Isolation and characterization of human microglia by flow cytometry

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Isolation and characterization of

human microglia by flow cytometry

Internship report Netherlands Institute for Neuroscience

November 2009 – Augustus 2010

Student: Tessa van der Maaden

Studentnr: 5888123

Master: Brain & Cognitive sciences, track neuroscience Supervisor: Jeroen Melief

Co-assessor: Dr. Inge Huitinga Examinor: Prof. Dr. Frank Baas

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List of abbreviations

CC - Corpus callosum

CD200R - CD200R

CNS - Central Nervous System

CP - Choroid plexus

FACS - Fluorescence Activated Cell Sort

GR - Glucocorticoid receptor

HLA - Human leukocyte antigen

IL - Interleukin

MRI - Magnetic resonance imaging

MR - Mannose receptor

MS - Multiple Sclerosis

NAWM - Normal appearing white matter

Q-PCR - Quantitative polymerase chain reaction

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Abstract

Although macrophages and microglia are both prominent populations of cells present in active expanding MS lesions many studies focus on macrophage biology only. Macrophages can be activated into different phenotypes. Activation patterns of microglia are unknown.

Microglia normally function by scanning the environment and contribute to homeostasis but in MS they appear to react differently. This study aims to validate the flow cytometric procedure used to isolate human micoglia and choroid plexus macrophages, to compare the activation patterns of human microglia to macrophages, and to compare the reactions of these cells between control and MS brain. It is hypothesized that unlike macrophages, microglia will not be alternatively activated after stimulation with anti-inflammatory stimuli. In MS microglia phenotype will be different, and microglia might be alternatively activated by anti-inflammatory stimuli. Human corpus callosum and choroid plexus tissue was obtained for isolation of microglia and macrophages. Cells were sorted by FACS, using CD11b expression. After stimulating and culturing cells with IL-4 and dexamethasone gene-expression of different activation markers was tested. Discarding autofluorescense enabled analysis of CD45 and CD11b positive populations in corpus callosum and choroid plexus cells. Unlike murine brain, human corpus callosum showed only one CD45 positive population. Microglia showed to be smaller and less granular than macrophages. Few differences were found in gene-expression between control microglia and macrophages.

When comparing MS to controls, only IL-10 showed differences in gene-expression.

Differences found between control microglia and macrophages and between controls and MS were minimal. However, the flow cytometric method used to isolate human microglia and macrophages has proved to be succesful, providing good prospects for future research on human microglia.

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Introduction

Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS). It is the most common neurological disease among young adults, starting usually round the age of 20-30. Worldwide, over 2.5 million individuals are affected.

The disease MS has a heterogeneous pathology. The pathological hallmarks of the disease are the destruction of the myelin layer around the axons in the white matter, axonal damage and neurodegeneration. Demyelination has direct impact on the axons’ signal conducting properties and leads to neurological symptops like sensory loss, paralysis and cognitive decline.

Although the etiology of MS is still unclear, it is apparent that multiple factors might play a role, such as viral infections, genetic predisposition and vitamin deficiency. Certain mechanisms are known to contribute to MS pathogenesis, among which the complement system and auto-reactive T-cells. Strong evidence from both animal and human studies points out that there is a prominent role for myeloid cells, like macrophages and microglia in disease pathogenesis [1-4].

Macrophages form the first line of defence againts infiltrating pathogens. The high expression of human leukocyte antigen (HLA) makes the macrophage a potent antigen- presenting cell.

Microglia are regarded as the resident macrophages of the brain, and form the largest population of immune cells in the CNS. In contrast to macrophages, microglia in healthy brain are considered to contribute to brain homeostasis. In unactivated state the cells show a highly ramified morphology, enabling them to scan the environment for abnormalities [5, 6]. Under normal conditions, microglia clear the environment by phagocytozing debris and apoptotic cells without inflammation [7]. Nonetheless, the cells respond quickly to signalling molecules released in pathogenic conditions, causing microglia activation. Upon activation, microglia loose their ramified morphology, and drastically change their phenotype. Immunoreactivity is induced in the cells by upregulation of molecules HLA, CD45 and CD11b; cells become capable of antigen presentation. Activated microglia might become involved in the initiation of acute inflammatory responses, and the phagocytosing of damaged tissue [8-10].

In MS the activated microglia cell is unable to keep homeostasis. The myelin phagocytozing cells originating from these activated microglia contribute to the maintenance of the inflammation [8].

Acute MS lesions are characterized by hypercellularity and perivascular infiltration of T- and B-cells and macrophages. Macrophages in this lesion type are actively participating in myelin breakdown [11]. The active MS lesion can change into a chronic active lesion, which

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is characterized by a hypocellular, demyelinated centre, surrounded by a rim with high amounts of myelin-laden macrophages. These macrophages originate from both resident microglia and infiltrating macrophages [9]. So-called foamy macrophages have a distictive morphology developed by the ingestion of myelin, and are characterized by an anti- inflammatory phenotype [12]. By unknown mechanims the inflammation resolves at some point, few macrophages remain present, and the chronic active lesion moves into an inactive stage.

Although many studies focus on macrohage phenotype in MS, the role of microglia was undefined.

Macrophage activation

Macrophages and microglia are the main populations of cells present in active expanding MS lesions [11, 13]. Macrophages originate from circulating peripheral-blood mononuclear cells, which in their turn are derived from myeloid progenitor cells. Once mature, macrophage functions include antigen presentation, phagocytosis and scanning the environment for foreign substances. Macrophages are the primary sensor for pathogens and changes in the microenvironment [14, 15]. Peripheral macrophages are able to migrate into other tissue, after which they further differentiate into resident macrophages, such as microglia in the CNS. While macrophages are able to activate into different phenotypes, microglia might show different activation patterns.

The phenotype of macrophages can be influenced by factors released following tissue injury, signals produced by antigen presenting cells, and autocrine stimuli. Previously, different macrophage phenotypes were classified into two main directions: classical activation (M1) and alternative activation (M2) [16]. Later, the M2 direction was further subdivided into the three phenotypes: M2a to c (see figure 1) [17]. An overview of the different types of macrophage activation and their markers is presented in table 1.

The M1 phenotype is most commonly seen during immune responses. These so-called classically activated macrophages are induced by interferon-γ (INFγ), in combination with microbial products like LPS or cytokines like tumor-necrosis factor (TNF). The M1 cells have a high antigen presenting capacity. Furthermore they produce high amounts pro- inflammatory cytokines, and reactive oxygen and nitrogen radicals, resulting in enhanced endocytic funtions and microbicidal activity [18]. This phenotype is critical in the elimination of pathogens, although uncontrolled inflammatory responses may lead to tissue injury. Taking into account the damaging effector functions and the release of pro- inflammatory cytokines, the reactive myelin phagocytozing cells in MS highly resemble M1 macrophages [11].

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Figure 1: Different phenotypes of activated macrophages [17]

In contrast to M1 macrophages, alternative activated (M2) macrophages are mainly involved in the supression and regulation of immune reactions. These M2 macrophages can be subdivided into three different phenotypes.

The first of these three: the M2a phenotype is also referred to as ‘wound-healing’. This type of activation is induced by interleukin 4 (IL-4) and interleukin 13 (IL-13); cytokines secreted by Th2 cells, basophiles and mast cells in response to tissue injury [19, 20]. IL-4 induces production of anti-inflammatory cytokines such as IL-10 and simultaneously inhibits expression of pro-inflammatory cytokines. Moreover, by secreting components of the extracelular matrix, M2a macrophages contribute to tissue repair. This specific phenotype is characterized by upregulated expression of the mannose receptor (MR) [21].

Other anti-inflammatory macrophages have differentiated into the M2b or M2c phenotype.

These cells are called ‘regulatory’, and although all M2 cells have anti-inflammatory properties, M2b and M2c effector functions differ from the ‘wound healing’ macrophages [22]. The M2b cells develop when stimulated with immune complexes, in combination with interleukin 1β (IL-1β) or lipopolysaccharide (LPS). Their phenotype is characterized by high interleukin 10 (IL-10) and low interleukin 12 (IL-12) production [17].

Cells differentiated into the M2c pathway are deactivated macrophages, in which the gene- expression of pro-inflammatory cytokines is downregulated. The M2c phenotype is induced by stimulation with, among others, glucocorticoids. Glucocorticoids are able to termitate immune responses by enhancing the clearance of foreign antigens, toxins and death cells.

They do so by enhancing opsonisation and the activity of scavenger systems. Furthermore, macrophage phagocytotic functioning and antigen uptake is stimulated [23, 24]. M2c macrophages are able to regulate, or even inhibit the duration and intensity of immune responses [25]. M2c macrophages show increased expression of the cytokine IL-10, and cell markers CD163 and CCL18 [25, 26].

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Although functions differ, all described pathways of macrophage activation are necessary in healthy brain, whether it is to form a line of defence to pathogens, promote wound healing, or regulate immune reactions.

Macrophage phenotype and biology is for a large extent elucidated and its activation patterns are described above. Although microglia are known to be easlily activated, it is unknown whether these CNS cells show activation patterns similar to those of peripheral macrophages.

Induced by Marker

Classical activation M1 IFNγ, TNF iNOS

IL-12

Alternative activation M2a IL-4, IL13 MR

CD200R CCL18 M2b Immune complexes with LPS or IL-1β IL-10 M2c IL-10, glucocorticoids, TGF-β IL-10 CD163 CCL18

Table 1: Different macrophage phenotypes, substances inducing the different phenotypes and markers charachterizing the activated macrophages.

Working model MS

In figure 2 a symplified model for the disease MS is shown, with coloured balls representing different stages of microglia activation. M1 is the reactive macrophage phenotype, resembling the phenotype of macrophages seen in MS lesions. These cells have a pro-inflammatory phenotype and actively phagocytoze myelin. Ingestion of myelin alters the cells so that they obtain a foamy appearance. These foamy macrophages are called M2 macrophages and are involved in wound healing and tissue repair. The M2 cell might be responsible for the inhibition of further lesion expansion in MS and contribute to the resolution of inflammation [12]. Due to unkown factors, cells might become reactivated into an M1 phenotype as is seen in chronic active MS lesions, or go back in to a resting M0 stage. What induces the differentiation steps to the various phenotypes is unsolved.

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Figure 2: Working model for MS, M0: healthy situation, M1: classical activation, M2:

alternative activation ‘wound healing’.

Isolation and charachterization microglia by flow cytometry

Macrophages are easily isolated from for example blood. However, isolation of microglia from human tissue is problematic because these cells are embedded in brain tissue and are easily activated. However, to learn about microglia differentiation in reaction to stimulation, it is necessary to obtain inactivated cells.

Studies with murine microglia were performed with cells obtained by extended culture of newborn CNS precursor cells [27-29]. Isolation methods were mainly based on adhesive properties, and purity was examined by assessment of the morphology and immunohistochemical analysis. The main disadvantage of these methods is the high risk for contamination with, among others; astrocytes and CNS associated peripheral macrophages. Furthermore, surface adherence of the cells in culture initiates microglial activation.

Isolation, and characterization of microglia from adult animal or human CNS tissue is particularly problematic due to the lack of specific cell surface markers. Although the morphology of microglia is well defined, the principal cell markers of this cell type are shared with that of other blood derived mononuclear cells [30].

Sedgwick and colleagues developed a method to isolate microglia from rodent, and later also human brain, based on the common leukocyte antigen CD45 and myeloid marker CD11b with fluorescence activated cell sort (FACS) [31-33].

After FACS analysis of CD45 expression on murine brain cells, a clear distinction between two populations of CD45 positive cells was found. Based on information from in situ immunohistochemical analyses it was considered that the major, CD45^low population probably consisted of microglia [31]. This assumption was later confirmed with results of irradiation bone marrow chimera experiments in CD45 congenic animals [32]. The other CD45 positive population found was CD45^high and comprised blood-derived or infiltrating macrophages. Both cell populations were CD11b positive (see figure 3) [34].

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Figure 3: FACS analysis of rat brain CD45 and CD11b expression. Three populatons were found.

R1: microglia (CD11b⁺CD45^low); R2:

macrophages, monocytes, granulocytes (CD11b⁺CD45^high); R3 lymphocytes (CD11b^- CD45^high) [34].

In our group, a functional procedure to isolate, sort, and culture resident microglia from post mortem human brain tissue was developed based on the method developed by Sedgwick and colleagues. However, CD45 low and high populations were hard to distinguish because autofluorescent cells overshadowed populations of interest (see figure 4) (unpublished data).

Figure 2: FACS analysis human blood, choroid plexus and corpus callosum cells. 1:

lymphocytes, 2: monocytes, 3: granulocytes [35].

Figure 4: FACS analysis human blood, choroid plexus and corpus callosum cells. 1:

lymphocytes, 2: monocytes, 3: granulocytes [35].

CD45 CD11b

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Aim of the study

Aim A

The first aim of the study is to validate the isolation method used, by excluding the possiblity that other cells contaminate the analysis. Flow cytometry is used to isolate and characterize macrophages and microglia from respectively post mortem choroid plexus and corpus callosum tissue, after which these cells are cultured and stimulated in vitro.

Aim B

The second aim is to examine whether microglia and macrophages’ gene expression patterns differ. Macrophage differentiation mechanisms are to a large extent established, and are known to play a prominent role in MS pathology. Although microglia are easily activated, and their reaction might escalate under pathologic events, their primary funtion in healthy brain is to maintain homeostasis. However, the microglia phenotype, and reactivity in healthy brain remains mysterious. Furthermore, it is unknown what signals cause the activation of microglia into myelin phagocytozing cells. Learning more about the phenotype of the unactivated microglia will lead to a better understanding of their role in neurological diseases like MS. By stimulating microglia isolated from healthy brain with anti-inflammatory stimuli, part of the microglia activation phenotype might be elucitated.

Aim C

Finally, the gene-expression of markers for alternative activation in microglia and macrophages from healthy brain will be compared to the same cell types isolated from NAWM derived from MS brain tissue. Microglia are prominently present in expanding MS lesions. By unknown mechanisms these cells can be activated into myelin phagocytozing macrophages. Using microglia from NAWM, it will be examined whether these microglia in healthy looking brain already act differently as compared to control brain microglia.

Hypothesis

Macrophages have shown to become alternatively activated upon stimulation with anti- inflammatory stimuli like IL-4 and the synthetic glucocorticoid dexamethasone, respectively into an M2a and M2c direction. Although microglia are regarded as the resident macrophages of the brain, their primary function is to tightly control and suppress local immune responses and mantain homeostasis. Using an infection model with transgenic mice overexpressing IL-13, IL-4 and IL-13 deficient mice, and wild type, it has been demonstrated that exclusively macrophages and not microglia were alternatively activated [36].

Taking into account these findings and the primary functions of microglia, the first hypothesis of this study is that microglia isolated from healthy white matter will exhibit differentiation patterns that differ from established macrophage differentiation pathways.

Microglia differentiated into activated macrophages are prominently present in expanding MS lesions and highly contribute to myelin phagocytosis. However, it is unknown why or

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when microglia switch from a normal to a pathogenic phenotype. Therefore, it is hypothesized that microglia in MS NAWM exhibit aberrant differentiation patterns possible due to an altered basal activation status compared to microglia from non-MS brain tissue.

This may lead to altered susceptibility and activation features in reaction to activating stimuli.

To test these hypotheses microglia are isolated using a protocol that was specifically developed to obtain a highly pure population of microglia using fluorescence activated cell sorting. Subsequently, microglia from white matter derived from non-MS brains and microglia from NAWM are stimulated in vitro with IL-4 and dexamethasone, both key anti- inflammatory stimuli that induce alternative activation, and compared to similarly stimulated choroid plexus macrophages, which represent brain-associated macrophages.

Finally, microglia differentation characteristics are assessed with the aid of various differentation markers to address several questions. Do microglia show the same differentation patterns as macrophages. More specifically, are they alternatively activated by anti-inflammatory stimuli? Is there a difference in the differentiation pattern of microglia from non-MS white matter and those from NAWM?

The microglia phenotype is compared to that of choroid plexus macrophages. Furthermore, human microglia isolated from non-MS brain are compared to the same cells isolated from MS brain. The primary subjects of this study are human microglia. However, also murine microglia were included in the study because of their well-established phenotype, and the limited availibity of human tissue.

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Materials and methods

Cell isolation and characterization

Tissue

The post mortem brain tissue from control donors, Alzheimer’s disease patients, and MS patients used in this study was provided by the Netherlands Brain Bank (NBB). Permission was given by the donor for brain autopsy and the use of tissue and clinical information for research purposes.

At the autopsy, normal appearing corpus callosum white matter and choroid plexus tissue was obtained, and stored in Hybernate A medium (Brain Bits LLC, Springfield, USA) at 4˚C. Corpus callosum from MS donors was normal appearing following macroscopic and on MRI observations. Tissue was used from 10 brain donors. One healthy control donor was excluded because its post mortem delay exceeded 15 hours. The mean age of the donors was 81 (range 72-99). Mean post mortem delay was 5.15 (range 2.55-10.00).

Three MS donors were included. The non MS group consisted of 2 healthy controls, 2 AD donors, 1 epilepsy, 1 LBD and 1 depression. Despite the variation in donors in the non MS group, their gene expression data were put together to form 1 ‘control group’. Table 2 and 3 lists the data of the brain donors used in this study.

NBB nr Diagnosis Sex Age PMD

08-100 MS f 77 10:00

09-008 MS f 81 7:17

09-036 MS m 72 7:55

Table 2: Data of used MS donors; Autopsy: number of autopsy, NBB nr: Netherlands Brain Bank number, PMD: post mortem delay, Br. Wgt: brain weight

NBB nr Diagnosis Sex Age PMD

08-076 Depression f 91 5:20

08/078 LBD m 63 6:05

08/081 Epilepsy m 91 6:50

08-104 AD f 82 7:15

09-021 Control f 99 4:15

09-022 Control f 77 2:55

09-033 Pick’s disease m 69 3:55

Table 3: Data of used non MS donors; Autopsy: number of autopsy, NBB nr: Netherlands Brain Bank number, PMD: post mortem delay, Br. Wgt: brain weight

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Human cell isolation

Corpus callosum (ongeveer 4-8 gram) and choroid plexus tissue were meshed through a metal sieve in a buffer containing 8 g/l NaCl, 0.4 g/l KCl, 1.77 g/l Na2HPO4.2H2O, 0.69 g/l NaH₂PO₄.H₂O, 2 g/l D-(+)-glucose and 3,3 g/l BSA (pH 7.4)). Disrupted choroid plexus tissue was collected and transferred into a 50 ml tube; disrupted corpus callosum tissue was divided amongst three 50 ml tubes. Sieves were then rinsed with GKN/BSA, and suspensions were collected in the same tubes and filled to 50 ml. Subsequently cell suspensions were centrifuged for 7 minutes, at 1400 RPM after which the supernatant was discarded. The pellet was resuspended in 4 ml dissociation buffer (4 g/l MgCl₂, 2.55 g/l CaCl₂, 3.73 g/l KCl, 8.95 g/l NaCl, pH 6-7), and subsequently enzymatically digested with 400 μl (150 U) Collagenase Type I (Worthington, Lakewood, NJ, USA) and 125 μl (200

g/ml) DNase I (Roche Diagnostics, Mannheim, Germany) for 1 h at 37°C. During incubation the tissue was further mechanically disrupted by passing it through a p1000 pipette tip with decreasing bore size every 15 minutes.

The cells were washed with GKN/BSA (centrifuging for 7 minutes at 1400 RPM and discarding the supernatant), and taken up into a percoll gradient; the cells were resuspended in 20 ml percoll (ρ1.03), then underlain with 10 ml percoll (ρ1.095), and overlain with 5 ml GKN/BSA. This gradient was centrifuged for 35 minutes at 2500 RPM, with slow acceleration and no break.

After the centrifugation, the layer of myelin formed on top of the percoll (ρ1.03) – GKN/BSA interface was discarded. The layer of cells located at the percoll (ρ1.095) - percoll (ρ1.03) interface was collected and transferred into a new 50 ml tube. The cells were cells were washed and resuspended in 50 μl GKN/BSA and subsequently counted in a cell counting chamber using tryptan blue.

FACS analysis

The cells were stained with phycoerythyrin (PE) labeled antibody against CD11b (Clone ICRF44, DakoCytomation, Glostrup, Denmark) and fluorescein isothiocyanate (FITC) labeled CD45 (Clone HI30, Dako), or with isotype matched controls in a concentration of 1:5 based on a total cell amount of 1*10⁶. The staining was incubated in the presence of 30 % human pool serum (HPS), for 30 minutes on ice. Subsequently, cells were washed by centrifuging for minutes at 1400 RPM and resuspended in 100 μl GKN/BSA. 15 Minutes before analysis, 2.5 μl per 100 μl cell suspension 7-Aminoactinomycin D (7AAD, BD Biosciences) was added. The cells are analyzed with a FACSCalibur flow cytometer (BD PharMingen, San Jose, CA, USA) and the data were analyzed using FlowJo software version 8.7.1 (Treestar, Inc. Ashland, OR, USA).

Cell sort

The cells were stained with phycoerythyrin (PE) labled CD11b (Clone ICRF44, DakoCytomation, Glostrup, Denmark) in a concentration of 1:5 in a total volume of 100 μl with addition of 30 % human pool serum (HPS). A number of cells varying between 2E5 - 1E6 was used per staining. This mixture was incubated for 30 minutes on ice. After

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washing, the cells were resuspended in 500 μl GKN/BSA and sorted on a FACS Aria cell sorter (BD Biosciences) to obtain a pure CD11b⁺ population to culture. The number of isolated CD11b⁺ cells ranged from 3.7 E+05 – 7.0 E+05 (corpus callosum) and 1.5 E+05 – 1.4 E+06 (choroid plexus).

Cell culture

The sorted CD11b⁺ cells from the corpus callosum and choroid plexus were cultured in a 96-wells culture plate with 60.000 cells per well in 200 μl RPMI with 1% heat-inactivated HPS for 72 hours. The cells were stimulated with IL-4 (5 ng/ml). Dexamethasone stimulation was done with at concentration of 2 µM in the same culture conditions.

Quantitative Polymerase Chain Reaction (Q-PCR)

RNA isolation

The cultured cells were washed in phosphate buffered saline (PBS) and lysed in 800 μl TRIzol reagent (Invitrogen). 160 μl Chloroform (Sigma) was added and the mixture was vortexed for 15 seconds, and centrifuged for 15 minutes at 12.000 g. For RNA isolation from cells of 2 donors (NBB: 09-021, 09-022, also see table 2) an alteration was applied in the RNA isolation method by using Phase Lock gel tubes for the separation of phases. 350 μl of the overlying aqueous phase is taken off and transferred into a new tube, after which an equal volume of 70 % ethanol is added carefully.

The mixture was then transferred into a RNeasy Mini Kit column (Qiagen, Hilden, Germany). The RNA isolation was further executed according to the protocol supplied by the manufactor. Finally, RNA was eluted in 30 μl RNAse free water. After isolation, the concentration of the isolated RNA was determined using a nanodrop (ND-1000, NanoDrop Technologies, Rockland, DE, USA). RNA quality was assessed using a Bioanalyzer (2100, Agilent Technologies, Palo Alto, CA).

cDNA synthesis

Reverse transcriptase was done using the Quantitect RT transcription kit (Qiagen Science).

1-12 μl RNA with a concentration varying between samples, and RNAse free water was added to 12 μl, together with 2 μl gDNA wipeout buffer (7x) was incubated at 42˚ C for 2 minutes. A master mix of RT (1 μl), RT buffer (4 μl), and RT primer mix (1 μl) was added.

The mixture was incubated at 42˚ C for 15 minutes, followed by incubation at 95˚ C for 3 minutes to inactivate the RT. cDNA samples were stored at –20 °C until used for further analysis.

Primer design

An overview of the forward and reverse primers used for Q-PCR analysis is given in appendix 1. Primers are intron-spanning and were designed with the PrimerS software.

Primer sequences were blasted with NCBI primer-BLAST to ascertain their specificity for the selected genes. Further testing of primers was done with Q-PCR on tonsils and human

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NAWM cDNA, by assessing amplification and dissociation curves. Predicted sizes amplicons were confirmed using polyacrylamide gel electrophoresis.

Q-PCR analysis

Q-PCR measurements were done with the 7300 Real Time PCR system (Applied Biosystems) using 5 ng cDNA per reaction in a final volume of 20 μl. Per reaction 10 μl SYBRgreen PCR Master Mix (Applied Biosystems), and 2 μl primermix (2 pmol/μl) was added.

Expression of selected genes was normalized to references genes 18s and EF-1α, efficiencies (E) of primerpairs were determined using LinRegPCR software. Absolute expression was calculated by: E-CT target / E-CT reference gene.

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Results

Cell isolation

FACS analysis

FACS analysis was done on choroid plexus and corpus callosum cells isolated from a Pick’s disease donor. Because of previous problems with autofluorescence, probably caused by death or dying cells [35] 7AAD viability staining was done to accomplish a negative selection for death or dying cells. Results of the 7AAD staining are shown in figure 5.

Figure 5: 7AAD viability staining of isolated choroid plexus cells (a,b) and corpus callosum cells (c,d), double negative staining CD11b and CD45 (a,c) and double positive staining (b,d).

Discarding 7AAD positive cells from the FACS analysis resulted in a disappearance of autofluorescent cells showing populations with a variety of CD11b and CD45 expression (figure 6).

7AAD

FSC-Height c

7AAD

a

FSC-Height

b

d

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Figure 6: a,b: isolated cells from choroid plexus; c,d:isolated cells from corpus callosum. a, c: double negative staining with isotypes for CD11b and CD45 in isolated cells from choroid plexus (a,b) and corpus callosum (c,d). b, d: double positive staining for CD11b and CD45 Circled population is CD11b and CD45 positive.

Negative control stainings for CD45 and CD11b corpus callosum (CC) cells showed no CD45 and CD11b positive populations in CC cells, demonstrating that no aspecific binding of antibodies has occurred. In CP cells however a population with a very low expression of both CD11b and CD45 was found.

Isolated CP and CC cells both show 2 distinct cell phenotypes: a CD45⁺CD11b^- lymphocyte and a CD45⁺CD11b⁺ population. The CD45⁺CD11b⁺ population displayed in CP cells shows a CD45 expression level similar to that of the CD11b^- population of lymphocytes, which are well known for their high expression of CD45. This indicates that the high CD45 expressing CD11b⁺ cells isolated from CP have CD45 expression levels similar to that of lymphocytes and may thus be considered to be CD45^high macrophages. The other CD11b positive population isolated from CP (see also figure 5b) has a lower CD45 expression intensity, and almost certainly consists of granulocytes. The circled cells in figure 5d have to be microglia, since they exhibit a relatively low expression of CD45 when compared to the CD45^high CP cells in figure 5b. After discarding the autofluorescent cells, only one population of CD45 positive cells is found, unlike what is seen in rat and mouse brain.

CD45 CD11b

Isotype

b

d a

c

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18

The lymphocyte population in the population cells from CC only consists of two events.

Nevertheless, it is clear that the CD45⁺CD11b⁺populationhas a CD45 expression intensity that is lower compared to CD45⁺ lymphocytes in both the CC and the CP cell population.

This indicates a phenotypical difference between CD45⁺CD11b⁺ cells isolated from CP and CC.

Cell sort

Sorting of the CC and CP cells was done by staining only with the marker CD11b. This was done because it was impossible to distinguish between different CD45 expression intensities due to autofluorescence when the technique was first set up. In figure 7 images are shown of the cell sort, with back-gating of CD11b positive populations. Forward scatter is a measure for cell size; side scatter provides information about cell granularity. Size and granularity between cells isolated from CP (sample macrophages) and CC (sample microglia) from a MS donor and a healthy control donor were compared.

Figure 7: Cell sort picture of macrophages (a,c) and microglia (b,d) isolated from a healthy control donor (a,b) and a MS donor (c,d). Green (P1) CD11b positive populations after back-gating. FSC: forward scatter, SSC: side scatter.

MS donor (NBB: 09-036) MS

MS donor (NBB 09-036) MS

Control donor (NBB: 09-022) Control donor (NBB: 09-022) MS

a b

c d

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When control macrophages were compared with control microglia, a distinct phenotype was found. Macrophages are bigger than microglia and show a much higher granularity.

Macrophages from the MS donor are bigger and less granular when compared to control macrophages. MS microglia show a much higher forward- and side scatter, indicating that they are both bigger and more granular than healthy microglia.

The total amount of isolated cells from choroid plexus and corpus callosum tissue varied strongly between different donors. This phenomenon can to some extent be explained by varying sizes of the tissue sample obtained. Other variations in cell numbers were possibly caused by isolation procedures. For example, when an unclear cell layer was found after centrifuging the percoll gradient the collection of the cells was hampered. Finally, cell densities in brain tissue might be subject to natural variation between individual donors leading to variations in the amount of isolated cells.

Cell culture

Human microglia and macrophage morphology was monitored during cell culturing. In figure 8 isolated microglia and macrophages are shown after 72 hours culture. Microglia show a small and rounded morphology, in contrast with the bigger and more elongated branched appearance of the macrophages.

Figure 8: Morphology of human microglia (left) and macrophages (right) after 72 hours of culture without stimulus. Donor NBB: 09-036.

Microglia Macrophages

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RNA quality and Q-PCR

Most RNA samples showed RNA integrity numbers (RIN) ranging between 7 and 9.9.

However, microglia samples of 2 control donors repeatedly showed RIN values of 1.

Because no degradation was seen in the bioanalyzer images, and the effect is only seen in microglia, it is expected that findings of low RIN values were caused by disturbed bioanalyzer measurements, possibly due to contamination of the RNA. For this reason samples were nonetheless included in the experiment.

The CT of CD200R and MR ranged from 30-36, implying a low expression of this molecule on isolated cells. In microglia and macrophages of the 2 healthy control donors CD200R expression was in most samples not detectable at all. Although initial expression in unstimulated sampels is very low, many samples upregulated CD200R expression after stimulation with IL-4. Although initial expression is very low, and absolute expression might be regarded as unreliable, it could still be concluded that CD200R was consistently upregulated.

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Gene expression

Data are presented as fold difference, making comparisons on three elements. First gene expression of cells with and without stimulation was compared. Secondly differences between macrophages and microglia in both the control group as the MS group were examined. Finally, expression data of control group and MS group were compared.

Appendix 1 shows an overview of the results.

Interleukin 4 stimulations

Data are shown for expression of IL-4 binding receptors (IL-4R, IL-13R), an immune acivation marker (HLA-DR) and three different M2a differentiation markers (MR, CD200R, CCL18) after 3 days of cell culture with or without the presence of 5 ng/ml IL-4. The concentration of the IL-4 stimulus was chosen based on those used in literature to activate macrophages [21, 37, 38].

Expression of IL-4R and IL-13R was determined to examine whether the initial expression levels could affect stimulus strength. Absolute expression of IL-4R and IL-13R after IL-4 stimulation is shown in figure 9 and 10. No differences were found in absolute expression levels of unstimulated cells between MS and control group.

Figure 9: Absolute gene-expression levels of IL-4R in microglia and macrophages macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Non-MS (left) and MS brain donors (right).

0 10 20 30 40 50

IL-4 - + - +

Microglia Choroid plexus macrophages

Relative ratio (AU * 106 )

0 20 40 60

IL-4 - + - +

IL-4R IL-4R

Non MS MS

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Figure 10: Absolute gene-expression levels of IL-13R in microglia and macrophages macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Non-MS (left) and MS brain donors (right).

Stimulation with IL-4 increased IL-4R expression in macrophages from the MS group compared to unstimulated cells (p = 0,021). In microglia and macrophages from the control group, only trends for downregulation (p=0,74; 0,34) were found as is shown in figure 11. Figure 12 shows that IL-13R was downregulated after IL-4 stimulation in control macrophages (p = 0,0027) and both MS microglia (p= 0,035) and macrophages (p = 0,048).

Figure 11: IL-4R gene-expression in microglia and macrophages from non-MS (left) and MS brain donors (right) before and after stimulation with IL-4. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right). Upregulation IL-4R in MS macrophages: p = 0,021.

*

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Figure 12: IL-13R gene-expression in microglia and macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right). Downregulation control macrophages: p = 0,0027. Downregulation microglia and macrophages p = 0,048, p = 0,035.

HLA-DR plays a central role in eliciting CD4⁺ T-cell responses and is upregulated in immune activation. The immune activation marker is typically found on antigen presenting cells like B-cells, dendritic cells and macrophages, but also on microglia. In microglia and macrophages in both groups, expression of HLA-DR was not upregulated after IL-4 stimulation (figure 13).

Figure 13: HLA-DR gene-expression in microglia and macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

MR is an established marker of the M2a macrophage activation, and is expected to be upregulated after IL-4 stimulation [21, 38, 39]. Its expression showed a tendency for 0

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upregulation in microglia and macrophages from both control (p = 0,33; 0,63) and MS brain (p = 0,42; 0,15) (also see figure 14).

Figure 14: MR gene-expression in microglia and macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

Expression of CD200R was determined, since our group found earlier that the molecule – which plays an essential role in suppression of immune responses – was strongly upregulated in peripheral macrophages in vitro after stimulation with IL-4 (unpublished results) [35]. Results of stimulations are shown in figure 14. After simulation with IL-4, its expression in control microglia remained unchanged. CD200R expression appears to be upregulated in control macrophages (p = 0,12), and MS microglia and macrophages (p = 0,45; 0,23).

Figure 15: CD200R gene-expression in microglia and macrophage after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

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CCL18 is a marker for both M2a and M2c macrophage differentiation [17, 40]. The cell marker tended to be upregulated in both microglia and macrophages in control (macrophages: p = 0,078) and MS group (p = 0,30; 0,23) after IL-4 stimulation. Fold differences however, were higher in the MS group, compared to the controls as is shown in figure 16.

Figure 16: CCL18 gene-expression in microglia and macrophages after 72 hours culturing with IL-4 stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

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Dexamethasone stimulations

Dexamethasone is a synthetic glucocortoid. Gene expression data are shown of the dexamethasone binding receptor glucocorticoid receptor (GR) and 3 different M2c differentiation markers (IL-10, CD163, CCL18) after culture with or without the presence of 2 μM dexamethasone. Concentration of the dexamethasone stimulus was chosen based on concentrations used in literature [40-42].

GR expression changes were measured to examine differences in initial expression between groups. The expression data are shown in figure 17. GR expression was significantly downregulated in macrophages in both control and MS group (p = 0,024;

0,035). No concrete differences were found in microglia.

Figure 17: GR gene-expression in microglia and macrophages after 72 hours culturing with dexamethasone stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right). Downregulation GR in control macrophages: p = 0,024. Downregulation GR in MS macrophages: p = 0,035.

The cytokine IL-10 earlier showed a downregulated gene expression in macrophages [43, 44]. The changes in IL-10 expression found in this experiment are shown in figure 18. In the control microglia and macrophages no differences in IL-10 expression were found. In the MS group the cytokine was upregulated in microglia from the MS group (p = 0,043).

This effect was significantly different from the reaction in controls (p = 0,017). In MS macrophages only showed a tendency for upregulation (p = 0,15).

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Figure 18: IL-10 gene-expression in microglia and macrophages macrophages after 72 hours culturing with dexamethasone stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

Upregulation IL-10 in MS microglia: p = 0,043. Difference MS and non MS: p = 0,017.

A hallmark for the M2c macrophage phenotype is an upregulation of the cell surface molecule CD163 [40]. In this study the molecule did tend to upregulate in both microglia and macrophages of control (p = 0,10; 0,27) and MS group (p = 0,40; 0,10) with an average fold difference of 5. No differences between cell types en groups were seen in CD163 expression (see figure 19).

Figure 19: CD163 gene-expression in microglia and macrophages after 72 hours culturing with dexamethasone stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

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CCL18 is a marker for both the M2a as the M2c pathway [17]. No reliable measurements were done of CCL18 expression in control microglia after dexamethasone simulation. The stimulation did not affect the expression of CCL18 in control macrophages. In the MS group, CCL18 gene expression tends to be upregulated in both cell types (p = 0,38; 0,45) (see figure 20).

Figure 20: CCL18 gene-expression in microglia and macrophages after 72 hours culturing with dexamethasone stimulation, compared to unstimulated cells. Results in fold difference, unstimulated cells: 1. Non MS group (left) and MS group (right).

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Discussion

The first aim of this study (aim A) was to validate the described procedure to isolate and characterize microglia and choroid plexus macrophages out of post mortem human brain tissue using FACS. The isolation method enables in vitro culture and stimulation of the cells after which their gene-expression patterns can be monitored. Using these isolated cells we aimed to examine the differentiation characteristics of human microglia compared to choroid plexus macrophages (aim B). The third aim (aim C) was to investigate whether microglia in NAWM from MS brain tissue exhibit different phenotypic features and differentiation characteristics when compared to microglia from non-MS brain tissue.

Aim A: Validation of the isolation protocol

In this study the autofluorescent cells overshadowing CD11b positive populations were discarded, revealing a single CD45 positive population in CC cells. After three days of culture microglia and macrophages showed a distinct morphology. It was concluded that sorting cells on CD11b expression only was sufficient to obtain a pure microglia population from corpus callosum tissue, and that the procedure used for isolation was succesful.

Sorting macrophages and microglia was based only on a high expression of the myeloid marker CD11b because distinguishing cells by their CD45 expression was not possible due to autofluorescent cells. However, by using 7AAD autofluorescence was discarded from the analysis, resulting in a clear view on CD45 positive populations. Choroid plexus CD11b positive population, which comprised macrophages, showed a CD45 expression similar to lymphocytes. Another population with a lower expression of CD45 consisted of granulocytes. However, granulocytes do not survive in culture [45], so gene expression data should not be affected. In contrast to choroid plexus cells, in corpus callosum cells only a population with low CD45 expression was found, indicating that the CD11b positive population in corpus callosum consisted only of microglia, which are well known for intermediate CD45 expression.

By discarding the autofluorescensce, FACS analysis showed a CD45^highpopulation in CP cells and a CD45^dim population in CC cells. This indicated that when CC cells were only stained with CD11b, a population microglia was obtained without contamination by other cells.

Isolated CP and CC cells showed differences in size, granularity and morphology.

Furthermore, microglia from the MS group showed much bigger size and higher granularity when compared to the control group (based on FSc-SSc). This indicates that microglia from MS corpus callosum might be activated. The macrophages from MS brain are bigger, but show a lower granularity compared to macrophages from non-MS brain tissue. This might be due to the ingestion of myelin, which induces foamy macrophages functioning differently as compared to the myelin phagocytozing cells.

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In this study, isolation of brain cells was done using 6 brain donors among which three MS and six non-MS donors. Including two AD donors was particularly riskfull, since the quiescent microglia phenotype might be disrupted due to inflammatory changes in AD brain. However, the tissue used for cell isolation was pure white matter from corpus callosum, an anatomical location where AD-pathology is hardly ever present. Therefore, including AD donors in the control group will most likely not bring about unreliable results.

Aim B: Differentiation characteristics – Microglia vs macrophages

It was hypothesized that, unlike macrophages, microglia isolated from healthy brain tissue would not be alternatively activated by anti-inflammatory stimuli.

Initial expression of IL-4 and dexamethasone binding receptors in the MS group was comparable to the controls, indicating that binding receptors did not influence the strength of the stimulus. When comparing unstimulated cells and stimulated cells from healthy brain significant differences were only found in receptor expression. In addition to these findings many tendencies for upregulation of activation markers were seen in both IL-4 and dexamethasone stimulated cells, indicating that included cell surface markers and cytokines were potent markers for alternative activation.

Only a few donors were used in the study, and this might be the reason that many markers only showed a tendency for upregulation. Increasing the power of the study may compensate for the variation in gene-expression between donors, leading to more robust data.

Alternative activation in macrophages into M2a direction is induced by stimulation with the cytokines IL-4 and IL-13. Differentiation into the M2c pathway is induced by glucocorticoids such as dexamethasone. The gene-expression of certain anti-inflammatory cytokines and cell markers, specific for M2a or M2c differentiation is induced in these activated macrophages [46]. Such a reaction to anti-inflammatory stimuli has not been demonstrated in microglia from healthy brain. Using IL-4 and IL-13 deficient mice, it has been demonstrated that exclusively macrophages and not microglia were alternatively activated [36], supporting the hypothesis.

The immune activation marker HLA-DR has demonstrated to be upregulated in macrophages of both the classical and alternative activation pathway [47, 48]. In this study, this clear upregulation after IL-4 stimulation was not found. Nonetheless, the receptor indeed tended to be upregulated after stimulation in macrophages. However, the expression of HLA-DR is also induced in stimulated microglia just as in macrophages, indicating that also microglia were activated. It was not possible to make a distinction between control microglia and macrophages based on this immune activation marker.

Furthermore, it was questioned whether an upregulation of HLA-DR in a donor was quantitatively related to the upregulation of specific differentiation markers such as MR and CCL18. However, when HLA-DR was strongly upregulated, no proportional upregulation

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was seen in those other activation markers, indicating there is no direct association between strenght of immune activation and expression of alternative activation markers.

CD200R upregulation was shown in peripheral macrophages in vitro after stimulation with IL-4 (unpublished results). It was therefore hypothesized that CD200R would be upregulated after IL-4 stimulation in macrophages but not in microglia. As expected, IL-4 stimulation tended to induce CD200R expression in choroid plexus macrophages. However, after stimulation of microglia from control group no upregulation of CD200R was found, implicating that microglia differentiation is different from that of macrophages.

The anti-inflammatory cytokine IL-10, has proved to be upregulated after stimulation with glucocorticoids, and can therefore be regarded as a marker for the M2c pathway [43, 44].

Upregulated was expected to be seen in choroid plexus macrophages but not in microglia.

In this study the cytokine was not upregulated in reaction to stimulation with dexamethasone in both control microglia and macrophages.

Other markers tested (MR, CCL18, CD163) were expected to be upregulated in macrophages after stimulation with anti-inflammatory simuli [17, 40]. Upregulation was not expected in control microglia. However, no distinction could be made between the two cell types or groups, based on these cell makers. Results indicate that microglia in healthy brain do have the potential to become activated by IL-4 or dexamethasone stimulation.

Aim C: Differentiation characteristics – MS vs Controls

The expression patterns of microglia and macrophages from healthy brain were furthermore compared to the same cells from MS brain. It was hypothesized that microglia from NAWM react differently to anti-inflammatory stimuli when compared to microglia from non-MS brain tissue. These MS microglia might be able to become alternatively activated. Significant differences between control and MS were only seen in expression of receptors and IL-10. Furthermore tendencies for differences between MS and controls were found, including an upregulation of CD200R expression in MS microglia. It can be concluded that even in NAWM the phenotype of MS microglia different from the control microglia. Further differences might be shown after increasing the power of the experiment.

In contrast to microglia from the control group, microglia from MS donors did appear to upregulate CD200R. By this upregulation MS microglia show a reaction similar to the established reaction of macrophages. Remarkably, this finding is in contrast to the finding healthy brain microglia which did not show any upregulation of CD200R after stimulation.

Both in microglia and macrophages from MS donors an upregulation was seen in IL-10 expression. In microglia this finding was significant. Apparently IL-10 expression is only affected in MS donors.

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Differences in gene expression between controls and MS of the other activation markers and M2a and M2c markers like MR, HLA-DR, CCL18 and CD163 were not found.

From these findings the conclusion can be drawn that in MS the microglia phenotype has changed, and the cells have become able to be alternatively activated. However, very few results were obtained from some of the markers in MS donors. The findings therefore might change when more donors are included.

Conclusion

This study aimed to validate the flow cytometric method to isolate human microglia and macrophages from post-mortem human brain tissue. By discarding autofluorescence, it was shown that the used isolation procedure was indeed successful, because pure population of microglia and macrophages were obtained.

Another goal was to compare the microglia differentiation features to those of choroid plexus macrophages. Furthermore, a comparison was made between microglia and macrophages from control and MS brain. It was hypothesized that in a control situation, unlike macrophages, microglia would not show alternative activation. However, in the case of MS the situation might be different. It is hypothesized that microglia in MS NAWM exhibit aberrant differentiation patterns possible due to an altered basal activation status compared to microglia from non-MS brain tissue. This may lead to altered susceptibility and activation features in reaction to activating stimuli.

In contrast to what was expected, no differences were found between microglia and macrophages in gene-expression of alternative activation markers in a control situation.

Furthermore, differences in gene-expression in control microglia and macrophages as compared to MS were only shown in IL-10 expression.

Nonetheless, several trends were found of upregulation of cellmarkers after stimulation, and reactions differing between microglia and macrophages, or between the control and the MS group. Only 1-4 brain donors were included in the study, which turned out to show a lot of variation basal gene-expression and upregulation of activation markers. This high variation between donors, in combination with the low power of the experiment hinders the robustness of the results. Increasing the power of the experiment will probably clarify whether there are indeed consistent differences in gene-expression patterns and thus diffentation pathways between microglia and macrophages.

Learning what induces microglial activation, and how the microglia phenotype relates to the macrophage, may lead to a better understanding of transitions into other phenotypes.

When is elucidated what causes the transition of a macrophage or microglia cell from for example a wound healing into a classically activated macrophage, possibly also their functioning in MS would be comprehended. Future studies should focus on the role of the microglial activation directions in MS in relation to a healthy situation.

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Acknowledgements

At first I would like to thank Inge Huitginga for inspiring me on the topic of microglia in MS during a presentation of my master course, and for giving me the opportunity to join her research group doing this specific internship project. Naturally I thank Jeroen Melief for his excellent supervision, for teaching me all laboratory techniques, and for always being prepared to help and answer all my questions. I thank Nathalie Koning setting up the method, and providing me all the extra information I needed, and Robert Hoek for usefull discussions and technical support. From the AMC departments biochemistry and experimental immunology I would like to thank Jörg Hamann and Marco van Eijk for usefull discussions and using the biochemistry culture lab. Last but not least I want to thank the Duch Brain Bank for providing the brain tissue used, and furthermore supplying me all the information I needed for this project.

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