3R-Project 137-13

Validation of a new human in-vitro model of microglia

Luis Filgueira
Department of Medicine, University of Fribourg, Switzerland
luis.filgueira@unifr.ch

Keywords: brain; immunology; neurology; validation

Duration: 1 year Project Completion: 2015

Background and Aim

Brain diseases are a burdensome problem for modern societies. Not surprisingly, therefore, substantial resources are invested in neuroscience research, which depends heavily on animal models. In this respect, microglia have assumed an important role. Animals are sacrificed for brain excision and the collection of cells of in-vitro studies. Animals are also used for in-vivo experiments. Depending on the aim of the study and on the nature of the procedure adopted, the in-vivo animal experiments involve different levels of invasiveness and suffering, which may include injuries to the brain and the spinal cord, if microglial response to trauma is being investigated, as well as exposure to toxic and infectious agents. Most of these experiments are conducted with human diseases in mind, even though animal microglia behave in many respects differently to human ones. The need to improve human in-vitro models of microglia is thus pressing.

Microglia are the unique resident immune cells of the brain. They play a crucial role in most brain diseases, being implicated in repair responses to injury, degeneration and infection-induced inflammatory reactivity. Being resident cells of the brain, they have to be isolated from fresh cerebral tissue for research purposes; only a few human microglial lines are available and for restricted applications only. We have developed a new human in-vitro model of microglia, which are derived from monocytes circulating in peripheral blood (Etemad et al. 2012). However, we now need to ascertain whether the properties of the human monocyte-derived microglia correspond to those of the brain-derived ones, which is a precondition for the validity of the in-vitro model.

Hypotheses:

The properties of human monocyte-derived microglia correspond to those of their brain-derived counterparts.
Human monocyte-derived microglia can be used to replace the brain-derived ones of animals for the in-vitro investigation of human cerebral diseases in which these cells play a key role.

Aims:

To compare the morphologies, phenotypes and functional activities of human monocyte- and brain-derived microglia.
To characterize the property-spectra of human monocyte- and brain-derived microglia with a view to establishing a gold-standard of this cell-type for research into human cerebral affections, taking Alzheimer’s disease as a pathological model and infection with the Japanese encephalitis virus as an infectious one.

Method and Results

In progress (present status)

Human microglia:
Human monocyte-derived microglia will be generated in-vitro using a published protocol (Etemad et al. 2012). Human brain-derived microglia will be isolated from 10 cadavers, not later than 8 hours post-mortem. According to published data, the brain-derived microglia should still be viable within this time-frame (Melief et al., 2012). In order to ascertain whether topographic differences in the properties of the brain-derived microglia exist, tissue blocks with a volume of 1-5 cm³ will be excised from the frontal, parietal, temporal and occipital lobes of the hemispheres, as well as from the cerebellum, thalamus, mesencephalon, pons and myelencephalon. The microglia will be isolated and cultured and then subjected to a comparative analysis. Monocyte- and brain-derived microglia will be cultured under the same conditions.
Comparative analysis of the properties of monocyte-and brain-derived microglia:
Various morphological and functional parameters, as well as viability, will be evaluated by flow cytometry (Etemad et al., 2012, Prabhakaranet al., 2012, Melief et al., 2012). The phagocytic activity of the microglia and their capacity to stimulate T-lymphocytes will also be assessed.
Influence of beta amyloid on human microglia:
There is an abundance of evidence indicating that microglia are responsive to treatment with beta amyloid, and for this reason, these cells have been implicated in the development of Alzheimer’s disease (Gentleman, 2013). In preliminary experiments, we have shown that the exposure of monocyte-derived microglia to beta amyloid induces significant changes in the expression pattern of chemokine receptor (Filgueira et al.: unpublished data). Hence, both monocyte- and brain-derived microglial populations will be exposed to beta amyloid, after which, their phenotypic characteristics and cytokine-secretion profile will be compared. We anticipate that the two microglial populations will respond similarly to treatment with beta amyloid.
Rates of infection with, and the influence of the Japanese encephalitis virus (JEV) on human microglia:
Microglia play an essential role in the inflammatory response of the brain to infections. In preliminary experiments, we have shown that human monocyte-derived microglia can be infected with JEV (Filgueira et al.: unpublished data). This virus is known to induce changes in the expression patterns of diverse surface markers, including the receptors for chemokines. We will thus compare the rates of infection of monocyte- and brain-derived microglia with the JEV, as well as changes in the cytokine-secretion profiles that are thereby induced.

Conclusions and Relevance for 3R

It is expected that by validating our new in-vitro human model of monocyte-derived microglia, and by applying it to two important areas of brain research, namely Alzheimer’s disease and viral infection, the prototype will gain wide acceptance in the implicated research community and will be used to replace animal experiments. In consequence, we anticipate a dramatic reduction in the consumption of animals for research in the field of neuroscience.

References

Etemad, S., Mohd Zamin, R., Ruitenberg, M.J., and L. Filgueira, A novel human invitro microglia model: Characterization of human monocyte-derived microglia. J Neurosci Meth, 2012. 2009: p. 79-89.
Gentleman, S.M., Review: Microglia in protein aggregation disorders: friend or foe? Neuropath Appl Neurobiol, 2013. 39: p45-50.
Melief, J., Koning, N., Schuurman, K.G., Van de Garde, M.D.B., Smolders, J., Hoek, R.M., Van Eijk, M., Hamann, J., and I. Huitinga, Phenotyping primary human microglia: Tight regulation of LPS responsiveness. GLIA, 2012. 60: p 1506-1517.
Prabhakaran, P., Hassioutou, F., Blancafort, P., Filgueria, L., Cisplatin induces differentiation of breast cancer cells. Frontiers Oncol, 2013. 3:134 doi:10.3389/fonc.2013.00134.

Figures

Figure 1: Human monocyte-derived microglia in culture.

Figure 2: Expression of Iba1, a marker specific for microglia, by human monocyte-derived microglia. A) Staining control (blue nuclei stained with DAPI). B) Iba1 expressing cells are shown in green. The cells were treated with a monoclonal mouse anti-Iba1 antibody, and a secondary donkey anti-mouse antibody labelled with AlexaFlour 488.

Figure 3: Influence of beta amyloid on human monocyte-derived microglia morphology. Left panel: light microscopy images of cells in culture before and after 48 hours of treatment with none or 25microgramm/ml of beta amyloid. Note the increased numbers of round shaped cells after 48 hours of treatment with beta amyloid indicating activation of microglia. Right panel: Quantification of activation of microglia after 0, 1, 8, 48 and 72 hours, after treatment with beta amyloid (0, 0.1, 1 and 25 microgramm7ml). There was a significant activation of the cells after 48 hours and treatment with 25microgramm of beta amyloid.

Figure 4: Down-regulation of expression of the chemokine receptor CX3CR1 in human monocyte-derived microglia after treatment with beta amyloid. Left panel: Representative histogram of flow cytometry measurements (black is staining control, green is CX3CR1 expression by control cells, red is expression of CX3CR1 after treatment with beta amyloid). Right panel: Quantitative analysis of CX3CR1 expression in cells treated with increasing concentrations of beta amyloid (0, 0.1, 1, 10, 25 and 50 microgramm/ml). n=4, ** is p<0.01, *** is p<0.005.