Monocyte dysfunction in Multiple Sclerosis (MS)

We have developed an interest over the years in monocyte dysfunction in
vascular disease and multiple sclerosis (MS), both recognized by inflammation.
In atherosclerosis particularly monocytes navigate to sites of endothelial
damage, adhere to the vessel wall and transmigrate the endothelial cell layer
to infiltrate the underlying tissue. Upon transendothelial migration, monocytes
differentiate into functionally defined subsets i.e. M1-macrophages
(inflammatory) or M2-macrophages (anti-inflammatory) and into dendritic cells,
depending on the stimulus they encounter in the local micro-environment of the
vessel wall (Wierda et al., 2010). Interestingly, the infiltration of monocytes
within the vasculature has a paradoxical dual role: continuous and prolonged
infiltration may drive chronic inflammation, which results in occlusion of the
vessel wall (stenosis), whereas transient recruitment promotes collateral
artery growth and tissue repair. Recent insights into the pathogenesis of MS
reveal parallels to the processes that occur in vascular diseases. Stenosis
affecting the cerebrospinal venous segment hampers venous flow, which could
trigger an inflammatory response. Subsequently, monocytes are then attracted by
and transmigrate the inflamed cerebrovascular endothelium, where they further
differentiate into defined subsets of macrophages/microglia in the
micro-environment of the brain. During monocyte differentiation remodeling of
the chromatin structure by modifications of histones and DNA is essential,
because they control transcription programs of genes that determine
lineage-specificity and cell activation. It is hypothesized that besides the
contribution of the inflamed (cerebro)vascular endothelium, also
inflammation-induced changes in differentiation-fate and function of monocytes
contributes to disease. Goal of this pilot grant application therefore is to
investigate disease-associated alterations in the expression of epigenetic
effectors in MS, and the bearing this has on monocyte differentiation and
transendothelial cell migration. A better understanding of the molecular
mechanisms responsible for monocyte differentiation and trafficking from the
periphery into the diseased CNS will ultimately help to intervene in these
processes in MS.

1). Evaluate the expression of the genes encoding the enzymes that modify
histones and DNA during monocyte differentiation in MS.

2). Consequences of monocyte dysfunction for migration and cytokine/chemokine
production.

1). Evaluate the expression of the genes encoding the enzymes that modify
histones and DNA during monocyte differentiation in MS.

To evaluate the mRNA expression characteristics of most of the currently known
lysine acetyltransferases, lysine deacetylases and sirtuins, lysine
methyltransferases and lysine demethylases, and also of DNA methyltransferases
in cultured cells of human origin, we have developed a real-time RT-PCR array
allowing the specific and quantitative monitoring of these expression levels.

For the purpose of these investigations, CD14+ monocytes will be isolated from
Fycoll gradient separated peripheral blood mononuclear cells (PBMCs) by
magnetic cell sorting (MACS® Technology) with the use of CD14+ Macs beads
(Miltenyi Biotec). For polarization into macrophage type 1 (M1,
pro-inflammatory), cultured CD14+ monocytes will be exposed to 50 U/ml
recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and
for polarization into macrophage type 2 (M2, anti-inflammatory) to 50 ng/ml
macrophage colony stimulating factor (M-CSF) for 5 days. For polarization into
immature dendritic cells (iDCs), cultured CD14+ monocytes will be exposed to
1000 U/ml (GM-CSF) and 500 U/ml IL-4 for 5 days. For activation, polarized
CD14+ monocytes subsequently will be exposed to 100 ng/ml LPS for 2 days.

mRNA will be isolated from unstimulated monocytes and from monocytes after
polarization and/or activation, which will be converted into cDNA for real-time
reverse transcriptase (RT)-PCR on an ICycler IQ system using the IQ SYBR-Green
Supermix. The culture media of the polarized monocytes will be stored for
analysis for levels of secreted cytokines/chemokines (see below).


2). Consequences of monocyte dysfunction for migration and cytokine/chemokine
production

Freshly isolated CD14+ monocytes obtained from PBMCs of MS patients will be
analyzed phenotypically by flowcytometry for the expression of the chemokine
receptors CCR5, CX3CR1 and CXCR4. Next, the migratory capacity of these
monocytes will be tested in chemotaxis assays in the response to CCL5 and CCL3
(ligands for CCR5), CX3CL1 (ligand for CX3CR1), CXCL12 (ligand for CXCR4) and
C5a (as control) in a 48-well microchemotaxis chamber as previously explored
for microglial cells (Kuipers et al., Glia 2006). Results will be compared with
CD14+ monocytes isolated from PBMCs of healthy control individuals.

To functionally examine whether the chemotaxis has altered the expression
characteristics of the epigenetic effectors, the migrated cells will be
collected and profiled as detailed in section 1. Results will be compared with
non-migrated cells.

For the determination of alterations in the secretion levels of cytokines and
chemokines during monocyte polarization in MS, collected culture media of
polarized and activated monocytes as detailed above will be analysed for
production of a variety of cytokines/chemokines by luminex technology with use
of a human 27-plex panel BioPlex cytokine assay kit (BioRad).

Statistical significance of the obtained results in sections 1 and 2 will be
determined by Student t-test or Mann-Whitney U-test for group variables.
Correlation will be determined by Pearson*s rank order or by Spearman*s rank
order correlation.

Collection of PBMCs (3 tubes of 10 mls) is a very benign burden with hardly any
risk.