No description available.

Obesity is increasing worldwide, and the associated health-risks are also on the rise. Eventually, obesity related tissue damage leads to complications such as chronic inflammation, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Adipose tissue expansion in obesity triggers specific mechanisms that cause tissue damage. The immune system is especially agitated with excessive fat accumulation, which triggers inflammation and subsequent immune cell infiltration of tissue. Neutrophils are a major immune cell that cause damage in obesity, and the protease neutrophil elastase (NE) is a major neutrophil released factor of tissue damage. The goal of this study is to use tissue extracted from neutrophil elastase knockout (NEKO) mice that have been fed a high-fat high-fructose diet (HFHFD), and compare them to wild-type (WT) mice fed a normal chow diet (NCD), high-fat diet (HFD), and HFHFD to understand the effect of neutrophil elastase damage in obesity. Tissue from aged (NEKO) mice will also be examined to evaluate the role of neutrophils and NE in tissue damage in aging and obesity. Mice in these experimental groups were sacrificed and had their tissue extracted for various staining protocols to discover the extent of tissue damage and immune cell infiltration between mice with and mice without NE. One experiment had 4 different diets fed to mice. The other experiment had mice aged for 2 years, and mice aged for 3 months and 4 months. Mice from the first experiment were fed for 4 months and separated into 4 groups based on diet, WT-NCD, WT-HFD, WT-HFHFD, and NEKO-HFHFD. Our data indicates that, in comparison with WT-HFHF mice, NEKO-HFHFD mice had less steatosis, fibrosis, immune cell infiltration, and apoptosis within the liver. Neutrophil infiltration into the liver is increased by the HFHFD diet. HFHFD diet also stimulates fibrosis, as indicated by collagen deposition in the liver. Neutrophil accumulation is also associated with the increase of macrophages and CD4 Th Cells in the liver, particularly in WT mice fed the HFHFD. Interestingly, the liver from NEKO-HFHFD mice had dramatically reduced infiltration of neutrophils, macrophages, and CD4+ Th cells. Our data suggests that NE is required for HFHFD induced inflammation and fibrosis in the liver. Mice from the second experiment were split into 3 groups based on age, WT-Young (3 months and 4 months), WT-Old, and NEKO-Old. All groups were fed the same normal chow diet, but WT Old and NE KO Old were both aged to 2 years old. Our data revealed that NE deletion in aged mice reduced fibrosis, elastin fragmentation, calcification, and presence of NE within the aorta. While part of the mechanism for neutrophil elastase related tissue damage has been explored through this one-year master degree research project, more work is needed to fully understand how NE is stimulated and causes tissue damage. Future work should examine the potential interaction between neutrophils and other immune cells in obesity and aging. / 2022-06-04T00:00:00Z

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis University of Porto Faculty of Pharmacy Department of Chemical Sciences Department of Biological Sciences Candidate: Kristína Morgošová Supervisor: assoc. prof. PharmDr. Radim Kučera, Ph.D. Consultants: prof. Maria da Conceição Branco da Silva, Ph.D. prof. Maria Alice dos Santos Silva Gomes Martins, Ph.D. Susana Maria Santos Rocha, Ph.D. Title of diploma thesis: Development and characterization of modified polysulfone hemodialysis membranes by means of immobilized neutrophil elastase inhibitors. Chronic kidney disease (CKD) is a major health and financial burden, mainly because of the costly renal replacement therapy and treatment associated. The last stage, end-stage renal disease, is associated with high morbidity and mortality rate, generally due to cardiovascular complications. Chronic inflammation is frequently present in CKD patients, which is enhanced by the long term intra-dialytic recurrent contact between blood and hemodialysis (HD) membrane and further contributes to development of atherosclerosis. Contact with the artificial material of HD membranes leads to oxidative stress and neutrophil activation with release of neutrophil serine proteases such as human...

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24798号 / 医博第4990号 / 新制||医||1066(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 萩原 正敏, 教授 妹尾 浩, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Dendritic cells (DC) are sentinels of the immune system that display an extraordinary capacity to present antigen to naïve T cells and initiate immune responses. DCs are distributed throughout the lungs in the conducting airways of the tracheobronchial tree and in the parenchymal lung, and play a pivotal role in controlling the immune response to inhaled antigens. The respiratory surface is continually exposed to potentially injurious particulates and pathogenic organisms, to which tightly regulated innate and adaptive immunological responses are made. The airways are usually sterile in healthy individuals. However, patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have increased susceptibility to microbial infections and increased neutrophil elastase (NE) in lung secretions. This thesis was designed to test the hypotheses that; (i) excess NE may result in a dysregulation of lung DCs function in pulmonary chronic diseases, and (ii) the natural NE inhibitors in the respiratory system are able to rescue the NE-mediated dysregulation of DCs and potentially enhance their antigen presenting activity. The data in this thesis demonstrate that purified human NE down-regulated murine bone marrow (BM)-derived DC co-stimulatory molecules (CSM; CD40, CD80 and CD86), which was due to its proteolytic activity. NE-treated LPS-matured DCs were less efficient at presenting ovalbumin (OVA) peptide to naïve OVAspecific transgenic (D011.10) T cells. In addition, immature DCs (iDC) simultaneously treated with LPS and NE failed to mature fully and produced significantly less IL-12 and TNF-α than DCs matured in the presence of LPS alone. Similarly, treatment of mature DC (mDC) with pooled and individual COPD and CF sputum samples caused a reduction in CD80 and CD86 levels (but not CD40) which positively correlated with the NE concentration present in the samples. The demonstration that NE could adversely affect DC phenotype and function suggested that augmentation of NE inhibitors could reverse this process and preserve DC function in inflammatory microenvironments. Over-expression of an NE specific inhibitor (elafin) in the lungs of mice (using either replication-deficient adenovirus [Ad] or elafin transgenic [eTg] mice) increased the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung DCs in in vivo models. Replication-deficient Ad vectors encoding NE inhibitors, namely elafin, secretory leukocyte protease inhibitor (SLPI) and α1-protease inhibitor (α1-PI), were also used to infect DCs in vitro, to further study the effect of these NE-inhibitors on DCs in isolation. These findings suggest that purified NE and NE-containing lung inflammatory secretions are powerful down-regulators of DC maturation, resulting in reduced capacity of these potent APCs to efficiently present antigens; whereas, NE inhibitors could boost immunity by increasing the activation state and/or number of DCs.

Fluorescent imaging technologies that offer new ways to visualise and quantify fluorescently labelled molecules are increasing, necessitating the development of fluorescent molecules that can efficiently and specifically label targets in vitro and in vivo. The first aim of this thesis was the study of human neutrophil elastase. Human neutrophil elastase is an important enzyme in the regulation of inflammation but if over expressed can become part of the cause of inflammation itself. To elucidate this dual function and have a greater understanding of this enzyme, an imaging probe for neutrophil elastase was designed. Firstly, the syntheses of fluorescently labelled three branched dendron core structures were optimised, and studied in neutrophils. The selected core structure was functionalised with an elastase specific peptide sequence and fluorescently labelled. The probe was specifically cleaved by neutrophil elastase in an enzymatic assay and in the presence of activated neutrophils (Chapter 1). Fluorescein and rhodamine are dyes that are readily available, are affordable and have convenient wavelengths for microscopy and flow cytometry. Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) is a commonly used fluorescein derivative, widely used in cell proliferation assay. It is mainly used as a mixture of isomers and its synthesis is not reported. Herein a short and simple synthesis of the two individual isomers of carboxyfluorescein diacetate N-succinimidyl ester as well as the equivalent rhodamine variation (carboxytetraethylrhodamine N-succinimidyl ester) is reported (Chapter 2). The labelling properties of these probes were studied in proliferation assays on mouse and human T lymphocytes. Finally, the nuclear penetration of the dendron structure combined with nuclear localisation sequences (NLS) was investigated. Attachment of nuclear localisation sequences to the probe in the presence of fluorescein demonstrated successful entry into the nucleus in human alveolar adenocarcinoma cell line (A549) (Chapter 3).

Alterations to sphingolipid metabolism are associated with increased pulmonary inflammation, but the impact of inflammatory mediators, such as neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. NE is a protease associated CF lung disease progression, and can be found in up to micromolar concentrations in patient airways. While sphingolipids have been investigated in the context of CF, the focus has been on loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we present a novel observation: oropharyngeal aspiration of NE increases airway ceramides in mice. Using a previously characterized mouse model of NE-induced inflammation, we demonstrate that NE increases de novo ceramide production, which is likely mediated via increased SPTLC2 levels. Inhibition of de novo sphingolipid synthesis using myriocin, an SPT inhibitor, decreases airway ceramide as well as the release of pro-inflammatory signaling molecules induced by NE. Furthermore, in a retrospective study of the sphingolipid content of CF sputum—the largest of its type in this patient cohort to date, we investigated the association between NE and sphingolipids. There were linear correlations between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA) positive culture and female gender both strengthened the association of NE and sphingolipids, but higher FEV1 % predicted weakened the association, and Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. These data suggest that NE may increase sphingolipids in CF airways as it did in our in vivo model, and that this association is stronger in patients that have worse lung function, are female, and whose lungs are colonized with MRSA. Modulating sphingolipid homeostasis could provide novel pharmacological approaches for alleviating pulmonary inflammation.

Alarmins and damage associated molecular pattern (DAMP) are endogenous proteins with distinct and various intracellular roles that when released extracellularly act as startingsignals for inflammatory immune responses. The endogenous protein High mobility group box 1 (HMGB1) acts as a DAMP and has been shown to drive progression of multiple inflammatory and autoimmune diseases. During homeostasis HMGB1 is localized in the nucleus of almost any cell, where its main function is organization of the DNA and regulation of transcription. Upon cell death or immune cell activation HMGB1 can be translocated into the cytoplasm for subsequent release into the extracellular space. Extracellular HMGB1 can act as a DAMP by activating several receptors of the immune system. Recent studies focus on HMGB1 release and functional regulation due to prost-translational modifications (PTMs) on cysteine residues. However, little is known about enzymatic regulation of HMGB1. The aim of this thesis was to investigate the possibility of proteolytic processing of HMGB1 by enzymes, which play a crucial role in inflammatory diseases and their progression. We utilized an in vitro model that mimics natural conditions of the autoimmune disease arthritis. Enzymatic digestion of HMGB1 was performed in kinetics studies using the neutrophilic enzymes cathepsin G, neutrophil Elastase as well as matrix metalloproteinase-3, which is released from tissues at the site of inflammation. We defined that HMGB1 is a novel substrate of all of the tested enzymes. All enzymes induced different cleavage pattern. In conclusion, my findings open up the possibility for future studies involving the observed fragments of HMGB1 and their functional features. It also demonstrated that HMGB1 is affected by protease modifications in a disease relevant environment.

BACKGROUND: Calcification is a natural process of bone formation or osteogenesis. However, calcium is able to be deposited abnormally in soft tissues such as the aorta, adipose tissue and liver, causing these to harden. Abnormal calcification in arteries is a common factor contributing to high blood pressure and, further, many severe cardiovascular diseases such as atherosclerosis and coronary disease. In liver and adipose tissue, calcification always takes place accompanied by excess extracellular matrix (ECM) accumulation which is called fibrosis, contributing to cirrhosis and metabolic disorders including insulin resistance. In addition, it is documented that severe calcification in adipose tissues is able to cause damage to the micro-vascular system, and calcification in perivascular adipose tissue (PVAT) is a key effector of arterial stiffness. Dystrophic calcification, one of the most common types of abnormal calcification, usually occurs as a reaction to tissue damage such as obesity-induced inflammation. Increasing numbers of studies indicate that abnormal calcification is the result of re-differentiation towards osteogenesis which occurs in the nascent resident cells under the stimulation of multiple factors. The BMP/Smad signaling pathway is commonly known to participate in bone formation and is implicated in mineralization as well as local induction of inflammation. Importantly, BMP/Smad signaling as an inducer of the osteochondrogenic phenotype in vascular calcification is fully appreciated. However, the molecular events of dystrophic calcification triggered by obesity-induced chronic inflammation still remain unclear. Our previous studies have identified that imbalance with increased activity of neutrophil elastase (NE), a Ser protease mainly released by neutrophils during inflammation, and decreased serum levels of the NE inhibitor α1-antitrypsin A1AT, contributes to the development of obesity-related metabolic complications including insulin resistance, fatty liver and chronic inflammation. This study explored the effects of NE on abnormal calcification in soft tissues, which may be mediated by BMP/Smad signaling pathway, and, furthermore, the molecular mechanism by which NE activates the BMP/Smad signaling pathway. METHODS: Wild-type mice were fed with either a high-fat high-fructose diet (HFHFD), a high-fat diet (HFD) alone or a normal chow diet (NCD), and NE-knockdown mice were fed with a HFHFD. Adipose tissue and liver were extracted from all mice. H&E staining and immunofluorescence staining (IF) detected the inflammation condition. Alizarin staining and von kossa staining were used to detect calcium deposits. 3,3′-Diaminobenzidine (DAB) staining was used to examine active phospho-Smad1/5 signaling. Regarding nascent resident cells which have potential ability of osteogenic re-differentiation, 3t3l1 fibroblast and human hepatic stellate cell (hHSC) were cultured in dishes and 6-well plates with coverslips. In our previous research, mouse aortic smooth muscle cells (mASMC) seeded in 6-well plates grew in an osteogenic medium (10mM β-glycerophosphate and 10mM Calcium chloride) in the presence or absence of NE (10nM). Calcium deposits were detected by Alizarin staining. 3t3l1 and hHSC was treated with NE (20nM, 30nM, 40nM), BMP2, TGFβ1 or NE combined with BMP2, TGFβ1 or NE inhibitor GW311616A (Axon). Further, we used specific chemical inhibitors, LDN-193189, BMP-ALK2/3 inhibitor, SB525334, TGFβ-ALK5 inhibitor, and I-191, PAR2 antagonist to investigate the molecular mechanism of NE’s effects on Smad signaling pathways. Cells in dishes were harvested, and the proteins were measured by western blot. Coverslips in 6-well plates were used for immunofluorescence. RESULTS: The most severe calcification was found in the adipose tissue of HFHFD fed wild-type mice and moderate calcification took place in the HFD mouse group while NCD mice rarely had calcium deposits. NE-knockdown significantly prevented calcium deposits in adipose tissue compared with HFHFD wild-type mice. Consistently, we found increased phospho-Smad1/5 (p-Smad1/5) signaling in the adipose tissues of mice on the HFHFD and HFD mice while p-Smad1/5 was prevented in the NE-knockout group. Furthermore, NE enhanced calcium deposits in mASMC cultured in osteogenic medium. NE significantly activated p-Smad1/5 signaling in hHSC in the dose-effect relationship and contributes to an additive effect on p-Smad1/5 in the presence of BMP2 or TGFβ1. Although p-Smad1/5 was only slightly aroused by NE in 3t3l1 fibroblast, NE was able to promote p-Smad1/5 activation tremendously and specifically in the presence of BMP2 or TGFβ1 but not p-Smad2/3 which is the main downstream signaling of TGFβ1. Chemical inhibition of ALK2/3, not ALK5 or PAR2, was able to completely block NE’s effects in hSHC on p-Smad1/5 activation. In addition, the cleavage of osteoblast-cadherin or CDH11 (OB-cadherin) was observed in hHSC, which may indicate a lower beta-catenin abundance in the hHSC cells which were treated with NE. CONCLUSION: NE has the potential to contribute to abnormal calcification in soft tissues including the liver, adipose tissue and aorta via activating canonical ALK2/3-BMP-Smad1/5 signaling pathway in the mesenchymal stem cell/MSC-lineage cells. In addition, NE is likely to break cell-cell adhesion which may contribute to cell proliferation and re-differentiation towards osteogenesis and fibrosis. / 2024-01-28T00:00:00Z

No description available.