cigarette smoking is a well-known risk factor for cardiovascular disease, including arterial diseases such as atherosclerosis and abdominal aortic aneurysm. However, our understanding of how exposure to cigarette smoke impacts arterial disease pathogenesis is not well known. Consequently, this doctoral thesis focuses on understanding the development of atherosclerosis and aortic aneurysm in the context of exposure to cigarette smoke. In particular, since monocytes and macrophage are key immune cells implicated in arterial pathology, this work concentrates on understanding the impact of cigarette smoke exposure on the function and kinetics of monocytes and arterial macrophages. Main Findings. Using a mouse model that combines two clinically relevant risk factors, hyperlipidemia and cigarette smoke, we showed that smoke exposure increases atherosclerosis and induces the spontaneous formation, progression, and rupture of abdominal aneurysms. We also provide experimental evidence that atherosclerosis strongly associates with regions of elastin damage and arterial dilation, suggesting atherogenesis may directly contribute to abdominal aneurysm formation. Given the importance of macrophages in arterial disease, we investigated arterial macrophage heterogeneity and function following exposure to cigarette smoke. We report that cigarette smoke exposure increased the abundance of arterial monocytes and macrophages, whereas heterogeneity was primarily driven by hypercholesterolemia in aneurysmal tissue. Specifically, hypercholesterolemia is associated with an increase in macrophage populations with putative functions in inflammation and tissue remodelling including Trem2 foamy macrophages, inflammatory macrophages, and interferon-inducible macrophages. Moreover, we demonstrated that arterial macrophages play a critical role in elastin fragmentation within the arterial wall of smoke exposed mice. Finally, we investigated the impact of cigarette smoke on kinetic factors that can contribute to arterial macrophage accumulation. We found that, despite increased development of arterial disease, exposure to cigarette smoke is associated with an overall suppression of circulating monocytes and pro-inflammatory cytokines. Using a parabiosis model, we show monocyte recruitment is significantly increased and is likely a key factor contributing to accumulation of arterial macrophages following exposure to cigarette smoke. We also present evidence suggesting that endothelial dysfunction, related to a loss of endothelial nitric oxide synthase, contributes to increased arterial monocyte recruitment following exposure to cigarette smoke. Conclusions and Significance. Overall, we provide evidence that atherosclerosis likely contributes to abdominal aneurysm pathology in a model of cigarette smoke-induced aneurysm formation. We further provide insight into how tobacco smoke promotes arterial disease development through increased local accumulation of arterial macrophages despite suppressed monopoiesis and systemic inflammation. We identify monocyte recruitment and endothelial dysfunction as key factors contributing to the increased accumulation of arterial macrophages, with no overall differences in macrophage heterogeneity, following smoke exposure. In addition to providing insight into the increased risk of arterial disease following exposure to cigarette smoke, this study also provides experimental evidence that atherogenesis can contribute to abdominal aneurysm pathology. Overall, this thesis furthers our understanding of arterial disease pathogenesis and can provide a foundation for further mechanistic or therapeutic focused research aimed at reducing the burden of cardiovascular disease. / Thesis / Doctor of Philosophy (PhD) / Diseases that affect the heart and major blood vessels are one of the leading causes of illness and death worldwide. Atherosclerosis is one such disease caused by the buildup of fatty deposits in the walls of major blood vessels called arteries. This buildup can eventually block the artery and lead to a heart attack or stroke. Abdominal aortic aneurysms are another type of disease that affects arteries. In this case, the walls of the artery grow weak and begin to balloon out until the artery eventually breaks causing severe internal bleeding and death. One of the most important cells involved in the development of atherosclerosis and aneurysms is the macrophage, a type of white blood cell that is an important part of the immune system and found in diseased arteries. Although we know that cigarette smoking is one of the most significant risk factors for developing atherosclerosis and abdominal aneurysms, we do not fully understand why. Therefore, the goal of this thesis project was to investigate how cigarette smoke affects the development of arterial disease with a focus on understanding how it impacts the movement and function of macrophages. Using a mouse model, we found that the development of atherosclerosis and aneurysm are likely related, and also identified ways that exposure to cigarette smoke increases the numbers of macrophages in arteries. This work advances our understanding of how arterial diseases may be related and provides insight into how smoking can increase the risk of developing arterial disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality, with the potential to afflict as many as half of the 1.1 billion smokers in the world. The inflammatory response to cigarette smoke is believed to mediate the progressive and irreversible loss of lung function that characterizes COPD. The greatest burden of the disease arises from episodes of worsened symptoms and inflammation, usually triggered by microbial infection. Currently, the mechanisms that drive cigarette smoke-induced inflammation are being elucidated but ambiguity remains about this response and the inflammatory response engaged in a smoke-exposed lung experiencing a microbial infection. This thesis sought to investigate inflammatory mediators induced by cigarette smoke and those induced by bacteria, the most common cause of infectious exacerbations of COPD, in the context of smoke exposure. In chapter two we investigated the role of Breast Regression Protein-39 (BRP-39), a gene commonly observed to be increased under inflammatory conditions, in the inflammatory response to cigarette smoke. In order to determine the mechanisms of BRP-39 induction, its expression and inflammation was assessed in IL-13, IL-18, and IL-1R1 deficient mice. BRP-39 was found to be redundant in cigarette smoke-induced inflammation, but these data confirmed that IL-1R1 was a crucial mediator of this response. After examining the inflammatory response elicited by smoke alone, we investigated the importance of IL-1 signaling in a model of bacterial exacerbation of cigarette smoke-induced inflammation. We found that the exacerbated neutrophilia that typifies the response of a smoke-exposed lung to bacteria was dependent on IL-1α-mediated production of the CXCR2 ligand CXCL5. This study identified the unique phenomenon that cigarette smoke primes alveolar macrophages to produce excessive amounts of IL-1α in response to bacterial stimuli. The purpose of the final study of the thesis was to more comprehensively characterize the extent to which cigarette smoke changes the phenotype of macrophages. Examining total gene expression by microarray found that smoke-exposed alveolar macrophages were in a proliferative state expressing a unique profile of inflammatory mediators. Further analysis revealed that this was likely the result of a pulmonary environment rich in growth factors. Taken together, these data provide detail to the understanding of the biological process of inflammation that drives the pathogenesis of COPD. These studies identify a phenomenon that predisposes smokers to experience more severe responses to bacteria and reinforces the targeting of IL-1 signaling in the treatment of COPD. / Thesis / Doctor of Philosophy (Medical Science)

Caffeine is one of the most widely used neurostimulants in the world and there is considerable debate on its effect in immune cells. One of its main targets is proposed to be adenosine receptors which mediate an anti-inflammatory switch in activated immune cells while another target is phosphodiesterase where it acts as an inhibitor. In macrophages, caffeine has been shown to cause both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. If the primary effect of caffeine on macrophages were to antagonize adenosine receptors we would expect cells exposed to caffeine to have a prolonged M1 response. However, we show that caffeine suppresses phagocytosis at physiological concentrations (an indicator of M2 phenotype). This suppression was reversed when macrophages were pretreated with protein kinase A inhibitor, suggesting that at physiological concentrations caffeine's phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an M2 fate. However, mRNA expression profiling suggests that caffeine can modulate A2A receptor expression and suppress MKP-1 expression, a hallmark of M1 macrophages. Caffeine is, therefore an immunomodulator that can suppress or prolong inflammatory responses in macrophages, which may account for the abundance of contradicting evidence in the literature. Additionally, these effects are complicated by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in macrophage activation.

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