title: Functional evaluation of the PTPase DEP-1 as a novel regulator of monocytes and macrophages in diabetes and inflammation
Creators: name: Obergassel, Julius
Publisher: name: Universitäts- und Landesbibliothek Münster
Year: 2022
Subjects: subject: 610 Medicine and health

description: Monozyten und Makrophagen spielen im Kontext von Diabetes mellitus und Atherosklerose eine wichtige Rolle. In der vorliegenden Arbeit wurde die Protein-Tyrosin-Phosphatase Density-enhanced phosphatase I (DEP-1) im Kontext von Diabetes in einem Hyperglykaemie- und Inflammationsmodell in Monozyten und Makrophagen charakterisiert. Es konnte gezeigt werden, dass die Expression von DEP-1 in Monozyten von Diabetikern im Vergleich zu Nicht-Diabetikern erhöht ist. In-vitro war DEP-1 in Monozyten unter Stimulation mit TNF-alpha, sowie unter hyperglykaemischen Bedingungen ebenfalls heraufreguliert. M1-Makrophagen zeigten die hoechste Proteinexpression, sowie eine erhoehte DEP-1 Phosphatase-Aktivitaet im Vergleich nicht-aktivierten und M2-aktivierten Makrophagen. siRNA basiertes Silencing von DEP-1 fuehrte zu reduzierter Migration von Makrophagen im Wundheilungsversuch. Die p65-Expression, die p65-Phosphorylierung und die TNF-alpha Expression waren in Makrophagen unter Silencing der Phosphatase erhoeht.
description: Monocytes and macrophages play crucial roles in the pathophysiological context of diabetes mellitus and atherosclerosis. The present work characterizes the protein tyrosine phosphatase Density-enhanced phosphatase 1 (DEP-1) in the context of diabetes by employing a model of hyperglycemia and inflammation in monocytes and macrophages. DEP-1 mRNA expression was upregulated in monocytes from diabetic versus non-diabetic patients. In monocytes, stimulation with TNF-alpha as well as culturing in hyperglycemia led to increased protein expression of DEP-1. M1-activated macrophages showed highest protein expression and a significantly increased phosphatase activity of DEP-1 in comparison with non-activated and alternatively activated M2-macrophages. siRNA-based silencing of DEP-1 led to reduced macrophage migration in a wound-heeling assay. Expression of p65 and p65-phosphorylation, as well as expression of TNF-alpha in macrophages was increased after silencing of the phosphatase.
description: 1 INTRODUCTION 4 1.1 Atherosclerosis is a major complication of diabetes mellitus 4 1.2 Monocytes, macrophages and their role in health and disease 5 1.2.1 Monocytes: Ontogenesis, subsets, and function 5 1.2.2 Monocyte to macrophage differentiation 8 1.2.3 Macrophage function and activation 8 1.2.4 Monocytes and macrophages in atherosclerosis 10 1.2.5 Macrophage migration 12 1.2.6 NF-κB signaling in macrophages 13 1.3 Protein tyrosine phosphatases (PTPs) 13 1.4 Density-enhanced phosphatase 1 (DEP-1) 14 1.4.1 DEP-1 as a regulator in cancer, metastasis, autoimmunity, and insulin resistance 14 1.4.2 Upstream regulation and downstream targets of DEP-1 15 1.4.3 DEP-1 regulates proliferation, adhesion, cell cycle and has impact on tumor metastasis, invasion and angiogenesis in cancer 16 1.4.4 Yet known aspects about DEP-1 ‘s role in immunity and inflammation 17 2 RESEARCH AIM AND QUESTIONS 18 3 MATERIALS AND METHODS 19 3.1 Materials 19 3.1.1 Chemicals 19 3.1.2 Solutions 20 3.1.3 Antibodies for Western Blot 20 3.1.4 Kits and transfection reagents 21 3.1.5 Consumables 21 3.1.6 Laboratory equipment 22 3.1.7 Software 22 3.2 Isolation of peripheral blood mononuclear cells (PBMCs) 23 3.2.1 Isolation of PBMCs from thrombocyte reduction filters 23 3.2.2 Patient recruitment 23 3.2.3 Isolation of PBMCs from whole blood 24 3.3 Isolation of primary human monocytes, CD14+CD16- from PBMCs 24 3.4 Cell culture of monocytes and macrophages 25 3.4.1 Cryo-preservation and thawing of freshly isolated monocytes 25 3.4.2 Differentiation, activation, and culturing of primary monocytes to monocyte-derived macrophages 26 3.4.3 Cell counting for experiments 26 3.5 Transfection of primary monocyte-derived macrophages with small-interfering RNA (siRNA) 27 3.5.1 Transfection protocol for Lipofectamine RNAiMAX 28 3.5.2 Transfection protocol for Viromer Blue 29 3.6 RNA biochemical methods and polymerase chain-reaction assays 30 3.6.1 RNA lysis and extraction 30 3.6.2 cDNA synthesis 31 3.6.3 Principles of real-time PCR with SYBR green 32 3.6.4 Implementation of rt-qPCR 33 3.6.5 rt-qPCR data analysis 35 3.7 Protein biochemical methods, SDS-PAGE and Western Blot 36 3.7.1 Protein lysis and protein determination assay 37 3.7.2 Preparation of polyacrylamide gels 38 3.7.3 Sample preparation 38 3.7.4 SDS-PAGE 39 3.7.5 Western Blot 39 3.8 Macrophage cell proliferation and viability assay 41 3.9 Immunoprecipitation and phosphatase activity assay 42 3.10 Scratch migration assay 42 3.11 Data collection and statistical analysis 43 4 RESULTS 45 4.1 DEP-1 is upregulated in monocytes from patients suffering from type 2 diabetes mellitus 45 4.2 Hyperglycemia and methylglyoxal treatment potentially lead to upregulation of DEP-1 in primary monocytes in-vitro 46 4.3 TNF-α induced pro-inflammatory signaling stimulates DEP-1 expression in primary monocytes 48 4.4 Inflammatory M1-macrophages show highest expression and activity of DEP-1 among non-activated and activated macrophage subpopulations 49 4.5 DEP-1 knockdown inflammatory in-vitro macrophage model 52 4.5.1 Knockdown efficiency 54 4.5.2 Knockdown toxicity 55 4.6 DEP-1 knockdown negatively influences macrophage migration 56 4.7 DEP-1 knockdown did not reveal significant differences in macrophage proliferation nor viability 60 4.8 DEP-1 knockdown mediates NF-kB transcription factor upregulation in inflammatory macrophages 62 5 DISCUSSION 65 5.1 DEP-1 protein expression can be efficiently reduced by siRNA-mediated knockdown in a time-efficient, reproducible and easily performable approach 65 5.2 DEP-1 expression is a target of metabolic stress and pro-inflammatory stimulation in type 2 diabetes mellitus 68 5.2.1 DEP-1 is elevated in the hyperglycemic diabetic disease 68 5.2.2 Inflammation and related pathways are potential mediators of elevated DEP-1 expression in diabetic individuals 70 5.2.3 TNF-α stimulation showed strongest capabilities of DEP-1 induction in monocytes in-vitro and the NF-kB pathway is a potential mediator in between 72 5.3 DEP-1 depletion impairs macrophage migration, possibly by dephosphorylating and activating SFKs 74 5.4 DEP-1 depletion enhances NF-kB expression and activation possibly as a feedback regulation 78 5.5 DEP-1’s role in atherosclerosis 82 6 CONCLUSIONS 83 7 REFERENCES 84 8 FIGURE DIRECTORY 102 8.1 Figure references 102 9 TABLE DIRECTORY 103 10 ABBREVIATIONS 104 11 ETHICAL APPROVALS 106 12 ACKNOWLEDGEMENT 110 13 CURRICULUM VITAE 111