Cigarette smoke contributes to 90% of lung cancer cases and 80% of COPD cases. These concerns loom large as lung cancer represents 13% of all cancer deaths and estimates report by 2020 COPD will be the third leading cause of death in the world. The master regulator of the ER stress response, IRE-1, in the context of cigarette smoke exposure lacks study. Interestingly, its downstream pathways are activated. In fact, the 2014 Surgeon General’s report on the health consequences of smoking highlighted the endoplasmic reticulum (ER) stress response as a potential mechanism leading to the development of lung cancer and Chronic Obstructive Pulmonary Disorder (COPD). Following acute cigarette smoke exposure, mouse lung homogenates exhibited increased levels of XBP-1 along with downstream mediators of IRE-1 activation— GRP-78 and CHOP. Specifically observing macrophages, an important immune cell and source of acute inflammation, cigarette smoke induced activation of IRE-1/XBP-1 pathway through splicing of XBP-1 mRNA. However, upon assaying for pro-inflammatory cytokines we were unable to determine that cigarette smoke directly caused inflammation in vitro. Furthermore, cigarette smoke inhibited the activation of M2 macrophages, an anti-inflammatory and tissue healing subset seen through CCL18 inhibition. A majority of M2 and M1 macrophage markers were decreased from IRE-1/XBP-1 inhibition. This suggests a different phenotype than classical M1 or M2 polarization being induced by cigarette smoke. In addition, it suggests the IRE-1/XBP-1 pathway having a robust role in controlling gene expression and balance of cellular proteomics. / Thesis / Master of Science (MSc) / Cigarette smoke exposure damages the lungs and over time places the user at risk for increased infections, progressive decreases in lung function and cancer. A specific cell of the immune system and found in the lungs, macrophages or “Big Eater” cells, responds first by picking up debris and responding to harmful foreign substances by releasing proteins signaling the immune system to become activated. Within all animal cells, an organelle called the Endoplasmic Reticulum (ER) manufactures a third of proteins produced allowing the cell to adapt to foreign substances, including cigarette smoke. Cigarette smoke could cause the ER, a plastic organelle, to change in size and function at a heightened level due to activation of a sensing protein integrated in the ER, Inositol Requiring Enzyme-1 (IRE-1). Both activation of the ER and cigarette smoke causes macrophages to behave as “tissue-healing” or M2 subsets that release factors promoting reconstruction of the lungs; alternatively, M1 macrophages fight diseases and promote further inflammation. Using genetic analysis of macrophages exposed to cigarette smoke in culture dishes and analyzing the proteins secreted, we determined cigarette smoke inhibits M1 macrophages and the “tissue-healing” subset, while increasing adhesion molecule expression. Overall, cigarette smoke affected the polarization of M1 and M2 phenotype, analyzed through proteins and genes expression. We observed an increase in sXBP-1, indicative of IRE-1/XBP-1 pathway activation, from cigarette smoke extract exposure in macrophages. However, the use of IRE-1 inhibitors increased ER stress markers while affecting M1 and M2 markers. This suggests ER compensation from the use of inhibiting one arm of the ER stress response.

Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages. / Thesis / Doctor of Philosophy (PhD) / The immune system is highly dependent on a specialized subset of white blood cells known as macrophages that are capable of clearing damaged and dead cells as well as a wide range of invading micro-organisms. Specific receptor proteins present on the membrane of macrophages are involved in the recognition of particles and subsequent signaling to recruit other immune cells or to promote healing and wound repair. To date, a variety of fluorescence-based microscopy methods have been used to study the dynamics of cell membrane components. The mobility of several membrane receptors in macrophages has been studied using microscopy techniques, which have provided valuable insights into their function. However, there is still insufficient information about the behavior of two key receptors (TLR2 and CD14) that participate in signaling in response to bacterial products. This thesis aims to answer three major questions with regard to receptor mobility (i.e., diffusion) within macrophage membrane: 1) Which type of fluorescence-based microscopy technique is more suitable for measuring the mobility of TLR2 and CD14 receptors on macrophage membranes? 2) What is the impact of different surface topographies on TLR2 diffusion in adhered macrophages, as well as cell shape, and the ability of macrophages to internalize particles? 3) Does aging alter TLR2 mobility in the membrane of macrophages? The following chapters provide detailed answers to these questions. In brief, we have demonstrated that TLR2 and CD14 diffusion measurements in adhered macrophages highly depend on the membrane section chosen. In addition, our results show that micro- and nanostructured surface topographies alter the shape of adhered macrophages and yield higher bacteria internalization, while the diffusion of TLR2 is not changed. When comparing macrophages derived from young and old mice, we find similar diffusion rate of TLR2 in macrophages of the two age groups.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24549号 / 薬科博第166号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Macrophages can be activated by a variety of extracellular signals to polarize to either the M1 (inflammatory and antimicrobial) or to the M2 (wound repair and inflammation resolution) phenotype. Expression of arginase 1 in macrophages is a key marker of the M2 phenotype. Arginase 1 expression is induced by interleukin 4 (IL-4), a cytokine secreted by Th2 helper cells. All-trans retinoic acid (ATRA) is a product of metabolism of dietary retinol (vitamin A). In a manner analogous to hormones, ATRA binds to nuclear receptors in cells and influences gene expression and cell physiology. ATRA is important in the resolution of inflammation systemically and on the cellular level, however it has not been linked to M2 activation or arginase 1 expression. Testing the hypothesis that ATRA can induce arginase 1 in macrophages either directly or indirectly, it was found that ATRA alone cannot cause murine bone marrow-derived macrophages (BMDM) to activate in the M2 phenotype (as indicated by arginase 1 expression), however it can dramatically potentiate induction of arginase 1 expression and activity by IL-4. This is the first observation positively linking ATRA to arginase 1. Lipopolysaccharide (LPS), is a conserved structural component of the outer membrane of Gram negative bacteria, and a potent pyrogen. In metabolic endotoxemia, LPS concentration in the blood is slightly elevated, and over the long term this contributes to diverse inflammatory diseases such as atherosclerosis, obesity, and diabetes. LPS promotes the M1 phenotype and suppresses the M2 phenotype, but its contribution at low doses such as those found in metabolic endotoxemia are not well studied. In order to investigate mechanisms of LPS suppression at low doses, mice deficient in IRAK1 and tollip, key mediators or proinflammatory LPS signaling, were used to study IL-4, ATRA, and LPS crosstalk. LPS suppression of arginase 1 was found to be dependent on IRAK1 and tollip, but only at low doses of LPS. / Ph. D.

Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS. / Ph. D.

Low dose endotoxemia is caused by several health conditions including smoking, alcohol abuse, high fat diets, and aging. Several studies have correlated low dose endotoxemia with increased risks of atherosclerosis, diabetes, and Parkinson's disease. Unlike high doses of endotoxin which induce a strong but transient induction of pro-inflammatory mediators, low doses of endotoxin result in a mild but chronic induction of pro-inflammatory genes. The central hypothesis of our study was that if low doses of endotoxin are capable of inducing mild prolonged inflammation, then a unique signaling circuit must be utilized. In the first study, the molecular mechanisms for the persistent induction of lipocalin 2 (LCN2) in response to 100 ng/mL of lipopolysaccharide (LPS) in kidney fibroblasts was examined. It appears that the intracellular signaling network responsible for the persistent induction of LCN2 requires both activator protein-1 (AP-1) and CCAAT/enhancer binding protein delta (C/ebpδ). Interleukin-1 receptor-associated kinase 1 (IRAK-1) is critical for LCN2 expression. In the second study, the molecular mechanisms governing the persistent induction of interleukin 6 (IL-6) upon a 50 pg/mL challenge of LPS in macrophages was examined. At this dose, only the persistent activation of cJun N-terminal kinase (JNK) and C/ebpδ was observed. IL-6 transcription requires the transient recruitment of activating transcription factor 2 (ATF2) and the persistent recruitment of C/ebpδ to the IL-6 promoter. In the third study, the molecular mechanisms that mediate LPS-induced priming was examined. The results demonstrate that macrophages are able to sense their prior history of exposure to LPS that result in either a priming or tolerance phenotype upon a secondary challenge of LPS. Results suggest that this sensing mechanism involves cross-talk between IRAK-1 and phosphoinositide-3-kinase (PI3K). Collectively, these studies indicate that JNK and C/ebpδ are the primary players responsible for the persistent expression of pro-inflammatory genes during low dose endotoxemia. IRAK-1 is a key intracellular signaling kinase that mediates signaling at low doses of LPS. IRAK-1 is not only critical for low dose induced expression, but also for LPS-induced priming. This research has revealed a novel signaling pathway that could provide new molecular targets for drug development against chronic inflammatory diseases. / Ph. D.

Alterations of immune parameters were investigated in fish exposed to non-overtly toxic levels of three different environmentally relevant chemicals, 7,12-dimethylbenzanthracene (DMBA), hexachlorocyclohexane (lindane), and 2,3,7,8- tetrachlorodibenzo-p -dioxin (TCDD). Each chemical agent was administered to tilapia in separate experiments by intraperitoneal injection for five consecutive days. Following the final dose, total cellularity and histology of the spleen and pronephros were assessed, as were activity of phagocytic celis contained in these hematopoietic organs. <p>Using chemical doses which produced no clinical toxicity, tilapia exposed to each chemical agent displayed a significant reduction in total cell number of both spleen and pronephros, in most cases in a dose-related manner. Consistent with this observation, splenic and pronephric hypocellularity was confirmed upon histological examination of chemical-treated fish. However, neither superoxide radical production or phagocytosis of splenic or pronephric macrophages was inhibited in either DMBA, lindane, or TCDD exposed fish. Results of this study indicate that depressed total cell number in fish hematopoietic organs may be a more sensitive indicator of exposure to these environmental contaminants than is the activity of macrophages contained within these organs. / Master of Science

Ischemic heart disease is a leading cause of death worldwide. Ubiquitin (UB), an evolutionary conserved protein, is found in all eukaryotic cells. Previous work has shown that treatment of mice with exogenous UB (eUB) reduces inflammatory response and preserves heart function 3 days following ischemia/reperfusion injury (I/R). This study investigated the long-term (28 days post-I/R) cardioprotective potential of eUB using a mouse model of myocardial I/R; and tested the hypothesis that eUB modulates phenotype and function of macrophages (key cells involved in inflammation post-I/R) using thioglycolate-elicited mouse peritoneal macrophages. Heart function measured at 3, 7, 14 and 28 days post-I/R using echocardiography showed that eUB improves heart function throughout the observation period, and decreases I/R-mediated increase in left ventricular dilation at 3, 14 and 28 days timepoints. Myocardial fibrosis, hypertrophy and apoptosis were lower in eUB-treated hearts 28 days post-I/R. These changes in the heart associated with decreased expression of fibrosis-related proteins (collagen-1α1 and MMP-2) and hypertrophy-related protein (MYH-7B) in UB-treated hearts. Activation of GSK3β (pro-apoptotic kinase) was lower (vs. Sham), while activation of anti-apoptotic kinases, ERK1/2 (vs. I/R) and Akt (vs. Sham), was higher in eUB-treated hearts 28 days post-I/R. Serum levels of IL-6, IL-2 and G-CSF were lower in I/R+UB vs. I/R group 28 days post-I/R. In peritoneal macrophages, eUB induced cytoskeleton reorganization in M1-polarized (IFNγ treatment for 72 hours; 100U/mL) and M2-polarized (IL-4 treatment for 72 hours; 20ng/mL) cells. eUB decreased secretion of IL-1β and TNFα in M1-polarized macrophages, while it decreased secretion of TNFα, IL-10 and GM-CSF in M2-polarized macrophages. Efferocytosis was lower in eUB-treated M2-polarized macrophages, which was reversed by CXCR4 receptor antagonist (AMD3100). eUB enhanced migration of M1-polarized macrophages, while it decreased the migration of M2-polarized macrophages. AMD3100 negated the effects of eUB on M1-polarized macrophage migration. eUB decreased activation of STAT1 and FAK, while increasing activation of ERK1/2 in M1-polarized macrophages. In M2-polarized macrophages, eUB decreased Akt activation. Thus, UB treatment preserves heart function and decreases adverse cardiac remodeling 28 days post-I/R. In polarized macrophages, eUB reduces secretion of inflammatory cytokines, and alters phenotype and function of M1- and M2-polarized macrophages.

The cellular microenvironment in vivo consists of both mechanical and chemical signals, which drive cell function and fate. These signals include the composition, architecture, and mechanical properties of the extracellular matrix (ECM), signaling molecules secreted by cells into their surroundings, as well as physical interactions between neighboring cells. Cells are able to interact with their surroundings through a number of different mechanisms such as remodeling of the ECM through adhesion, contraction, degradation, and deposition of proteins, as well as the secretion of pro- or anti-inflammatory molecules. In diseased states, where homeostasis has been perturbed, inflammatory signals are secreted which can modify the cellular microenvironment. Diseased states such as cancer and fibrosis are often associated with the excessive production of ECM proteins that subsequently lead to an increase in tissue stiffness and changes to ECM architecture. Such changes to the mechanical properties of the cellular microenvironment affect the cytoskeletal arrangement, migration and adhesion of both the parenchymal cells, as well as immune response cells, which migrate to the sites of injury. Further understanding of the inflammatory responses and their relationships to tissue stiffness and ECM architecture could aid in the development of novel strategies to predict diseases as well as to target and monitor therapies. Since inflammation and mechanical properties of the affected tissue are closely interlinked, obtaining a detailed understanding of the interplay between the properties of the microenvironment and the cells that reside within it will be very beneficial to obtain physiologically relevant information. We have investigated the combinatorial effects of matrix stiffness, and architecture in the presence of co-cultures of cells to determine the overall effect on cellular responses and phenotypes. We have conducted studies on co-cultures of cells in 2D and 3D environments to identify how cellular behavior is affected by dimensionality. / PHD / The cellular microenvironment in vivo consists of both mechanical and chemical signals, which drive cell function and fate. These signals include the composition and organization of the extracellular matrix (ECM), signaling molecules secreted by cells into their surroundings, as well as physical interactions between neighboring cells. Cells are able to interact with their surroundings through reorganization of the ECM and secretion of pro- or anti-inflammatory molecules. In diseased states, inflammatory signals are secreted which can modify the cellular microenvironment. Diseased states such as cancer and fibrosis are often associated with the excessive production of ECM proteins that subsequently lead to an increase in tissue stiffness and changes to ECM architecture. Such changes to the mechanical properties of the cellular microenvironment affect the function and behavior of cells within a given tissue. Further understanding of the inflammatory responses and their relationships to tissue stiffness and ECM architecture could aid in the development of novel strategies to predict diseases as well as to target and monitor therapies. Since inflammation and mechanical properties of the affected tissue are closely interlinked, obtaining a detailed understanding of the interplay between the properties of the microenvironment and the cells that reside within it will be very beneficial to obtain physiologically relevant information. We have investigated the combinatorial effects of matrix stiffness, and architecture in the presence of co-cultures of cells to determine the overall effect on cellular responses and phenotypes. We have conducted studies on co-cultures of cells in 2D and 3D environments to identify how cellular behavior is affected by dimensionality.

The establishment of non-resolving inflammation underlies the pathogenesis of chronic inflammatory diseases in humans. Super low dose (SLD) endotoxin has been associated with exacerbating inflammation and the pathogenesis of chronic inflammatory diseases. However, the underlying molecular mechanisms are not well studied. In this study, I tested the hypothesis that SLD endotoxin may potentiate non-resolving innate immune cell inflammation through disrupting cellular endoplasmic reticulum (ER) homeostasis. We chose to study the dynamics of ER homeostasis in macrophages stimulated with SLD endotoxin. In naïve cells, ER stressor such as tunicamycin (TM) not only will induce cellular stress and inflammation through JNK and NFkβ activation, but also will cause subsequent compensatory homeostasis through inducing homeostatic molecules such as XBP1 and GRP78/BiP. We observed that cells challenged with SLD endotoxin have significantly reduced expression of homeostatic molecules XBP1 and BiP. Mechanistically, we observed that SLD-LPS increases phosphorylated HCK expression in TM treated cells. Phosphorylated HCK activation resulted in the phosphorylation of Golgi protein GRASP, leading to unstacking of Golgi cisterna and overall dysfunction of the Golgi apparatus. Dysfunctional Golgi apparatus and its effect on protein transport and secretion, may account for decreased levels of Site 2 Protease, reduced generation of ATF6 and its transcriptional target BiP. Taken together, our study reveal that super low dose endotoxin exacerbates low grade inflammation through increasing phosphorylation of HCK, inducing Golgi dysfunction, and decreasing BiP /homeostatic protein expression in innate immune cells. / Ph. D.