Verslag – Rapport – Bericht – Report - GSKE - FMRE

containing 2 (NOD2) receptor responses. Interestingly, A20/TNFAIP3 has been identified in humans
as a susceptibility locus for multiple immunopathologies including Crohn’s disease, systemic lupus
erythematosus and rheumatoid arthritis (RA) (reviewed by Vereecke et al., 2009). Importantly, we could
recently confirm these associations using mice with a conditional A20 knockout allele, allowing tissuespecific
A20 deletion (Vereecke et al., 2010; Kool et al., 2011; Matmati et al., 2011). These findings clearly
indicate a crucial and cell type specific role for A20 in controlling inflammatory immune responses.
Interestingly, genome-wide association studies also suggested A20/TNFAIP3 as a susceptibility gene
for multiple sclerosis (De Jager et al., 2009; Gilli et al., 2011).
One of the aims of our GSKE-funded project is to understand the function, activation and regulation of
A20 in the development and progression of central nervous system inflammation and demyelination.
The basis approach is to genetically manipulate the A20 gene in mice in specific neuronal populations
and immune effectors and to determine the effects of such mutation in development and inflammatory
disease pathogenesis.
1.1 A20/TNFAIP3 in MS/EAE pathology
To study the CNS-specific role of A20 in the immunopathology of MS, we make use of the experimental
MS model EAE, which can be induced by immunization of mice with myelin oligodendrocyte
glycoprotein (MOG) or other encephalogenic agents. To evaluate the cell specific contribution of A20
in the pathogenesis of EAE, different tissue-specific A20 knockout mice (all-CNS-, neuron-, astrocyte-,
oligodendrocyte-specific, T cell-, myeloid cell- and DC-specific) were generated from a conditional
‘floxed’ A20 knockout mouse (Vereecke et al., 2010), and subjected to MOG-peptide-induced EAE. The
clinical course for disease initiation and progression is followed and spinal cord sections are evaluated
for inflammatory infiltrates and demyelination. Furthermore, inflammatory cytokine and chemokine
production is measured by quantitative real time PCR. The capacity of macrophages and DCs in T cell
activation is further analysed in vitro using primary cultures isolated from respective A20 knockout mice
and control littermates. Similarly, primary neuronal cultures are used in vitro to establish the impact of
A20 deficiency on inflammatory challenge. Next to the EAE model of MS, brain-specific demyelination
can also be induced by putting mice on a diet containing the neurotoxicant cuprizone. In this model,
administering cuprizone for 6 weeks causes complete demyelination of the corpus callosum in the absence
of an immune reaction. Furthermore, when this administration is terminated, complete remyelination of
the corpus callosum occurs, rendering this model useful to study both de- and remyelination. CNS
demyelination, and astro- and microgliosis are evaluated by histology on corpus callosum sections, and
inflammatory cytokine and chemokine expression by qPCR (Mc Guire et al., ongoing studies).
1.2 A20/TNFAIP3 in cerebral ischemia
Cerebral ischemia is characterized by the activation of glial cells, causing a rapid and massive local
inflammatory reaction leading to tissue damage and neuronal cell death (Ridder and Schwaninger, 2009).
Over the past decade, it has become increasingly clear that NF-kB plays a central role in the pathogenesis
of cerebral ischemia. NF-kB gets activated in the ischemic hemisphere shortly after permanent middle
cerebral artery occlusion (MCAO). Furthermore, NF-kB deficient mice show a reduction in ischemic
damage after transient or permanent MCAO, suggesting a cell death promoting role for NF-kB in this
model. In line with these, mice lacking IKK2 in all neuroectodermal cells or specifically in neurons show
a decreased infarct volume 48 hours after pMCAO, whereas constitutive activation of IKK2 increased
infarct size (Herrmann et al., 2005). However, NF-kB may also act beneficial in conditions of ischemic
preconditioning which was shown to protect against a subsequent prolonged ischemic insult through
transcriptional activition of NF-kB.
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Verslag – Rapport – Report 2012