Obergassel, Julius: Functional evaluation of the PTPase DEP-1 as a novel regulator of monocytes and macrophages in diabetes and inflammation. 2022
Inhalt
- 1 Introduction
- 1.1 Atherosclerosis is a major complication of diabetes mellitus
- 1.2 Monocytes, macrophages and their role in health and disease
- 1.2.1 Monocytes: Ontogenesis, subsets, and function
- 1.2.2 Monocyte to macrophage differentiation
- 1.2.3 Macrophage function and activation
- 1.2.4 Monocytes and macrophages in atherosclerosis
- 1.2.5 Macrophage migration
- 1.2.6 NF-κB signaling in macrophages
- 1.3 Protein tyrosine phosphatases (PTPs)
- 1.4 Density-enhanced phosphatase 1 (DEP-1)
- 1.4.1 DEP-1 as a regulator in cancer, metastasis, autoimmunity, and insulin resistance
- 1.4.2 Upstream regulation and downstream targets of DEP-1
- 1.4.3 DEP-1 regulates proliferation, adhesion, cell cycle and has impact on tumor metastasis, invasion and angiogenesis in cancer
- 1.4.4 Yet known aspects about DEP-1 ‘s role in immunity and inflammation
- 2 Research aim and questions
- 3 Materials and Methods
- 3.1 Materials
- 3.1.1 Chemicals
- 3.1.2 Solutions
- 3.1.3 Antibodies for Western Blot
- 3.1.4 Kits and transfection reagents
- 3.1.5 Consumables
- 3.1.6 Laboratory equipment
- 3.1.7 Software
- 3.2 Isolation of peripheral blood mononuclear cells (PBMCs)
- 3.2.1 Isolation of PBMCs from thrombocyte reduction filters
- 3.2.2 Patient recruitment
- 3.2.3 Isolation of PBMCs from whole blood
- 3.3 Isolation of primary human monocytes, CD14+CD16- from PBMCs
- 3.4 Cell culture of monocytes and macrophages
- 3.4.1 Cryo-preservation and thawing of freshly isolated monocytes
- 3.4.2 Differentiation, activation, and culturing of primary monocytes to monocyte-derived macrophages
- 3.4.3 Cell counting for experiments
- 3.5 Transfection of primary monocyte-derived macrophages with small-interfering RNA (siRNA)
- 3.5.1 Transfection protocol for Lipofectamine® RNAiMAX
- 3.5.2 Transfection protocol for Viromer® Blue
- 3.6 RNA biochemical methods and polymerase chain-reaction assays
- 3.6.1 RNA lysis and extraction
- 3.6.2 cDNA synthesis
- 3.6.3 Principles of real-time PCR with SYBR green
- 3.6.4 Implementation of rt-qPCR
- 3.6.5 rt-qPCR data analysis
- 3.7 Protein biochemical methods, SDS-PAGE and Western Blot
- 3.7.1 Protein lysis and protein determination assay
- 3.7.2 Preparation of polyacrylamide gels
- 3.7.3 Sample preparation
- 3.7.4 SDS-PAGE
- 3.7.5 Western Blot
- 3.8 Macrophage cell proliferation and viability assay
- 3.9 Immunoprecipitation and phosphatase activity assay
- 3.10 Scratch migration assay
- 3.11 Data collection and statistical analysis
- 4 Results
- 4.1 DEP-1 is upregulated in monocytes from patients suffering from type 2 diabetes mellitus
- 4.2 Hyperglycemia and methylglyoxal treatment potentially lead to upregulation of DEP-1 in primary monocytes in-vitro
- 4.3 TNF-α induced pro-inflammatory signaling stimulates DEP-1 expression in primary monocytes
- 4.4 Inflammatory M1-macrophages show highest expression and activity of DEP-1 among non-activated and activated macrophage subpopulations
- 4.5 DEP-1 knockdown inflammatory in-vitro macrophage model
- 4.6 DEP-1 knockdown negatively influences macrophage migration
- 4.7 DEP-1 knockdown did not reveal significant differences in macrophage proliferation nor viability
- 4.8 DEP-1 knockdown mediates NF-κB transcription factor upregulation in inflammatory macrophages
- 5 Discussion
- 5.1 DEP-1 protein expression can be efficiently reduced by siRNA-mediated knockdown in a time-efficient, reproducible and easily performable approach
- 5.2 DEP-1 expression is a target of metabolic stress and pro-inflammatory stimulation in type 2 diabetes mellitus
- 5.2.1 DEP-1 is elevated in the hyperglycemic diabetic disease
- 5.2.2 Inflammation and related pathways are potential mediators of elevated DEP-1 expression in diabetic individuals
- 5.2.3 TNF-α stimulation showed strongest capabilities of DEP-1 induction in monocytes in-vitro and the NF-κB pathway is a potential mediator in between
- 5.3 DEP-1 depletion impairs macrophage migration, possibly by dephosphorylating and activating SFKs
- 5.4 DEP-1 depletion enhances NF-κB expression and activation possibly as a feedback regulation
- 5.5 DEP-1’s role in atherosclerosis
- 6 Conclusions
- 7 References
- 8 Figure directory
- 9 Table directory
- 10 Abbreviations
- 11 Ethical approvals
- 12 Acknowledgement
- 13 Curriculum vitae