Research Debbie Le Blon

Abstract

The host's laboratory's promising results of IL13 treatment in a mouse model for MS, encourage us to further investigate this potential therapeutic strategy in a human setup. Therefore, we propose to develop a human in vitro demyelination model to study the effect of IL13-induced M2 macrophages and microglia. We will do so by (I) differentiating hiPSC towards oligodendrocytes; (II) evaluating the myelinating potential by oligodendrocyte-neuron co-culture; (III) optimizing a toxin-induced demyelination procedure; and (IV) evaluating the effect of macrophage/microglia addition with or without IL13 stimulation.

Researcher(s)

• Promoter: Le Blon Debbie
• Co-promoter: Ponsaerts Peter

Research team(s)

• Laboratory for Experimental Hematology (LEH)
• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Varicella-zoster virus (VZV) causes chickenpox in children and remains latent in neural ganglia afterwards. VZV can cause encephalitis or cerebellitis during both the acute and subacute phases of chickenpox. After resolution of chickenpox, VZV can reactivate from its latent state and cause herpes zoster. Moreover, VZV reactivation is believed to be able to cause stroke in children. The pathophysiology underlying all of these central nervous system VZV complications remains largely unknown so far. In this project, we aim to deepen our understanding regarding two factors that might cause a genetic predisposition in humans for the development of chickenpox-associated neurological complications. Preliminary data from our lab previously showed that mutations in RNA polymerase III (POL III) cause a defect in VZV sensing (via AT-DNA "recognition") in blood cells and consequently cause a reduced control of VZV proliferation. In this project, we first aim to show that following primary VZV infection, glial cells, which are immune-responsive cells in the central nervous system, recognize VZV and subsequently produce protective cytokines. Moreover, we will assess whether mutations in AT-DNA sensor POL III in children with encephalitis, cerebellitis or stroke/vasculitis due to chickenpox have a defective recognition of VZV and subsequently increased VZV proliferation in central neurons. We will do this by differentiating induced pluripotent stem cells (iPSC) from patients and controls into neurons and glial cells, and subsequently infecting these with VZV. This will lead to a simultaneous analysis of VZV dynamics in neurons and cytokine production by glial cells. Preliminary data from our labs and others have shown that the cellular process called autophagy, important for protein processing, might be involved in cellular VZV dynamics as inhibition of autophagy led to reduced VZV proliferation. In this project, we aim to further address this potential pathogenic route by experimentally inhibiting autophagy in iPSC-derived neurons from healthy controls and measuring the subsequent effects on VZV dynamics. In addition, we noted that 3/9 cerebellitis patients had a mutation in the autophagy-associated gene TBC19DB. In a first exploration, we will assess how autophagy is affected by this mutation and whether this influences VZV proliferation in monocytes from these patients and controls.

Researcher(s)

• Promoter: Ogunjimi Benson
• Co-promoter: Le Blon Debbie
• Fellow: Buyle-Huybrecht Tamariche

Research team(s)

• Centre for Health Economics Research and Modelling of Infectious Diseases (CHERMID)

Project type(s)

• Research Project

Abstract

Neuroinflammation occurs in all central nervous system (CNS) pathologies and can be defined as the activation of local and peripheral infiltrating immune cells, with the key players being brain-resident microglia and blood-borne infiltrating macrophages. While growing evidence ascribes different roles to microglia and macrophages in neuro-inflammation, the main interest in both phagocytes, with regard to therapeutic strategies, is their ability to obtain different activation states, ranging from pro-inflammatory (M1) to anti-inflammatory (M2) activation. These new revelations led to many studies nowadays, which are investigating immune modulation as a potential therapeutic strategy to treat CNS pathologies. However, since existing pre-clinical models for the study of neuro-inflammation are based on either human cell lines or rodent models, this new and potential therapeutic strategy creates the need for more reliable pre-clinical models for human neuro-immune research. Therefore, with this project, we aim to develop an in vitro assay to study and modulate activation states in human neuro-inflammation by using human induced pluripotent stem cell (hiPSC)-derived microglia and macrophages. For this, we will introduce and validate in vitro differentiation protocols for hiPSC-derived microglia and macrophages. Phenotypical characterization will be performed by using known markers for immunocytochemistry and flow cytometry. Next, functional analyses of the developed hiPSC-derived microglia and macrophages will include (i) migration assays for chemokines CX3CL1 and CCL2, known to attract, respectively, microglia and macrophages; (ii) phagocytosis assays; and (iii) M1-M2 priming experiments, determining the polarising capacity of both microglia and macrophages by flow cytometry and ELISA. With this research project, our main aim is to meet the urgent need for novel in vitro human neuro-inflammation models, but with a successful outcome, we will also achieve a major step forward towards less animal testing.

Researcher(s)

• Promoter: Le Blon Debbie

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Due to the current understanding that multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), where mononuclear cell infiltration in brain and spinal cord is a major contributor to demyelination, gliosis, axonal loss and eventually loss of neuronal function, we investigated over the past 6 years whether local modulation of CNS lesions with the immune-modulating cytokine interleukin (IL)13 might ameliorate detrimental disease progression. While our pre-clinical studies in the cuprizone (CPZ)-induced CNS inflammation/demyelination mouse model for human MS have demonstrated proof-of-principle for this approach, currently we do not know whether human microglia and macrophages are equally well susceptible to IL13-mediated immunomodulation in the pro-inflammatory MS environment. Using advanced human induced pluripotent stem cell (hiPSC) derived 3D cell culture models, we aim to provide further pre-clinical rationale for the use of IL13 as an additional treatment approach in advanced stage MS.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: Le Blon Debbie

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Development of three-dimensional (3D) in vitro cell culture models for human neuro-immunological research is currently a hot topic in medical cell biology research. Although multiple protocols have been described for generating human 3D brain organoids starting from pluripotent stem cells, current models display several limitations, including the lack of extracellular matrix (ECM), the absence of multiple types of immune cells and a functional blood-brain-barrier (BBB). With this project we aim to develop and optimize a new method for generating 3D neuro-immune cell culture models to study and modulate human neuro-inflammatory responses. For this, isogenic 3D cell cultures comprising human embryonic stem cell (hESC)-derived neurons, astrocytes and microglia will be established on decellularized mouse brain sections in order to provide growth and organizational support by original brain ECM proteins. In addition, hESC-derived astrocytes and endothelial cells will be used to create a BBB model for physical separation of hESC-derived macrophages. Further inclusion of genetic engineering strategies, to allow for real time bioluminescence imaging and (live cell) confocal microscopy, will be applied to ensure profound validation and high throughput screening applications. Once established, we will use this technology to further extend our research efforts to optimize therapeutic strategies based on interleukin (IL)13-mediated immunomodulation for cerebrovascular disease.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: Berneman Zwi
• Fellow: Le Blon Debbie

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

This project represents a research agreement between the UA and on the onther hand IWT. UA provides IWT research results mentioned in the title of the project under the conditions as stipulated in this contract.

Researcher(s)

• Promoter: Berneman Zwi
• Co-promoter: Le Blon Debbie

Research team(s)

Project type(s)

• Research Project