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Implementation of primary cells for mechanistic investigations of inflammatory and metabolic diseases

Marbet, Philippe. Implementation of primary cells for mechanistic investigations of inflammatory and metabolic diseases. 2018, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12626

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Abstract

Cell based in vitro experiments consist an essential tool in many research fields as they offer a far less complex and easier to manipulate system compared to in vivo models. In most cases, investigators can either use established continuous cell lines or opt for primary cells directly isolated from the tissue of interest. While cell lines are cost effective and easy to obtain in high numbers, continuous growth is facilitated by their cancer background or by genetic manipulation, both of which often limit their ability to simulate the physiology of the tissue of origin. Primary cells on the other hand require labor-intensive isolation procedures but therefore much closer resemble the in vivo situation in terms of sensitivity, transporter expression and physiological behavior. To choose the appropriate tool for each experiment a detailed knowledge of the abilities and limitations of both systems is essential, whereas it is always recommendable to repeat at least some key experiments in a primary cell system. The present thesis aims to assess the implementation of bone marrow-derived macrophages (BMDMs), a 3D model of rat brain as well as primary proximal tubular cells (PTCs) for mechanistic investigations of inflammatory and metabolic diseases in different research projects.
The first project implements BMDMs in the assessment of hexose 6 phosphate dehydrogenase (H6PD) function in macrophage differentiation and metabolism. Macrophages are phagocytic cells present in essentially all tissues fulfilling various functions vital for tissue repair, homeostasis and immunity. During pathological and homeostatic inflammatory reactions they arise from the bone marrow under the influence of macrophage-colony stimulating factor, patrol the body and eventually enter compromised tissue. Presence of lipopolysaccharide and interferon gamma will differentiate them into a pro-inflammatory M1 phenotype producing toxic effector molecules and inflammatory cytokines whereas interleukin 4 induces an anti-inflammatory M2 phenotype involved in resolution of inflammation and promotion of tissue remodeling. For the first study, BMDMs were derived from bone marrow progenitor cells flushed from the femurs of wild-type (WT) and H6pd knockout (KO) mice. The H6PD produces the cofactor NADPH for the activation of glucocorticoids by oxo-reduction activity of 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) in the endoplasmic reticulum (ER). In macrophages this could influence the phenotypic and functional differentiation and therefore also their metabolism. Absence of H6pd was reported to cause a switch in the bidirectional 11β HSD1 towards an inactivation of glucocorticoids. Comparing BMDMs of WT and H6pd KO mice we found no such switch but only a decrease in 11β HSD1 oxo-reduction activity by 40 50 %, indicating an alternative source of NADPH. Furthermore, H6pd KO did not cause a major disturbance in macrophage phenotypic differentiation although it caused a slightly exaggerated M1 phenotype as well as an overall reduced glucose consumption. This study showed the suitability of BMDMs to study macrophage differentiation and to perform a variety of assays to assess characteristic macrophage parameters like phagocytosis, nitric oxide production or metabolism. Most importantly, by using animal derived cells, we could use a H6pd KO mouse which circumvents an incomplete gene knockdown by siRNA.
In the second project, BMDMs were implemented to investigate the contribution of secondary calciprotein particles (CPPs) to the process of vascular calcification frequently observed in chronic kidney disease (CKD) patients. The formation of primary CPPs is a physiological process in which serum proteins prevent the precipitation of calcium and phosphate as hydroxyapatite by forming spherical complexes instead. In CKD patients, primary transform into secondary CPPs, which were shown to activate macrophages. Nuclear factor erythroid 2 related factor 2 (NRF2), a master regulator of oxidative cell defense, was reported to play an important role in CPP-induced inflammation in CKD. Using BMDMs derived from WT and Nrf2 KO mice we showed the induction of macrophages by secondary CPPs at concentrations measured in CKD patients. Mechanistic studies suggested a TLR4-mediated CPP response, which could be reduced or exacerbated by induction or knockdown of Nrf2. Whereas the use of Nrf2 KO mice facilitated complete absence of the target gene, the relevance of the study would benefit from the use of a human model like human PBMCs.
A primary cell system that includes a specific type of macrophage, the microglia found in brain, can be obtained as part of an in vitro 3D brain model derived from rat embryonic brain tissue. These 3D cultures contain all cell types of the brain, including neurons, oligodendrocytes, astrocytes and microglia cells. The latter two are involved in neuroinflammation, which can be caused by heavy metals such as trimethyltin. The project investigates the combination of three risk factors of neurodegenerative diseases, namely the metabolic syndrome characterized by low brain insulin and high glucocorticoid levels as well as trimethyltin exposure. Therefore, an approach consisting of the described 3D rat brain model, the murine microglia cell line BV 2 as well as a mouse model of diabetes was used to report the absence of an additive effect of the risk factors to neurodegeneration but an increased neuroinflammatory response. The implementation of models with various levels of complexity allowed to address mechanistic questions using the BV 2 cell line but also to draw more in vivo relevant conclusions by using a primary 3D model and in vivo mouse experiments.
In a fourth project, the implementation of primary PTCs in the investigation of metabolic acidosis was assessed. Within the functional unit of a kidney, the nephron, the proximal tubule is responsible for 65 % of the total sodium reabsorption as well as most solutes, amino acids and low molecular weight proteins. In addition, the proximal tubule plays an important role in counteracting metabolic acidosis via a process that involves the uptake of glutamine by the glutamine transporter SNAT3 which is mainly expressed in the second and third proximal tubule segment. To test the involvement of Nrf2 in the upregulation of SNAT3 in response to metabolic acidosis, we exposed primary PTCs isolated from mouse kidneys to acidified medium. We could observe an upregulation of Snat3 mRNA, which was prevented by siRNA knockdown of Nrf2. Studies in Nrf2 KO mice fed with high acid diet confirmed the in vitro findings but also revealed a compensatory adaption of other transporters not detected in the primary cell model.
Overall, the four projects revealed many abilities but also some disabilities of the implemented primary cell systems. As the availability of human material is very limited, inducible pluripotent stem cell technology will grow more and more important as it increases translational relevance of in vitro experiments. However, the described primary cell models will probably remain of great use, especially in basic research.
Advisors:Odermatt, Alex and Huwyler, Jörg
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Pharmazie > Molecular and Systems Toxicology (Odermatt)
UniBasel Contributors:Odermatt, Alex and Huwyler, Jörg
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12626
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (178 Blätter)
Language:English
Identification Number:
Last Modified:13 Jun 2018 04:30
Deposited On:12 Jun 2018 14:38

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