Hannover 2015
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH
35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: [email protected] · Internet: www.dvg.de
MUHAMMAD AKRAM KHAN
ISBN 978-3-86345-246-9
Institut für Pathologie
Stiftung Tierärztliche Hochschule Hannover
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1. Auflage 2015
© 2015 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH,
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ISBN 978-3-86345-246-9
Verlag: DVG Service GmbH
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0641/24466
[email protected]
www.dvg.de
University of Veterinary Medicine Hannover
Department of Pathology
Expansion of regulatory T cells in Theiler’s murine encephalomyelitis virusinfected C57BL/6 mice
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
Awarded by the University of Veterinary Medicine Hannover
By
Muhammad Akram Khan
Born in Mianwali, Punjab/Pakistan
Hannover, Germany 2015
Supervisor:
Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.
Supervision Group: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.
Prof. Dr. Martin Stangel
Prof. Dr. Andrea Tipold
1st Evaluation:
Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.
Department of Pathology,
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Martin Stangel
Clinical Neuroimmunology and Neurochemistry,
Department of Neurology,
Hannover Medical School Hannover, Germany
Prof. Dr. Andrea Tipold
Department of Small Animal Medicine and Surgery,
University of Veterinary Medicine Hannover, Germany
2nd Evaluation
Dr. med vet. Anna Oevermann Dipl. ECVP
Abteilung Experimentelle Klinische Forschung
Universität Bern, Switzerland
Date of final exam
13-03-2015
Muhammad Akram Khan has received financial support from the HEC Islamabad,
Pakistan and DAAD Germany. This work was supported by the German Research
foundation (Deutsche Forschungsgemeinschaft, DFG; FOR 1103).
To my family
PUBLICATIONS AND PRESENTATIONS
Publications
HERDER, V., ISKANDAR, C.D., HANSMANN, F., ELMARABET, S.A., KHAN, M.A.,
KALKUHL, A., DESCHL, U., BAUMGÄRTNER, W., ULRICH, R., BEINEKE, A
(2014):
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s
murine encephalomyelitis.
Brain Pathol. DOI: 10.1111/bpa.12238.
QESKA, V., BARTHEL, Y., ISERINGHAUSEN, M., TIPOLD, A., STEIN, V.M., KHAN,
M.A., BAUMGÄRTNER, W., BEINEKE, A (2013):
Dynamic changes of Foxp3 (+) regulatory T cells in spleen and brain of canine
distemper virus-infected dogs.
Vet Immunol Immunopathol. 156, 215-222, DOI: 10.1016/j.vetimm.2013.10.006
Poster presentation
KHAN, M.A., HERDER, V., HUEHN, J., TEICH, R., BAUMGÄRTNER, W.,
BEINEKE, A (2014):
Expansion of regulatory T cells in Theiler’s murine encephalomyelitis virus-infected
C57BL/6 mice. ‘‘2nd International Workshop of Veterinary Neuroscience’’, Hannover,
Germany, March 20-22, 2014.
Oral presentation
KHAN, M.A., HERDER, V., HUEHN, J., TEICH, R., BAUMGÄRTNER, W.,
BEINEKE, A (2014):
Expansion of regulatory T cells in Theiler’s murine encephalomyelitis virus-infected
C57BL/6 mice. ‘‘2nd Annual European Veterinary Pathology Congress’’, Berlin,
Germany, August 27-30, 2014.
I
Contents
Contents
1. Introduction ....................................................................................... 1
1.1
Aim of the study ............................................................................................. 2
1.2
Multiple sclerosis and animal models for demyelinating disorders ................ 3
1.2.1
Theiler’s murine encephalomyelitis virus ................................................ 7
1.2.2
Persistence of Theiler’s murine encephalomyelitis virus in the central
nervous system ..................................................................................... 10
1.2.3
Theiler’s murine encephalomyelitis virus-induced neuropathology ....... 12
1.2.4
Clinical findings in Theiler’s murine encephalomyelitis ......................... 14
1.3
T cells in demyelinating disorders................................................................ 15
1.3.1
CD8+ T cells .......................................................................................... 15
1.3.2
CD4+ T cells .......................................................................................... 18
1.3.3
Regulatory T cells ................................................................................. 20
1.4
Regulatory T cells and immune homeostasis .............................................. 21
2. Materials and Methods.................................................................... 25
2.1
Experimental design and tissue preparation ................................................ 26
2.2
Histological examination of brain and spinal cord ........................................ 28
2.3
Immunohistochemistry................................................................................. 29
2.4
Flow cytometry ............................................................................................ 32
2.4.1
Spleen samples..................................................................................... 32
2.4.2
Blood samples ...................................................................................... 33
2.5
Reverse transcription quantitative polymerase chain reaction ..................... 35
2.5.1
RNA isolation and reverse transcription ................................................ 35
2.5.2
Polymerase chain reaction .................................................................... 35
2.6
Statistical analyses ...................................................................................... 37
3. Results ............................................................................................. 39
3.1
Clinical findings in C57BL/6 mice following regulatory T cell expansion and
depletion of CD8+ T cells ............................................................................. 40
3.2
Effects of regulatory T cell expansion and depletion of CD8+ T cells upon
the peripheral immune system .................................................................... 41
3.2.1
Spleen weight........................................................................................ 41
3.2.2
Quantification of Foxp3+ cells in the blood by flow cytometry ............... 41
3.2.3
Measurement of CD4/CD8 ratio in the blood by flow cytometry ............ 43
3.2.4
Quantification of Foxp3+ cells in the spleen by flow cytometry .............. 44
3.2.5
Measurement of CD4/CD8 ratio in the spleen by flow cytometry .......... 45
Contents
3.3
II
Effects of regulatory T cell expansion and depletion of CD8+ T cells in the
brain ......................................................................................................... 47
3.3.1
Virus load quantification in the brain by real time polymerase chain
reaction ................................................................................................. 47
3.3.2
Virus load quantification in the brain by immunohistochemistry ............ 48
3.3.3
Histological examination of the hippocampus ....................................... 49
3.3.4
Quantification of CD3+ T cells in the hippocampus ............................... 51
3.3.5
Quantification of Foxp3+ regulatory T cells in the hippocampus ............ 52
3.3.6
Quantification of CD45R+ B cells in the hippocampus .......................... 53
3.3.7
Quantification of CD107b+ microglia/macrophages in the hippocampus
.............................................................................................................. 55
3.4
Effects of regulatory T cell expansion and depletion of CD8+ T cells in the
spinal cord ................................................................................................... 56
3.4.1
Virus load quantification in the spinal cord by immunohistochemistry ... 56
3.4.2
Histological examination of the spinal cord ........................................... 58
3.4.3
Myelin basic protein expression ............................................................ 59
3.4.4
Axonal β-amyloid precursor protein expression .................................... 60
3.4.5
Quantification of CD3+ T cells in the spinal cord ................................... 62
3.4.6
Quantification of Foxp3+ regulatory T cells in the spinal cord ................ 64
3.4.7
Quantification of CD45R+ B cells in the spinal cord .............................. 66
3.4.8
Quantification of CD107b+ microglia/macrophages in the spinal cord... 67
4. Discussion ....................................................................................... 69
4.1
Effect of interleukin-2 immune complex treatment and antibody mediated
CD8-depletion upon regulatory T cells in C57BL/6 mice ............................. 71
4.2
Lesion development in the brain of Theilervirus-infected C57BL/6 mice
following interleukin-2 complex treatment and depletion of CD8+ T cells..... 72
4.3
Lesion development in the spinal cord of Theilervirus-infected C57BL/6
mice following interleukin-2 complex treatment and depletion of CD8+ T
cells .......................................................................................................... 76
4.4
Interaction between regulatory T cells and cytotoxic CD8+ T cells............... 80
4.5
General aspects of regulatory T cells in infectious disorders of the central
nervous system ........................................................................................... 82
4.6
Conclusion ................................................................................................... 84
5. Summary.......................................................................................... 85
6. Zusammenfassung ......................................................................... 89
7. References....................................................................................... 93
8. Annex ............................................................................................. 119
8.1
Results of statistical analyses ..................................................................... 119
III
Contents
Table 8.1.1 Clinical examination .......................................................................... 119
Table 8.1.2 Spleen weight ................................................................................... 119
Table 8.1.3 Foxp3+ cells in the blood determined by flow cytometry .................. 120
Table 8.1.4 CD4/CD8 ratio in the blood determined by flow cytometry .............. 120
Table 8.1.5 Foxp3+ cells in the spleen determined by flow cytometry ................ 121
Table 8.1.6 CD4/CD8 ratio in the spleen determined by flow cytometry ............ 121
Table 8.1.7 Theilervirus RNA concentration in the brain determined by real time
polymerase chain reaction............................................................... 122
Table 8.1.8 Amount of Theilervirus-infected cells in the brain determined by
immunohistochemistry ..................................................................... 122
Table 8.1.9 Severity of hippocampal inflammation in the hippocampus
determined by histology................................................................... 123
Table 8.1.10 Number of CD3+ T cells in the hippocampus determined by
immunohistochemistry .................................................................... 123
Table 8.1.11 Number of Foxp3+ regulatory T cells in the hippocampus
determined by immunohistochemistry ............................................. 124
Table 8.1.12 Number of CD45R+ B cells in the hippocampus determined by
immunohistochemistry .................................................................... 124
Table 8.1.13 Number of CD107b+ macrophages/microglia in the hippocampus
determined by immunohistochemistry ............................................ 125
Table 8.1.14 Amount of Theilervirus-infected cells in the spinal cord determined
by immunohistochemistry ............................................................... 125
Table 8.1.15 Severity of spinal cord inflammation determined by histology ........ 126
Table 8.1.16 Quantification of myelin basic protein expression in spinal cord by
immunohistochemistry .................................................................... 126
Table 8.1.17 Quantification of axonal β-amyloid precursor protein expression in
the spinal cord by immunohistochemistry ....................................... 127
Table 8.1.18 Number of CD3+ T cells in the spinal cord determined by
immunohistochemistry .................................................................... 127
Table 8.1.19 Number of Foxp3+ regulatory T cells in the spinal cord determined
by immunohistochemistry ............................................................... 128
Table 8.1.20 Number of CD45R+ B cells in the spinal cord determined
by immunohistochemistry ............................................................... 128
Table 8.1.21 Number of CD107b+ microglia/macrophages in the spinal cord
determined by immunohistochemistry ............................................ 129
8.2
Materials used for animal infection ............................................................ 130
8.3
Reagents, chemicals and antibodies ......................................................... 130
8.3.1
Solutions for immunohistochemistry ................................................... 131
8.3.2
Equipment and disposable items ........................................................ 133
8.3.3
Materials used for RT-qPCR ............................................................... 134
Contents
8.3.4
IV
Materials used for flow cytometry........................................................ 138
9. Acknowledgments....................................................................... 140
V
Abbrviations
Abbreviations
Ab
Antibody
Ag
Antigen
APC
Antigen presenting cell
BBB
Blood brain barrier
CD
Cluster of differentiation
cDNA
Complementary DNA
CDV
Canine distemper virus
CNS
Central nervous system
CSF
Cerebrospinal fluid
CTL
Cytotoxic lymphocytes
CTLA-4
Cytotoxic T-lymphocytes antigen 4
DA
Daniel’s strain
DC
Dendritic cells
DNA
Deoxyribonucleic acid
EAE
Experimental autoimmune encephalomyelitis
FOXP3
Forkhead box P3
HE
Hematoxylin-eosin
IFN-α
Interferon-alpha
IFN-β
Interferon-beta
IFN-γ
Interferon-gamma
Ig
Immunoglobulin
IL
Interleukin
MBP
Myelin basic protein
MHC I
Major histocompatibility complex class I
MHC II
Major histocompatibility complex class II
Abbreviations
mRNA
Messenger RNA
MS
Multiple sclerosis
PCR
Polymerase chain reaction
PPMS
Primary progressive multiple sclerosis
PRMS
Progressive relapsing multiple sclerosis
PVI
Perivascular infiltrate
RNA
Ribonucleic acid
RRMS
Relapsing-remitting multiple sclerosis
SPMS
Secondary progressive multiple sclerosis
TCR
T-cell receptor
TGF-β
Transforming growth factor
Th1
T-helper cell type 1
Th2
T-helper cell type 2
Th17
T-helper cell type 17
TME
Theiler’s murine encephalomyelitis
TMEV
Theiler’s murine encephalomyelitis virus
TNF
Tumor necrosis factor
TO
Theiler’s virus original strains
Treg
regulatory T cells
VP
Viral protein
VI
Chapter 1 - Introduction
Chapter 1 Introduction
1
2
Chapter 1 - Introduction
1. Introduction
1.1
Aim of the study
Multiple sclerosis, one of the most frequent central nervous system (CNS) diseases
in young adults, is a chronic demyelinating disease of unknown etiology and possibly
multifactorial causes. Due to clinical and pathological similarities, Theiler’s murine
encephalomyelitis (TME) represents a commonly used infectious animal model for
the chronic-progressive form of human MS. Inadequate viral clearance in susceptible
SJL mice leads to persistence of Theiler’s murine encephalomyelitis virus (TMEV) in
the CNS and immune mediated spinal cord demyelination, respectively. In contrast
resistant C57BL/6 mice eliminate the virus from the CNS by specific cellular
immunity.
CD8-mediated cytotoxicity plays a central role for virus elimination in different
infectious CNS diseases, while regulatory T cells (Treg) have the ability to reduce
inflammatory responses in the brain by inhibiting effector T cells, microglia,
macrophages and astrocytes. This has led to the hypothesis that Treg reduce
antiviral immunity in TME, which represents a prerequisite for virus persistence and
immune mediated demyelination. Referring to this, Treg have been shown to
efficiently reduce antiviral immune responses in TME in susceptible mice strains
(RICHARDS et al., 2011; MARTINEZ et al., 2014). However, manipulations of the
Treg-compartment have failed to influence the disease course in resistant C57BL/6
mice, demonstrating the complexity of immune responses in this infectious MS
model.
To get further insights into role of Treg and their interaction with other leukocyte
subsets in persistent viral infections, the aim of the present study was to investigate
the impact of Treg-expansion and CD8+ T cell depletion upon TMEV-induced disease
progression in C57BL/6 mice. Findings will potentially support the development of
Chapter 1 - Introduction
3
novel therapeutic strategies for chronic inflammatory disorders and provide a base for
future studies upon virus-specific immune responses and immunopathology in
transgenic mice with a C57BL/6 genetic background.
1.2
Multiple sclerosis and animal models for demyelinating disorders
Multiple sclerosis (MS) is a progressive inflammatory disorder of the brain and spinal
cord which leads to demyelination. Inflammatory plaques with myelin loss can be
diagnosed
by magnetic
resonance
imaging
or
histopathology,
respectively
(FLETCHER et al., 2010). Moreover, detection of oligoclonal bands (KARUSSIS,
2014) and other biomarkers such as CXCL13 and neurofilament protein subunits in
the cerebrospinal fluid are also helpful diagnostic tools to detect MS (TEUNISSEN
AND KHALIL, 2012). MS has a worldwide distribution with a high incidence in
Europe, North America, Canada, Australia and New Zealand, while its occurrence is
comparatively low in Asia and Africa (CROXFORD et al., 2002). Clinical signs of MS
patients include loss of coordination, ataxia, hyperreflexia, spasticity, and fatigue as
well as sensory and visual impairment (GOVERMAN, 2009). Myelin-specific
autoimmunity is supposed to induce myelin loss. However, the precise etiology of MS
is still unknown. Different genetic and environmental factors as well as viral infections
(e.g. Epstein-Barr virus) are currently discussed as triggering events (figure 1.1;
SALVETTI et al., 2009).
The most common clinical course of MS is the relapsing-remitting form (ROSCHE et
al., 2003; DITTEL, 2008). This disease form is characterized by sporadic
neurological episodes (relapses) followed by periods of recovery (remissions)
resulting in a wide spectrum of disabilities. The common late phase of neurological
disability, which follows relapsing-remitting MS in approximately 40% of patients, is
4
Chapter 1 - Introduction
the secondary progressive MS form, characterized by continuous irreversible
neurological deficits (RAMSARANSING and DE KEYSER, 2006). The less common
manifestation is primary progressive MS, in which disability progresses continuously
without phases of remission (TRAPP and NAVE, 2008; SATO et al., 2011).
Several animal models have been established for the investigation of myelin
disorders, including infectious and toxic models, as well as autoimmune (e.g.
experimental autoimmune encephalomyelitis [EAE]) and genetic models (RADDATZ
et al., 2014; ULRICH et al., 2014; BEINEKE et al., 2009; OLESZAK et al., 2004;
RODRIGUEZ, 2007). A summary of viral MS models is given in table 1.1
Figure 1.1. Factors potentially involved in the development of multiple sclerosis (MS) lesions. Myelinspecific autoimmunity is supposed to cause progressive white matter lesions, but initiating events are
largely undetermined. Virus infection might represent a triggering event for neuroinflammation and
demyelination in MS patients. In addition, several genetic factors and gender have an influence upon
MS pathogenesis (modified from Sato et al., 2011).
Chapter 1 - Introduction
5
Table 1.1. Experimental and spontaneous viral animal models for multiple
sclerosis*
Virus
Virus family
Host
Visna virus
Retroviridae
Sheep
Canine distemper virus
Paramyxoviridae
Dog
Theiler`s murine encephalomyelitis virus
Picornaviridae
Mouse
Mouse hepatitis virus
Coronaviridae
Mouse
*adapted from Lipton et al., 2007
MS lesions have been divided into four distinct types (OLESZAK et al., 2004;
HICKEY et al., 1999).
1. Acute lesions show an infiltration of lymphocytes, particularly T cells and
macrophages, associated with damage or reduction of oligodendrocytes.
Macroscopically, acute lesions show a pink to white or yellowish discoloration
due to hyperemia, inflammation and lipid destruction.
2. Chronic active lesions are characterized by myelin damage and phagocytosis
by
macrophages
(myelinophagia)
as
well
as
reduced
numbers
of
oligodendrocytes. In addition, axonal damage might be present.
3. Chronic inactive lesions are well demarcated (burnt-out) and show
axonopathies, loss of oligodendrocytes and damage of the blood brain barrier.
4. Shadow plaques might be demyelinated to some extent or remyelinated. They
can be observed frequently in chronic stages of MS but they might be absent
in some chronic cases.
In addition, MS lesions can be sub-classified according to the scheme described by
(LUCCHINETT et al., 2000). Pattern I and II of this classification show many
similarities, such as an infiltration of T lymphocytes and macrophages. In pattern I
there is marked accumulation of immunoglobulin (Ig)G. Both patterns exhibit Ig
reactivity in the cytoplasm of astrocytes and considerable damage of the blood brain
6
Chapter 1 - Introduction
barrier. In pattern II a significant Ig reactivity within macrophages and myelin
destruction at the edges of active plaques can be observed. Pattern I and II are
concentrated around venules with distinct sharp boundaries. Pattern III also
comprises T cells, macrophages, and activated microglia. The lesion protrudes into
the
surrounding white
matter.
Oligodendrocyte
damage
can
be
observed
predominantly at the borders of active lesions and occasionally in the normal
appearing white matter. Similar to pattern III lesions no accumulation of Ig can be
found in pattern IV lesion. Infiltrates consist of T lymphocytes and macrophages.
Oligodendrocyte
death
near
active
lesions
results
in
demyelination.
DNA
fragmentation can be observed in altered oligodendrocytes but typical features of
apoptosis cannot be found in these cells.
Although the primary etiology of MS is still unknown, different infectious agents have
been considered to play a role in its pathogenesis (table 1.2). Due to similarities in
the pathogenesis and clinical findings with MS, Theiler`s murine encephalomyelitis
(TME) is a widely used mouse model for human demyelinating disorders (RADDATZ
et al., 2014; GRANT et al., 1992).
Chapter 1 - Introduction
7
Table 1.2. Selected viruses associated with multiple sclerosis
Family
Virus
Human herpes virus type 6
Herpes complex virus
Herpesviridae
Varicella zoster virus
Epstein-Barr virus
Marek`s disease virus
Retroviridae
Paramyxoviridae
Coronaviridae
Reference
KNOX et al., 2000
PERRON et al., 1993
BRETTSCHNEIDER
et
al., 2009
LUNEMMAN et al., 2010
BOUGIOUKLIS, 2006
Human T cell leukaemia virus type 1
OGER, 2007
Human endogenous retrovirus
PERRON et al., 2012
Measles virus
FUJINAMI et al., 1983
AHLGREN et al., 2012
Mumps virus
TOBLER et al., 1982
Parainfluenza virus type 1
RAUCH et al., 1975
Canine distemper virus
HAILE et al., 1982
Simian virus type 5
GOSWAMI et al., 1984
Coronavirus
BOUCHER et al., 2007
1.2.1 Theiler’s murine encephalomyelitis virus
Theiler’s murine encephalomyelitis virus (TMEV) was first discovered by Max Theiler
(THEILER, 1937). The virus has the ability to cause chronic infection of the central
nervous system (CNS). A serological survey showed that TMEV is commonly present
in wild mice (Mus musculus) and several types have been isolated from wild and
laboratory mice. Although CNS infection via the oral route is a rare event,
spontaneous paralyses can occur following natural infection (BRAHIC et al., 2005).
TMEV is a non-enveloped, single-stranded, positive-sense RNA virus that belongs to
the picornaviridae family and cardiovirus genus (OLESZAK et al., 1995; BRAHIC et
al., 2005; TSUNODA and FUJINAMI, 2010). The genome consists of 8100
nucleotides and encodes for 12 proteins (figure 1.2). VP1, VP2, VP3, and VP4
8
Chapter 1 - Introduction
represent capsid proteins and 2A, 2B, 2C, 3A, 3B, 3C, and 3D are required for viral
RNA replication (OLESZAK et al., 2004; TSUNODA and FUJINAMI, 2010). The
genome is similar to the poliovirus genome, except for the presence of the L and L*
proteins at the 5´end. The L protein contains 76 amino acids. This zinc finger protein
inhibits the IFN-α/ß pathway at the transcriptional level which leads to reduced host
immune defense mechanisms. The L* protein is translated from an alternative open
reading frame. It was detected initially in cell processes and somata of infected
neurons and plays an important role for the infection of macrophages and virus
persistence. Accordingly, the L* protein can be found in all persistent TMEV strains.
Translation of the polyprotein and the L* protein strongly depends on the presence of
an internal ribosome entry site at the 5´-noncoding region (figure 1.2). It enables the
replication of the virus in macrophages and microglial cells (BRAHIC et al., 2005). In
addition, capsid proteins have been demonstrated to play a significant role for viral
persistence (OLESZAK et al., 2004; McALLISTER et al., 1990).
Figure 1.2. Theiler’s murine encephalomyelitis virus (TMEV) genome consists of a large open reading
frame which encodes for a 2300 amino acid protein. The polyprotein is cleaved into 12 proteins. Viral
proteins 2B, 2C, 3A, 3B, 3C, and 3D are responsible for replication. VP1, VP2, VP3, VP4, are the
capsid proteins which are responsible for virus tropism. The L protein inhibits IFN-α/ß and cytokine
gene transcription in infected cells. The L protein facilitates the infection of macrophages. Internal
ribosome entry sites (IRES) are responsible for translation of the polyprotein and L protein. CRE =
cis-acting replication signal, IFN=interferon (modified from Brahic et al., 2005).
Chapter 1 - Introduction
9
TMEV strains can be discriminated based on their neurovirulence. The GDVII and FA
strains are extremely neurovirulent and cause an acute polioencephalomyelitis
following intracerebral (i.c.) infection. Due to early and massive neuronal damage
infected mice usually die before the development of white matter demyelination (Fu
et al., 1990). In contrast, the Theiler’s original (TO) subgroup is less neurovirulent
and causes persistent CNS infection with demyelination following experimental i.c.
infection. Comparison of disease courses induced by the two TMEV subgroups is
shown in figure 1.3. The TO subgroup includes the BeAn and DA strain, which exhibit
93% homology of their amino acid sequences (OLESZAK et al., 2004; FU et al.,
1990). The TO and GDVII strains are 90% identical at the nucleotide level and show
a 95% similarity at the amino acid level (TSUNODA and FUJINAMI, 2010).
Strains of the TO subgroup cause a biphasic disease course which consists of an
early acute polioencephalitis and subsequent chronic inflammation, associated with
virus persistence in the brain stem and spinal cord white matter. During the
demyelinating
phase
microglia/macrophages
and
oligodendrocytes
are
the
predominantly infected cell types (KUMMERFELD et al., 2012). Although both TO
strains induce demyelination in the chronic phase, the DA strain causes a more
severe grey matter disease compared to the BeAn strain (DAL CANTO et al., 1996).
10
Chapter 1 - Introduction
Figure 1.3. The neurovirulent GDVII strain causes an acute fatal polioencephalomyelitis, while TO
strains induce a biphasic disease course. In the acute phase, virus infects mainly neurons of the brain
gray matter. Subsequently, inflammatory demyelination can be observed in the spinal cord white
matter of TO strain-infected mice (modified from Tsunoda and Fujinami, 2002).
1.2.2 Persistence of Theiler’s murine encephalomyelitis virus in the central
nervous system
Different viruses persist in macrophages, which enables their transportation within
tissues and protects them from host defense mechanisms (SHAW-JACKSON and
MICHIELS, 1997). The L* protein of TMEV helps to infect microglia and
macrophages and prevents their apoptosis, which favors virus persistence. Thus,
with disease progression an increasing number of infected macrophages can be
observed in TMEV-infected SJL mice. The importance of macrophages for disease
progression and TMEV persistence is also stressed by experiments, which
demonstrate
that
chemical
depletion
of
CNS-infiltrating
macrophages
by
mannosylated liposomes leads to viral elimination and maintained myelin integrity
(ROSSI et al., 1997). The role of oligodendrocytes for lesion development during the
late disease stages has been demonstrated also in myelin basic protein-deficient
mice (shiverer mice) that do not develop viral persistence following TMEV-infection
(BRAHIC et al., 2005). Microglia and macrophages engulf infected oligodendrocytes
Chapter 1 - Introduction
11
which is important for virus propagation during the chronic disease phase. Activated
macrophages and microglia produce proteolytic enzymes that degrade proteins of
myelin sheaths (OLESZAK et al., 2004; MECHA et al., 2012). Thus, the
perpetuating interaction between virus and microglia/macrophages might induce a
vicious circle which causes continuous inflammation and impaired repair in the spinal
cord. The presence of virus antigen within macrophages might be the result of
phagocytosis rather than a consequence of viral replication (ROSSI., et al 1997;
ZHENG et al., 2001). TMEV replication can be observed more frequently in
astrocytes
as
compared
to
microglia/macrophages
and
oligodendrocytes.
Accordingly, although discussed controversially, astrocytes are supposed to serve as
the main target cell for virus replication at all disease stages (ZHENG et al., 2001).
The role of macrophages in the pathogenesis of TME and virus persistence is
illustrated in figure 1.4.
Figure 1.4. Persistence of Theiler’s murine encephalomyelitis virus (TMEV) in mice requires
macrophage to macrophage spread, which is supposed to be achieved by apoptosis of macrophages
and/or oligodendroglial lysis with release of viral particles, followed by an uptake by macrophages
(modified from Lipton et al., 2005).
12
Chapter 1 - Introduction
1.2.3 Theiler’s murine encephalomyelitis virus-induced neuropathology
The BeAn-strain of TMEV initially infects neurons predominantly in the brain, which
causes an acute but mild encephalomyelitis. A subsequent switch of cell tropism
leads to a preferential infection of glial cells and macrophages in the white matter of
the brain stem and spinal cord, which represents a prerequisite for demyelination
during the chronic disease phase. Myelin loss in TME is caused by virus-induced
immunopathology (ZHANG et al., 2013; KUMMERFELD et al., 2012).
Susceptibility to TMEV-induced demyelinating disease depends on the genetic
background of the mouse strain (figure 1.5). For instance, susceptible mouse strains
(e.g. SJL mice) develop Th1-mediated immune responses and delayed type
hypersensitivity against viral epitopes. Similar to human MS patients, susceptibility in
mice is controlled by different genetic factors, such as genes involved in major
histocompatibility complex (MHC) expression (DAL CANTO et al., 1996). A list of
mouse strains with different degrees of susceptibility to TMEV-induced demyelination
is given in table 1.3 (DAL CANTO et al., 1996; GHADGE et al., 2011).
Table 1.3. Susceptibility to Theiler’ murine encephalomyelitis virus-induced
demyelination in different mouse strains
Resistant mouse strain Susceptible mouse
Intermediate susceptible
strain
mouse strain
C57BL/6
SJL/J
C3H
C57BL/10
DBA/1, DBA/2
CBA
C57/L
SWR
AKR
BALB/c
PL/J
C57BR
129/J
NZW
-
Chapter 1 - Introduction
13
An increasing activation of macrophages and microglia potentiates the inflammatory
process within the CNS of infected mice by the production of pro-inflammatory
cytokines. In addition, activated macrophages and microglial cells secrete
myelinotoxic substances (e.g. proteases), which lead to myelin damage (bystander
demyelination; LIUZZI et al. 1995). Phagocytized myelin fragments also lead to an
activation of macrophages which might lead to immune mediated tissue damage.
CNS-infiltrating macrophages and microglia also have the ability to present myelin
proteins to CD4+ T helper T cells, which initiates autoimmune demyelination, as
discussed for human MS (OLESZAK et al., 2004).
Besides myelin loss, axonal damage has attracted attention in demyelinating
disorders, because it is supposed to account for permanent functional deficits in MS
patients (SEEHUSEN et al., 2010). Similarly, axonopathies due to disturbance of
axonal transport mechanisms can be observed in the spinal cord of TMEV-infected
mice (KREUTZER et al., 2012). There are two proposed models for the
pathogenesis of axonal damage and myelin loss: the inside-out and outside-in model.
The outside-in model proposes that the lesion starts from the myelin sheath (outside).
Accordingly, the primary target is the myelin or oligodendrocytes, followed by axon
(inside) damage. The inside-out model suggests that the lesion develops from the
axon (inside) to the myelin (outside), so the primary target is the axon or its cell body.
Here, the axonopathy is supposed to trigger secondary demyelination (TSUNODA
and FUJINAMI, 2002). Axonal damage is known to cause a release of neuroantigens
which potentially induces autoimmune responses and attack of the myelin from the
outside, which leads to a vicious circle and disease progression (SATO et al., 2011).
14
Chapter 1 - Introduction
Figure 1.5. Comparison of immune responses and the disease course in susceptible SJL and resistant
C57BL/6 mice following Theiler`s murine encephalomyelitis virus (TMEV) infection of the DA strain.
Resistant mice develop a strong antiviral immunity, while susceptible strains are unable to clear the
virus and develop persistent infection with chronic demyelinating leukomyelitis (adopted from Oleszak
et al., 2004).
1.2.4 Clinical findings in Theiler’s murine encephalomyelitis
In C57BL/6 mice seizures can be observed, which represents an interesting model
for the investigation of epilepsy and the impact of virus infection upon brain
hyperexcitability (STEWART et al., 2010). Seizures are associated with the
development of hippocampal sclerosis with neuronal damage and the presence of
reactive astrocytes. Innate immune responses and particularly an increased
expression of interleukin-6 and tumor necrosis factor contribute to the development of
seizures in TMEV-infected C57BL/6 mice (KIRKMAN et al., 2010). In contrast,
TMEV-infection of SJL mice usually leads to mild neurological signs during the acute
phase. With the onset of white matter lesions (approximately 4 weeks post infection)
infected susceptible mice develop wobbling gait, progressive weakness of posterior
limbs and spastic paralysis. Clinical worsening is a consequence of prolonged
lymphocytic infiltration and ongoing demyelination of the spinal cord white matter
(DAL CANTO et al., 1996).
Chapter 1 - Introduction
1.3
15
T cells in demyelinating disorders
MS is considered to occur in genetically predisposed individuals in combination with
an as yet undetermined environmental factor which activates myelin-specific T cells
in the periphery. Due to the release of chemokines and the expression of endothelial
adhesion molecules in the CNS, these activated peripheral T cells and memory T
cells cross the blood brain barrier, enter the neuroparenchyma and become
reactivated. This process leads to further recruitment of immune cells to the CNS with
an enhancement of demyelination and axonal damage (GOVERMAN et al., 2009).
Similarly, EAE is mediated by encephalitogenic T cells which are primed in peripheral
lymphoid organs, cross the blood brain barrier and migrate into the brain where they
become reactivated by antigen presenting cells (MECHA et al., 2012). This process
leads to inflammatory demyelination and axonal damage (FLETCHER et al., 2010).
However, dependent upon their polarization, besides detrimental effects also
beneficial and neuroprotective effects can be exerted by CNS-infiltrating T cells (e.g.
regulatory T cells).
1.3.1 CD8+ T cells
Adequate and timely activation of the immune system determines the outcome of
viral CNS diseases. CD8+ T cells recognize viral antigen via MHC class I, which leads
to antiviral cytotoxicity (GRIFFIN, 2011). Under inflammatory conditions particularly
due to the influence of IFN-γ, oligodendrocytes can present viral antigen or myelin
antigen via MHC class I, which fosters CD8-mediated cytotoxicity and myelin damage
(SATO et al., 2011). The pivotal but also ambiguous role of CD8+ T cells in infectious
demyelinating disorders has been demonstrated in the coronavirus mouse model,
where CD8+ T cells produce IL-10 during the acute infection stage. This initial
secretion of the anti-inflammatory cytokine decreases the severity of immune
16
Chapter 1 - Introduction
responses which prevents immunopathology but on the other hand results in
inadequate antiviral immunity and virus persistence (PUNTAMBEKAR et al., 2011).
CD8+ T cells are also important for the pathogenesis of acute and chronic
demyelination caused by TMEV (McDOLE et al., 2006; JOHNSON et al., 2001;
MILLER et al., 1995). Thymectomized susceptible TMEV-infected SJL and CBA mice
show reduced numbers of CD8+ T cells associated with an early disease onset and
more profound clinical signs (BORROW et al., 1992). It has been observed that
genetic depletion of CD8+ T cells in mice with a resistant C57BL/6 background
causes TMEV persistence and hence the development of demyelinating disease.
This underlines the importance of CD8+ cytotoxic T cells for virus clearance in TME
(OLESZAK et al., 2004; MILLER et al., 1995). TMEV elimination in C57BL/6 mice is
a consequence of an effective CD8-mediated cytotoxicity directed against viral
epitopes VP2121-131, VP2165-173, and VP2110-120 (RICHARD et al., 2011). In
comparison, infected SJL mice show CD8+ T cells responses to VP3173-181, VP111-20,
and VP3159-166. At this, VP111-20-specific CD8+ T cells have been demonstrated to be
unable to lyse TMEV-infected cells. Moreover, an unfavorable ratio of CD8 + and
regulatory T cells in SJL mice is supposed to be responsible for disease susceptibility
and TMEV persistence, respectively (RICHARD et al., 2011). The protective role of
CD8+ T cells was also proven by experiments using β2M-deficient SJL mice. Here,
β2M-deficient mice exhibit high levels of virus as well as an increased macrophage
infiltration and pro-inflammatory cytokine production in the CNS, demonstrating a
protective role of CD8+ T cells in TMEV-induced demyelinating diseases (BEGOLKA
et al., 2001). Transfer of CD8+ T cells specific for VP3159-166 results in viral clearance
and prevention from TMEV-induced demyelinating disease in susceptible SJL mice.
This study shows that early CD8-mediated responses prevent disease development
in TME (GETTS et al., 2010). On the other hand, motor neuron function is preserved
Chapter 1 - Introduction
17
in CD8-depleted mice and neurological defects are reduced in MHC class I-deficient
mice, showing the complexity of immune responses in infectious CNS disorders
(JOHNSON et al., 2001; RIVERA-QUINONES et al., 1998). Similarly, ablation of
antiviral CD8+ T cells in IFN-γ receptor-depleted mice results in the preservation of
motor neuron function and maintenance of axonal transport mechanism following
TMEV-infection (HOWE et al., 2007).
A genetic association with certain MHC class I alleles has been described for MS. In
MS lesions an up-regulation of MHC class I can be observed on endothelial and
microglial cells, which enhances the activation of CD8 + T cells. Intact neurons do not
express MHC class I, but as a consequence of cell damage or under the influence of
pro-inflammatory cytokines neurons express this surface molecule and become a
target for cytotoxic T cells. Similar to EAE, cross-presentation of myelin epitopes by
antigen presenting cells (e.g. dendritic cells) is required for priming of CD8 + T cells at
initial stages and their retention within the CNS in MS (figure 1.6; FRIESE and
FUGGER, 2005; KARMAN et al., 2004).
18
Chapter 1 - Introduction
+
Figure 1.6. Mechanisms of invasion, activation and expansion of CD8 T cells in multiple sclerosis and
experimental autoimmune encephalomyelitis. Dendritic cells (DC) present autoantigens via MHC class
+
+
II to CD4 T cells. DC also activates CD8 T cells by cross-presentation via MHC class I. Besides
+
cytotoxic T cells, a portion of CD8 T cells differentiate into a regulatory phenotype to restrict immune
responses by IL-10 production. On the other hand, autoreactive cytotoxic T cells which invade the
central nervous system and interact with macrophages and microglia which express co-stimulatory
+
molecules (CD80, CD86, CD40) and MHC class I. Upon this, CD8 T cells become reactivated,
expand clonally and attack neurons and oligodendrocytes presenting antigens via MHC class I. This
process leads to tissue damage and lesion development (adopted from Friese and Fugger, 2005).
1.3.2 CD4+ T cells
Antigen presenting cells, such as dendritic cells in secondary lymphoid organs and
within inflammatory lesions present self or pathogen antigens via MHC class ΙΙ
molecules to naïve CD4+ T cells. Based on the stimulus or inflammatory environment,
CD4+ T cells differentiate into Th0, Th1, Th2, Th17 or regulatory T cells (figure 1.7).
Th1 cells produce interferon (IFN)-γ, interleukin (IL)-2 and TNF, while Th2 cells
produce IL-3, IL-4, IL-5, IL-10 and IL-13 (SWAIN et al., 2012; SAKAGUCHI, 2000).
Thus, CD4+ T cell play a pivotal role for cellular immune responses and humoral
immune responses. Besides their helper cell function, CD4 + T cells also act as
effector T cells and contribute to antiviral immune responses. They contribute to T
cell-mediated cytotoxicity and macrophage activation as demonstrated in a variety of
infectious diseases, such as experimental West Nile virus, influenza virus,
Chapter 1 - Introduction
19
Venezuelan equine encephalitis virus, dengue virus, Sendai virus and corona virus
infection (SWAIN et al., 2012). The effector function of CD4+ T cells is induced by
the production of IFN-γ and TNF as well as by the activation of cytolytic pathways,
including perforin release (SWAIN et al., 2012). For instance, the absence of CD4+ T
cells leads to compromised generation of cytotoxic T cells in influenza virus and
vaccinia virus infection (SWAIN et al., 2012). In TME, while C57BL/6 wild type mice
eliminate the virus and do not develop demyelination, CD4-deficient C57BL/6 mice
show inadequate antiviral immune responses and demyelinating disease. In contrast,
depletion of CD4+ T cells in TMEV-infected SJL mice decreases the severity of
demyelination (OLESZAK et al., 2004; NJENGA et al., 1996).
Th17 cells produce IL-17 and contribute to autoimmunity, as discussed for MS and
EAE. IL-17-mRNA has been detected in perivascular lymphocytes, oligodendrocytes
and astrocytes within active lesions of MS (TZARTOS et al., 2008). Experimental
transfer of Th1 and Th17 cells in Rag
-/-
mice which are deficient of B and T cells
show that these cells enter the CNS after 7 days. Th17 cells have been
demonstrated to disrupt the blood brain barrier which facilitates the immigration of
additional CD4+ T cells. Receptors for IL-17 and IL-22 are expressed on brain
endothelial cells of MS patients (KEBIR et al., 2007). Referring to this, CD4+ T cell of
MS patients secrete more IL-17 then cells of healthy individuals. A distinct population
of natural regulatory T cells expressing CD4, CD25, CD39, and Foxp3 is able to
suppress IL-17 production of responder T cells (FLETCHER et al., 2009).
20
Chapter 1 - Introduction
+
Figure 1.7: T cell polarization. Antigen presentation leads to the differentiation of naïve CD4 T cell into
different T cell subsets. The differentiation process is controlled by the cytokine milieu and expression
+
of specific transcription factors. Protective pathogen-specific immunity mostly depends on CD4 T cell
responses, which mediate lysis of infected cells and B cell responses, but potentially also initiates
immune mediated tissue damage (adopted from Swain et al., 2012).
Foxp3 = forkhead box p3, RORγt = retinoic acid receptor-related orphan receptor-γt, GATA3 = GATAbinding protien3, BCL-6 = B cells lymphoma-6,TFh = follicular helper T cells T-bet = T-box expressed
in T cells, EOMES = eomesodermin, TGF-β = transforming growth factor-β, TNF = tumor necrosis
factor.
1.3.3 Regulatory T cells
MS patients are supposed to have functional defects of regulatory T cells (Treg)
probably as a consequence of reduced levels of Foxp3 mRNA and protein.
Furthermore, disturbed thymic generation of Treg and expansion of memory Treg
might lead to an impaired homeostasis of the immune system and hence favor the
progression of autoimmunity in MS patients (LOWTHER and HAFLER, 2012). In
agreement with this, in vivo expansion of Treg and the adoptive transfer of in vitro
expanded Treg reduce the severity of demyelination in EAE (KORN et al., 2007; JEE
et al., 2007).
Treg have the ability to reduce inflammatory responses in the brain by inhibiting
effector T cells, microglia, macrophages and astrocytes. However, in contrast to
primary autoimmune myelin loss disorders, in infectious demyelinating diseases Treg
Chapter 1 - Introduction
21
can exhibit both beneficial effects by reducing immune mediated tissue damage and
detrimental effects due to their immunosuppressive properties, causing disease
exacerbation or viral persistence, respectively (MARTINEZ et al., 2014; RICHARDS
et al., 2011).
1.4
Regulatory T cells and immune homeostasis
Initially Treg were recognized as a CD4+CD25+high T cell population with strong
suppressive effects on effector T cells in vitro following antigenic stimulation. Later
the transcription factor forkhead box P3 (Foxp3) was identified as a specific marker
for Treg. A mutation of the FOXP3 gene leads to immune dysfunction,
polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is a fatal disease
in humans. The syndrome is associated with a dysfunction of Treg and the induction
of autoimmunity. Similarly, in mice, Foxp3 mutation is responsible for the scurfy
phenotype that results in early death of hemizygous males. As a consequence of
impaired immune regulation, scurfy mice show an increased proliferation of CD4+ T
cells and excessive cytokine responses. In contrast, over-expression of the FOXP3
gene in mice leads to reduced numbers of T cells, which demonstrates the
importance of Foxp3 for proper Treg function and maintenance of immune regulation
(LOWTHER and HAFLER, 2012).
The population of Treg includes natural Treg and inducible Treg. It is assumed that
together with effector T cells pathogen-specific Treg expand during virus infection.
These cells might represent thymus-originated natural Treg (figure 1.7) or induced
Treg originating from virus-specific effector CD4+ T cells (SWAIN et al., 2012).
Natural Treg develop within the thymus and express CD25 as well as Foxp3. They
are also characterized by the expression of glucocorticoid induced tumor necrosis
22
Chapter 1 - Introduction
factor receptor (GITR), cytotoxic T lymphocyte antigen-4 (CTLA-4), CD152, CD103,
CD45RO and neurophilin (NANDAKUMAR et al., 2009). In the thymus immature T
cells exhibit a CD4-CD8- phenotype (double negative) and subsequently a CD4+CD8+
phenotype (double positive). Due to interactions with self-peptides a subpopulation of
thymocytes develops into Treg with immunomodulatory properties, which maintain
immune homeostasis in the periphery (NANDAKUMAR et al., 2009). Inducible Treg
include T regulatory-1 (Tr1) cells, Th3 cells and CD8+ Treg. Tr1 cells secrete
increased amounts of IL-5, IL-10 and TGF-β and develop from naïve CD4+ T cells
due to chronic stimulation in infectious or neoplastic diseases. Th3 cells are induced
by oral administration of antigen and secrete TGF-β (NANDAKUMAR et al., 2009;
FLETCHER et al., 2010).
Central tolerance is induced in the thymus, where developing thymocytes that
recognize self-antigens with a high affinity are deleted. However, the process of
negative selection and elimination of autoreactive T cells is incomplete in the thymus.
Control of self-reactive T cells in the periphery is termed peripheral tolerance, which
is maintained by different mechanisms. For instance, a lack of co-stimulatory
molecules (CD80 and CD86) on antigen presenting cells lead to T cell anergy. T cell
suppression is also induced by the interaction between co-stimulatory molecules and
CTLA-4 (NANDAKUMAR et al., 2009; ROMAGNANANI, 2006). In addition,
peripheral tolerance is a consequence of T cell depletion by activation induced cell
death. Here, interaction between Fas and Fas-ligand lead to T cell apoptosis. This
process occurs when T cells are exposed to a high amount of antigen
(ROMAGNANANI, 2006). Furthermore, Treg diminish the availability of IL-2 for other
T cells by their increased density of IL-2 receptors (IL-2 deprivation) and release
immunosuppressive cytokines such as interleukin-10 and transforming growth factorβ (NANDAKUMAR et al., 2009). Certain Treg also suppress the production of IL-17
Chapter 1 - Introduction
23
and Th17 cells, respectively, with the help of the ectonucleotidase CD39. This
enzyme hydrolyzes extracellular ATP to ADP or AMP and finally to adenosine which
exhibits immunosuppressive effects on T cells. The mechanism requires direct
contact between CD39+ Treg and IL-17 producing Th17 cells (FLETCHER et al.,
2009).
In summary, the generation of Treg represents a physiological process to prevent
immune mediated tissue damage (MACDONALD et al., 2002; SAKAGUCHI, 2003;
VIGNALI et al., 2008). However, it has become evident that Treg not only influence
self-antigen specific immune responses but also dampen foreign antigen specific
immunity in infectious diseases, including CNS disorders (NANDAKUMAR et al.,
2009).
24
Chapter 2 – Materials and Methods
Chapter 2 Materials and Methods
25
26
Chapter 2 - Materials and Methods
2.
Materials and Methods
2.1
Experimental design and tissue preparation
Five-week old, female C57BL/6 mice JOla Hsd (Harlan) were divided into one control
group and three different treatment groups. All mice were inoculated in the right
cerebral hemisphere with 1.63x106 plaque forming units of the BeAn strain of TMEV
diluted in 20µl Dulbecco’s modified Eagle Medium (PAA Laboratories) with 2% fetal
calf serum and 50 µg/kg gentamicin. The virus was kindly provided by Prof. Dr. H.L.
Lipton (Department of Neurology, Northwestern University Medical School Chicago,
USA). For intracerebral injection, mice were anesthetized with medetomidine
(0.5mg/kg, Domitor, Orion Pharma) and ketamine (100mg/kg, Ketamine 10%, WDT
eG). Additionally, the control group received intraperitoneal PBS injections (group I,
TMEV). Group II animals (Treg _TMEV) were treated with interleukin-2 immune
complexes (IL-2C; 12µg/mouse) to expand regulatory T cells (WEBSTER et al.,
2009, BOYMAN et al., 2006). IL-2C consist of 10µg of anti-IL-2 antibodies
(eBiosciences, clone JES6-1A12) mixed with 2µg murine recombinant IL-2 proteins
(eBiosciences, catalog number 34-8021), incubated at 37˚C for 30 minutes before
injection. IL-2C were administered intraperitoneally on three consecutive days (day 3, -2, -1) before TMEV-infection on day 0 (figure 2.1). For depletion of CD8+ T cells,
group III animals (CD8 _TMEV) received anti-CD8 antibodies (CHANG et al., 2001;
BioXCell, clone 53-6.72, 500µg/mouse) on three consecutive days (day -3, -2, -1)
intraperitoneally before virus infection at day 0. In order to determine a synergistic
effects of these two treatments, group IV animals (Treg _CD8 _TMEV) received a
combined treatment with IL-2C and anti-CD8 antibodies on three consecutive days
(day -3,-2,-1) before TMEV-infection. IL-2C and anti-CD8-antibodies were injected
with an interval of 6 hours.
Chapter 2 - Material and Methods
27
Animals were weekly examined for the presence of gait abnormalities: 0=normal gait,
1 = mild ataxia with inconsistent waddling gait, 2 = moderate ataxia with consistent
waddling gait or stiff gait and paddling tail, 3 = severe ataxia with stiff or sliding gait
and reduced righting response, and 4 = severe ataxia and spastic paresis of the hind
legs, as described previously (ULRICH et al., 2006). Treated and control animals
were euthanized at 3, 7, 14, 42 days post infection (dpi) and necropsied (figure 2.1).
For necropsy animals were first anesthetized as described above and then
euthanized with an overdose of medetomidine (2.0 mg/kg) and ketamine (400
mg/kg). Animals were perfused via the left ventricle of the heart with PBS and brain,
spinal cord, and spleen were removed. Spleen weight was measured before dividing
into threeparts for formalin fixation and paraffin embedding (histology and
immunohistochemistry) and flow cytometric analysis, respectively. Blood samples
were taken before perfusion and collected for flow cytometric analysis after adding 50
units of heparin (Heparin-Natrium-5000-ratiopharm® GmbH).
The spinal cord was divided into three parts (cervical, thoracic and lumbar spinal
cord), formalin fixed for 24 hours and subsequently embedded in paraffin for
histology and immunohistochemistry. The brain was divided into four parts. The
caudal half of the cerebrum and cranial part of the cerebellum were fixed in 10%
formalin for 24 hours and then embedded in paraffin wax, while the cranial half of
cerebrum and caudal half of the cerebellum were snap frozen in liquid nitrogen
stored at -80˚C.
The animal experiment was approved and authorized by the local authorities
(Niedersächsisches Landesamt für Verbraucherschutz- und Lebensmittelsicherheit
[LAVES], Oldenburg, Germany, permission number: 33.9-42502)
28
Chapter 2 - Materials and Methods
Figure 2.1. Experimental design. On three consecutive days (-3d, -2d, -1d) prior to
Theiler’s murine encephalomyelitis virus (TMEV) infection, mice received an
intraperitoneal injection of interleukin-2 (IL-2) complexes (group II), CD8-depleting
antibodies (group III) or a combined treatment with IL-2C and CD8-depleting
antibodies (group IV). Control animals (group I) consist of infected animals which
received only PBS intraperitoneally. At day 0 (0d), TMEV was injected into right
cerebral hemisphere under general anesthesia. Necropsies (†) were performed at 3,
7, 14, and 42 days post infection (3d, 7d, 14d, 42d).
2.2
Histological examination of brain and spinal cord
Transversal sections of formalin-fixed, paraffin-embedded brain and spinal cord
segments (cervical, thoracic, lumbar) were stained with hematoxylin and eosin (HE).
Inflammatory responses in the hippocampus were graded based upon the degree of
perivascular infiltrates and hypercellularity using a semiquantitative scoring system: 0
= no changes, 1 = scattered perivascular infiltrates, 2 = two to three layers of
perivascular inflammatory cells, 3 = more than three layers of perivascular
inflammatory cells. Hypercellularity within the brain parenchyma was graded
Chapter 2 - Material and Methods
29
semiquantitatively as follows: 1 = 1-25 cells; 2 = 26-100 cells (moderate); 3 = > 100
cells per high power field.
Grading of the inflammatory response in the spinal cord was measured by a semiquantitative scoring system based on the degree of perivascular infiltration (0 = no
changes, 1 = one layer, 2 = two to three layers, 3 = more than three layers if
inflammatory cells in the meninges) and hypercellularity (0 = no change, 1 = 1-25
cells, 2 = 26-50 cells, 3 = >50 cells) as described previously (HERDER et al., 2012,
GERHAUSER et al., 2007).
2.3. Immunohistochemistry
In order to detect viral antigen (TMEV), myelin basic protein (MBP), β-amyloid
precursor protein (β-APP), CD3, CD45R, CD107b, and the transcription factor
forkhead box P3 (Foxp3), a standard avidin-biotin-peroxidase complex (Vector
Laboratories)
method
was
used
as
described
(HERDER
et
al.,
2012;
KUMMERFELD et al., 2009). A monoclonal rat Foxp3-specific antibody (NatuTec)
was used for the detection of Tregs, a monoclonal rat anti-CD45R/B220-specific
antibody (BD Biosciences) for the detection of B cells and a monoclonal rat CD107bspecific antibody (AbD Serotec) for the detection of macrophages/microglia (table
2.1). Additionally, a polyclonal rabbit anti-CD3-antibody (DakoCytomation) for the
detection of T cells was used (table 2.1). Damaged axons were labeled with a
monoclonal mouse β-amyloid precursor protein (β-APP)-specific antibody (table 2.1;
Chemicon International Inc.). For blocking of the endogenous peroxidase, formalinfixed, paraffin-embedded tissue sections were treated with 0.5% H2O2 diluted in
methanol for 30 minutes at room temperature. For the demonstration of Treg, T cells,
B cells, macrophages/microglia and β-APP sections were heated in 10 mM Na-citrate
buffer pH 6.0 for 20 min in a microwave oven (800 W). Thereafter, sections for the
30
Chapter 2 - Materials and Methods
detection of Treg and B cells were incubated with 20% rabbit serum, while sections
for the detection of β-APP-expressing axons were incubated with 20% goat serum
each for 30 minutes to block non-specific binding sites prior to incubation with
primary antibodies.
Subsequently, slides were incubated with the respective primary antibody overnight
at 4°C. Used antibody dilutions are given in table 2.1. Biotinylated goat-anti-rabbit
IgG diluted 1:200 (Vector Laboratories) was used as a secondary antibody for the
labeling of T cells and macrophages/microglia for one hour at room temperature.
Demonstration of Foxp3- and β-APP-specific binding was performed by a biotinylated
rabbit-anti-rat antibody (Vector Laboratories) and a goat-anti-mouse antibody (Vector
Laboratories), respectively, as secondary antibodies. No secondary antibody was
necessary for detection of B cells via the biotinylated monoclonal rat antiCD45R/B220-specific antibody (BD Biosciences) that was used at a dilution of
1:1000. Sections used as negative controls for CD3-immunohistochemistry were
incubated with rabbit normal serum at a dilution of 1:3000 (Sigma-Aldrich Chemie
GmbH). Additionally negative controls for Foxp3 and CD45R/B220 were stained with
rat-IgG2-isotype control antibody (R&D Systems GmbH), for CD107b with a rat-anti
IgG1-isotype control antibody (R&D Systems GmbH) and for β-APP with a mouseanti IgG1-isotype control (Millipore). Slides were subsequently incubated with the
peroxidase-conjugated avidin-biotin complex for 30 minutes at room temperature.
After
positive
antigen-antibody
reaction
visualization
by
incubation
with
3.3-diaminobenzidine-tetrachloride in 0.1M imidazole, sections were counterstained
with Mayer’s hematoxylin.
The absolute numbers of TMEV-infected cells labeled by immunohistochemistry as
well as CD3+, CD45R+, CD107b+, Foxp3+ cells were counted in cross section of the
cerebrum (hippocampus) and spinal cords of infected mice. The obtained brown
Chapter 2 - Material and Methods
signal
following
incubation
with
31
β-APP-specific
antibodies
was
evaluated
quantitatively by counting the number of positive axons in spinal cord cross sections.
Table 2.1: Antibodies used for immunohistochemistry
Antigen
Specificity
Company;
Blocking
Pre-
product
serum
treatment
Dilution Secondary Reference
antibody
Microwave, 1:1000
Goat anti- HERDER et al.,
number
CD3
T cells
Dako/Agilent
Goat
Technologie;
20
A0452
minutes,
rabbit
2012
citrate
buffer
Foxp3
Regulatory T cells
eBioscience;
Rabbit
14-5773
Microwave,
1:20
20
Rabbit anti- HERDER et al.,
rat
2012
minutes,
citrate
buffer
TMEV
TMEV
None
CD107b Macrophages/microglia Abd serotec;
Goat
Rat
MCA 2293B
None
1:2000
Microwave, 1:200
Goat anti- KUMMERFELD
rabbit
et al., 2009
None
HERDER et al.,
2012
20
minutes,
citrate
buffer
β-APP
Axonal damage
Chemicon;
Mouse
MAB348
Microwave, 1:2000
20
Goat anti-
KREUTZER et
mouse
al., 2012
minutes,
citrate
buffer
MBP
Myelin sheaths
Merck/Millipore;
Goat
None
1:500
AB980
CD45R
B cells
BD
None
Microwave, 1:1000
Biosciences;
20
550286
minutes,
Goat anti- HERDER et al.,
rabbit
2013
None
HERDER et al.,
2012
citrate
buffer
TMEV = Theiler’s murine encephalomyelitis virus, MBP = myelin basic protein, β-APP = beta-amyloid
precursor protein, Foxp3 = Forkhead box P3
32
2.4
Chapter 2 - Materials and Methods
Flow cytometry
2.4.1 Spleen samples
Spleen cells were dissolved to single cell suspension in phosphate buffered saline
(PBS; Gibco) containing 0.2% bovine serum albumin (BSA, Sigma Aldrich) using a
100µm sieve at room temperature. Cells were centrifuged for 10 minutes at 20°C
(1400 rounds per minutes [rpm]) and erythrocyte lysis was performed by adding 1ml
ACK-buffer containing 0.01M KHCO3, 0.155M NH4Cl and 0.1mM EDTA (pH7.5) for
3.5 minutes at room temperature. PBS/BSA was added and cells washed again
(1400 rpm, 10 minutes, 20°C). The cell pellet was re-suspended in PBS/BSA and
filtered (30µm). Subsequently, cells were stored on ice until further proceeding.
The cell number was determined using trypan blue solution (Sigma Aldrich) in a
counting chamber. 2 x 106 cells were pipetted into each tube. 600µl PBS was added
and cells washed again (1400 rpm, 4 minutes, 4°C). For FcγR blockade, 100 µl of
Fcy block medium (BioXcell, 2.4G2, 1:100, diluted in PBS) were added to each tube.
Samples were incubated for 5 minutes at 4°C. Subsequently, 600µl PBS was added
and washing procedure was repeated (1400 rpm, 4 minutes, 4°C). Live/dead staining
was performed using the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen,
1:500, diluted in PBS). 100 µl were added to each tube and samples were incubated
for 30 minutes at 4°C. Following, 600µl PBS/BSA were added and washing
procedure was repeated (1400 rpm, 4 minutes, 4°C).
For cell surface staining, 100µl PBS/BSA and antibodies (aCD4-HV450 [BD
Horizont], aCD8-APC [Biolegend]) were added (table 2.2). Dilution of antibodies
depends on titration of lot. Samples were incubated for 15 minutes at 4°C. Cells were
washed by adding 600µl PBS/BSA (1400 rpm, 4 minutes, 4°C). For fixation, cells
were re-suspended in 200µl fixation/permeabilization concentrate (one part of fixation
Chapter 2 - Material and Methods
33
permeabilization concentrate with three parts fixation permeabilization diluent) and
incubated for 30 minutes at 4°C. Subsequently, 600µl PBS/BSA was added and
washing procedure was repeated (1400 rpm, 4 minutes, 4°C). Finally, the cell pellet
was re-suspended in 600µl PBS/BSA and stored at 4°C in the dark.
Intracellular staining for the detection of Foxp3 was performed using the
Foxp3/transcription
factor
staining
buffer
set
(eBioscience). Samples
were
centrifuged (1400 rpm, 4 minutes, 4°C) and 200µl 1x working solution of the
permeabilization buffer was added. Centrifugation was repeated (1400 rpm, 4
minutes, 4°C). 50µl permeabilization buffer, including rat IgG (40µg/ml) were added
and samples were incubated for 15 minutes at 4°C in the dark. Subsequently, without
washing 50µl permeabilization buffer and the anti-Foxp3 antibody (aFoxp3-PE
[eBioscience]) were added and incubated for 30 minutes at 4°C in the dark (table
2.2). Followings, 100 µl permeabilization buffer were added and samples were
centrifuged (1400 rpm, 4 minutes, 4°C). In the next step, 200 µl permeabilization
buffer was added and samples were incubated for 5 minutes at room temperature in
the dark. The centrifugation step was repeated (1400 rpm, 4 minutes, 4°C) and cells
were re-suspended in PBS/BSA and stored at 4°C in the dark until measurement.
2.4.2 Blood samples
For erythrocyte lysis, 75µl whole blood were mixed with 2ml ACK-buffer and
incubated for 4 minutes at room temperature. Lysis reaction was stopped by adding 3
ml RPMI medium 1640. Samples were centrifuged (300xg, 8 minutes, 4°C) and
supernatants were discarded. Following cells were washed in 3ml PBS/BSA.
For cell surface staining a four-fold concentrated antibody mix was added to the
residual volume, including Fcy block medium (1:150, diluted in PBS). Samples were
incubated for 15 minutes at 4°C in the dark. Following cells were washed in 3ml
34
Chapter 2 - Materials and Methods
PBS/BSA.
For
intracellular
fixation/permeabilization
staining
concentrate
cells
(one
part
were
of
re-suspended
fixation
in
1ml
permeabilization
concentrate and 3 parts fixation permeabilization diluent) and incubated for 30
minutes at 4°C in the dark. After centrifugation (300xg, 8 minutes, 4°C) cells were resuspended in 600 µl PBS/BSA at 4°C in the dark until further processing. According
to the staining protocol used for spleen cells, blood cells were washed twice with 2ml
of 1x working solution of the permeabilization buffer and supernatants were
discarded. 50µl permeabilization buffer, including rat IgG (40µg/ml) were added and
samples were incubated for 15 minutes at 4°C in the dark. Subsequently, without
washing 100 µl permeabilization buffer and the anti-Foxp3 antibody were added and
incubated for 30(-45) minutes at 4°C in the dark. Afterwards 400 µl permeabilization
buffer were added and samples were centrifuged (1400 rpm, 4 minutes, 4°C). Adding
of 400µl permeabilization buffer was repeated and samples were incubated for 5
minutes at room temperature in the dark. Finally, cells were re-suspended in
PBS/BSA and stored at 4°C in the dark until measurement. Used antibodies are
listed in table 2.2.
Cells were sorted on the BD LSR-II SORP cytometer (BD bioscience) and data were
analyzed using FlowJo software
Table 2.2. Antibodies used for flow cytometry
Antibody
Company
Clone
αCD4 HV450
BD Horizont
RPA-T4
αCD8 APC
BioLegend
SK1
Foxp3 PE
eBioscience
FJK-16s
Chapter 2 - Material and Methods
2.5
35
Reverse transcription quantitative polymerase chain reaction
2.5.1 RNA isolation and reverse Transcription
Snap frozen coronal brain slices were cut and RNA was isolated from 10-40 mg
tissue of each brain using an Omni’s PCR Tissue Homogenizing Kit (SüdLaborbedarf GmbH), QIAzol® lysis reagent (Qiagen) and RNeasy® Lipid Tissue Mini
Kit (Qiagen). RNA sample quality and amount of RNA was measured by applying a
NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific). Equal amounts of
RNA were subsequently transcribed into cDNA with the Omniscript TM RT Kit
(Quiagen
GmbH),
RNaseOUT™
Recombinant
Ribonuclease
Inhibitor
(life
technologies), and random primers (Promega). For the reverse transcription, the
following thermo cycler program was used: 10 minutes at 25°C, followed by 1 hour at
37°C and at the end 5 minutes at 93°C.
2.5.2 Polymerase chain reaction
RT-qPCR for the quantification of TMEV and three housekeeping genes
(glyceraldehyde 3-phosphate dehydrogenase (GAPDH) β-actin and hypoxanthineguanine phosphoribosyltransferase (HPRT) in brain tissues was performed using the
Mx3005P™ Multiplex Quantitative PCR System (Agilent Technologies) and Brilliant
III SYBR® Green Mastermix (Agilent Technologies). Used primer sequences are
listed in table 2.3. For each PCR, 40 cycles were performed starting with
denaturation with 30 seconds at 95°C, followed by 20 seconds annealing and
elongation using the annealing temperature indicated in table 2.3. For each gene,
ten-fold serial dilution standards ranging from 108 to 102 copies/µl were used to
quantify the results. In order to compare the results of each gene, which were
performed in two different PCR-runs, a multiple experiment analysis was performed
applying the MxPro QPCR Software (Agilent Technologies). A normalization factor
36
Chapter 2 - Materials and Methods
achieved from the three housekeeping genes was calculated using the geNorm
software version 3.4 to correct for experimental variations (VANDESOMPELE et al.,
2002). Specificity of each reaction was controlled by melting curve analysis
(GERHAUSER et al., 2005; ULRICH et al., 2006).
Table 2.3. Summary of primer pairs used for the quantification of viral RNA and
housekeeping genes in the brain of infected C57BL/6 mice
Gene
β-Actin
Accession
Annealing
Primer
number
temperature
direction
(GeneBank)
[°C]
NM_007393.2
60
HPRT
NM_013556.2
60
GAPDH
XM_001476683.1
60
TMEV
GI_335239
65
Sequence of primer from 5 → 3
Forward
GGC TAC AGC TTC ACC ACC AC
Reverse
ATG CCA CAG GAT TCC ATA CC
Forward
GGA CCT CTC GAA GTG TTG GA
Reverse
TTG CGC TCA TCT TAG GCT TT
Forward
GAG GCC GGT GCT GAG TAT GT
Reverse
GGT GGC AGT GAT GGC ATG GA
Forward
GAC TAA TCA GAG GAA CGT CAG C
Reverse
GTG AAG AGC GGC AAG TGA GA
HPRT: hypoxanthine phosphoribosyl transferase; GAPDH: glyceraldehyde-3-phosphate
dehydrogenase; TMEV: Theiler´s murine encephalomyelitis virus
Chapter 2 - Material and Methods
2.6
37
Statistical analyses
For statistical analysis Mann-Whitney U-test was performed to determine any
difference between treatment groups and control groups. A p-value of ≤ 0.05 was
considered as being statistically significant. Analyses were performed using SPSS for
windows (version 17.0, SPSS Inc.).
38
Chapter 3 - Results
39
Chapter 3 Results
40
Chapter 3 - Results
3.
Results
3.1
Clinical findings in C57BL/6 mice following regulatory T cell expansion
and depletion of CD8+ T cells
Animal were clinically examined weekly as described in materials and methods.
Clinical findings, including mild gait abnormalities were observed in three animals of
the combined treated group (group IV, Treg _CD8 _TMEV) at 42 dpi. No changes,
suggestive of neurological deficits were observed in other groups or time points,
respectively.
Statistical analysis revealed significantly elevated clinical scores in combined treated
mice (group IV, Treg _CD8 _TMEV) at 42 dpi (figure 3.1) compared to untreated
virus-infected mice (group I, TMEV). Results of statistical analyses are listed in the
annex (table 8.1.1).
Figure 3.1. Clinical evaluation (clinical scores) of Theilervirus-infected C57BL/6 mice
with different treatments (TMEV = no treatment; Treg _TMEV = expansion of
regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
Chapter 3 - Results
3.2
41
Effects of regulatory T cell expansion and depletion of CD8 + T cells upon
the peripheral immune system
3.2.1 Spleen weight
An increase of spleen weights together with organ enlargement was found
predominantly in combined treated mice (group IV, Treg _CD8 _TMEV) and Tregexpanded mice (group II, Treg _TMEV) at 3 dpi. At later time points adjustment of
spleen weights was observed in the different groups.
Statistical analysis revealed significantly increased spleen weights of combined
treated mice (group IV, Treg _CD8 _TMEV) compared to CD8-depleted mice
(group III, CD8 _TMEV), Treg-expanded mice (group II, Treg _TMEV) and
untreated mice at 3 dpi. In addition, Treg-expanded animals (group II, Treg _TMEV)
showed significantly increased spleen weight compared to untreated virus-infected
mice (group I, TMEV) and CD8-depleted mice (group III, CD8 _TMEV). At 7 dpi
CD8-depleted mice (group III, CD8 _TMEV) exhibited significantly decreased
spleen weights compared to Treg-expanded mice (group II, Treg _TMEV; figure
3.2). Results of statistical analyses are listed in the annex (table 8.1.2).
Figure 3.2. Spleen weights in Theilervirus-infected C57BL/6 mice with different
treatments (TMEV = no treatment; Treg _TMEV = expansion of regulatory T cells
(Treg) by interleukin (IL)-2 immune complexes; Treg _CD8 _TMEV = expansion of
Treg by IL-2 immune complexes and antibody-mediated CD8-depletion). Box and
whisker plots display median and quartiles together with minimum and maximum
values. Significant differences (p 0.05) are labeled with an asterisk.
42
Chapter 3 - Results
3.2.2 Quantification of Foxp3+ cells in the blood by flow cytometry
After necropsy blood samples were immediately processed for flow cytometric
analyses to determine the relative amount of Foxp3+ Treg. Increased levels of Foxp3+
cells were observed in Treg-expanded mice (group II, Treg _TMEV) and combined
treated mice (group IV, Treg _CD8 _TMEV), showing the efficacy of IL-2 immune
complexes to expand Foxp3+ Treg.
Statistical analyses revealed significantly higher percentages of Foxp3 + cells in Tregexpanded mice (group II, Treg _TMEV) and combined treated mice (group IV,
Treg _CD8 _TMEV) compared to untreated virus-infected mice (group I, TMEV)
and CD8-depleted mice (group III, CD8 _TMEV) at 3 and 7 dpi. At 14 dpi combined
treated mice (group IV, Treg _CD8 _TMEV) exhibited higher percentages of
Foxp3+ cells compared to all other three groups, including Treg-expanded mice
(group II, Treg _TMEV), indicative of a prolonged effect of the combined treatment
upon peripheral blood Treg elevation. Strikingly, a significant reduction of Foxp3 +
cells was observed in IL-2 immune complex treated mice (group II, Treg _TMEV)
compared to all other groups at 14 dpi. Similarly, at 42 dpi a significant decrease of
the relative portion of Foxp3+ cells was observed in the blood of Treg-expanded mice
(group II, Treg _TMEV) compared to other groups (figure 3.3). Results of statistical
analyses are listed in the annex (table 8.1.3).
Chapter 3 - Results
43
Figure 3.3. Quantification of Foxp3+ cells in the blood (percentage of Foxp3+ cells of
total CD4+ T cells) by flow cytometry in Theilervirus-infected C57BL/6 mice with
different treatments (TMEV = no treatment; Treg _TMEV = expansion of regulatory T
cells (Treg) by interleukin (IL)-2 immune complexes; Treg _CD8 _TMEV =
expansion of Treg by IL-2 immune complexes and antibody-mediated CD8depletion). Box and whisker plots display median and quartiles together with
minimum and maximum values. Significant differences (p 0.05) are labeled with an
asterisk.
3.2.3 Measurement of CD4/CD8 ratio in the blood by flow cytometry
Blood samples were analyzed by flow cytometry to determine the amount of CD4+
and CD8+ T cells. As a consequence of antibody mediated reduction of CD8 + T cells,
highest CD4/CD8 ratios were observed in CD8-depleted mice (group III,
CD8 _TMEV) and combined treated animals (group IV, Treg _CD8 _TMEV).
Interestingly, a prolonged effect upon the CD4/CD8 ratio till the end of the
observation period (42 dpi) was present in these two groups, with highest values
observed in combined treated mice (group IV, Treg _CD8 _TMEV; figure 3.4).
Statistical analyses revealed a significant increase of the CD4/CD8 ratio in combined
treated mice (group IV, Treg _CD8 _TMEV) and CD8-depleted mice (group III,
CD8 _TMEV) compared to untreated virus-infected mice (group I, TMEV) and Tregexpanded mice (group II, Treg _TMEV) at all investigated time points. Significantly
elevated CD4/CD8 ratios were also detected in Treg-expanded mice (group II,
Treg _TMEV) compared to untreated mice (group I, TMEV) at 7 and 14 dpi. While
significantly reduced values were found in combined treated mice (group IV,
Treg _CD8 _TMEV) compared to CD8-depleted mice (group III, CD8 _TMEV) at 7
44
Chapter 3 - Results
dpi, significantly increased CD4/CD8 ratios were present in combined treated mice
(group IV, Treg _CD8 _TMEV) compared to CD8-depleted mice (group III,
CD8 _TMEV) at later time points (14 and 42 dpi; figure 3.4). Results of statistical
analyses are listed in the annex (table 8.1.4).
Figure 3.4. Measurement of the CD4/CD8 ratio in blood samples by flow cytometry in
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
3.2.4
Quantification of Foxp3+ cells in the spleen by flow cytometry
At necropsy, spleen samples were immediately processed for flow cytometric
analyses. Similar to blood samples, elevated percentages of splenic Foxp3 + Treg
were observed following IL-2 immune complex treated groups with (group IV,
Treg _CD8 _TMEV) and without CD8-depletion (group II, Treg _TMEV). However,
in contrast to blood samples, no elevated values were found in combined treated
mice (group IV, Treg _CD8 _TMEV) at 14 dpi.
Statistical analyses revealed a significantly higher percentage of Foxp3 + Treg among
CD4+ T cells in the spleen of Treg-expanded mice (group II, Treg _TMEV) and
combined treated mice (group IV, Treg _CD8 _TMEV) compared to untreated
Chapter 3 - Results
45
virus-infected mice (group I, TMEV) and CD8-depleted mice (group III,
CD8 _TMEV) at 3 and 7 dpi (figure 3.5). The percentage was significantly reduced in
combined treated mice (group IV, Treg _CD8 _TMEV) compared to Treg-expanded
mice (group II, Treg _TMEV) at 7 dpi. At 14 dpi, no differences were observed
between groups. At 42 dpi, a significant reduction of the relative portion of Foxp3+
cells was found in combined treated mice (group IV, Treg _CD8 _TMEV) compared
to Treg-expanded mice (group II, Treg _TMEV). Results of statistical analyses are
listed in the annex (table 8.1.5).
Figure 3.5. Quantification of Foxp3+ cells in the spleen (percentage of Foxp3+ cells of
total CD4+ T cells) by flow cytometry in Theilervirus-infected C57BL/6 mice with
different treatments (TMEV = no treatment; Treg _TMEV = expansion of regulatory T
cells (Treg) by interleukin (IL)-2 immune complexes; Treg _CD8 _TMEV =
expansion of Treg by IL-2 immune complexes and antibody-mediated CD8depletion). Box and whisker plots display median and quartiles together with
minimum and maximum values. Significant differences (p 0.05) are labeled with an
asterisk.
3.2.5
Measurement of CD4/CD8 ratio in the spleen by flow cytometry
Spleen samples were taken at necropsy and immediately processed for flow
cytometric analyses. Similar to blood samples, highest values were observed in CD8depleted animals (group III, CD8 _TMEV) and combined treated animals (group IV,
Treg _CD8 _TMEV).
46
Chapter 3 - Results
Statistical analyses revealed significantly increased CD4/CD8 ratios in the spleen of
combined treated (group IV, Treg _CD8 _TMEV), CD8-depleted (group III,
CD8 _TMEV) and Treg-expanded (group II, Treg _TMEV) mice compared to
untreated virus-infected mice (group I, TMEV) at 3 and 7dpi (figure 3.6). At later time
points (14 and 42 dpi), significantly elevated values were only observed in CD8depleted mice (group III, CD8 _TMEV) and combined treated mice (group IV,
Treg _CD8 _TMEV). Moreover, significantly increased CD4/CD8 ratios were found
in combined treated animals (group IV, Treg _CD8 _TMEV) compared to CD8depleted mice (group III, CD8 _TMEV), indicative of a prolonged and profound
effect of the combined treatment upon splenic T cell populations. Results of statistical
analyses are listed in the annex (table 8.1.6).
Figure 3.6. Measurement of the CD4/CD8 ratio in spleen samples by flow cytometry
in Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no
treatment; Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2
immune complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune
complexes and antibody-mediated CD8-depletion). Box and whisker plots display
median and quartiles together with minimum and maximum values. Significant
differences (p 0.05) are labeled with an asterisk.
Chapter 3 - Results
3.3
47
Effects of regulatory T cell expansion and depletion of CD8+ T cells in the
brain
3.3.1 Virus load quantification in the brain by real time polymerase chain
reaction
Real time PCR was performed to quantify the virus load in the brain. Viral RNA was
detected in all treatment groups.
Statistical analyses revealed a significantly higher amount of viral RNA in Tregexpanded mice (group II, Treg _TMEV) as compared to CD8-depleted mice (group
III, CD8 _TMEV) and combined treated mice (group IV, Treg _CD8 _TMEV) at 7
dpi. Contrary combined treated mice (group IV, Treg _CD8 _TMEV) at 14 dpi had
significantly higher viral RNA levels compared to Treg-expanded mice (group II,
Treg _TMEV), CD8-depleted mice (group III, CD8 _TMEV) and untreated virusinfected mice (group I, TMEV). CD8-depleted mice (group III, CD8 _TMEV) showed
significantly higher concentrations of viral RNA as compared to Treg-expanded mice
(group II, Treg _TMEV) mice at 14 dpi. At 42 dpi combined treated mice (group IV,
Treg _CD8 _TMEV) exhibited significantly higher concentrations of viral RNA
compared to Treg-expanded mice (group II, Treg _TMEV), CD8-depleted mice
(group III, CD8 _TMEV) and untreated virus-infected mice (group I, TMEV; figure
3.7). Results of statistical analyses are listed in the annex (table 8.1.7).
48
Chapter 3 - Results
Figure 3.7: Quantification of Theilervirus RNA in the brain of infected C57BL/6 mice
with different treatments (TMEV = no treatment; Treg _TMEV = expansion of
regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion) by real time PCR. Box and whisker plots display median
and quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
3.3.2 Virus load quantification in the brain by immunohistochemistry
The number of Theilervirus-infected cells was determined in the cerebrum by
immunohistochemistry. A preferential infection of cells with neuronal morphology
within the hippocampus was found (figure 3.8).
Statistical analyses revealed significantly higher numbers of infected cells in
combined treated mice (group IV, Treg _CD8 _TMEV) at 14 dpi compared to Tregexpanded
mice
(group
II,
Treg _TMEV),
CD8-depleted
mice
(group
III,
CD8 _TMEV) and untreated virus-infected mice (group I, TMEV). At 14 dpi Tregexpanded mice (group II, Treg _TMEV) had significantly more infected cells in the
cerebrum than untreated virus-infected mice (group I, TMEV). Similarly, at 42 dpi
combined treated mice (group IV, Treg _CD8 _TMEV) and Treg-expanded mice
(group II, Treg _TMEV) showed significantly higher numbers of infected cells as
compared to untreated virus-infected mice (group I, TMEV; figure 3.9). The increase
of infected cells in combined treated mice (group IV, Treg _CD8 _TMEV) and Tregexpanded mice (group II, Treg _TMEV) at 14 and 42 dpi is indicative of reduced
antiviral immunity. Results of statistical analyses are listed in the annex (table 8.1.8).
Chapter 3 - Results
49
Figure 3.8. Theilervirus-infected cells in the hippocampus of a C57BL/6 mouse
following expansion of regulatory T cells by interleukin-2 complex application and
depletion of CD8+ T cells (combined treatment) at 7 days post infection. Figure A
(Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.9: Quantification of infected cells in the cerebrum of Theilervirus-infected
C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV =
expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.3.3
Histological examination of the hippocampus
Transversal sections of formalin-fixed, paraffin-embedded brain tissue were HEstained for histological examination and scored as described in materials and
50
Chapter 3 - Results
methods. Lesions in the brain were dominated by hypercellularity and perivascular
infiltrations of the hippocampus (figure 3.10).
Statistical analyses revealed a significantly higher degree of inflammation in
combined treated mice (group IV, Treg _CD8 _TMEV) compared to untreated
virus-infected mice (group I, TMEV), at 14 and 42 days post infection (figure 3.11).
Results of statistical analyses are listed in the annex (table 8.1.9).
Figure 3.10: Inflammation of the hippocampus in a Theilervirus-infected C57BL/6
mouse following expansion of regulatory T cells by interleukin-2 complex application
and depletion of CD8+ T cells (combined treatment) at 14 days post infection. Figure
A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.11. Quantification of inflammatory responses in the hippocampus of
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
Chapter 3 - Results
3.3.4
51
Quantification of CD3+ T cells in the hippocampus
CD3+ T cell were observed in the hippocampus of all investigated C57BL/6 mice.
Immunoreactivity was characterized by a predominantly membrane bound staining of
cells with lymphocyte morphology (figure 3.12).
Statistical analyses revealed a significantly higher infiltration of CD3 + T cell in Tregexpanded mice (group II, Treg _TMEV) compared to combined treated (group IV,
Treg _CD8 _TMEV), CD8-depleted mice (group III, CD8 _TMEV) and untreated
virus-infected mice (group I, TMEV) at 3 dpi. Similarly at 7 dpi combined treated
mice (group IV, Treg _CD8 _TMEV) showed significantly less CD3+ T cell in the
hippocampus as compared to Treg-expanded mice (group II, Treg _TMEV) and
untreated virus-infected mice (group I, TMEV). At 14 dpi significantly higher numbers
of
CD3+
T
cell
were
present
in
combined
treated
mice
(group
IV,
Treg _CD8 _TMEV) than in untreated virus-infected mice (group I, TMEV; figure
3.13). Results of statistical analyses are listed in the annex (table 8.1.10).
Figure 3.12. CD3+ T cells in the hippocampus of a Theilervirus-infected C57BL/6
mouse following expansion of regulatory T cells by interleukin-2 complex application
and depletion of CD8+ T cells (combined treatment) at 14 days post infection. Figure
A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
52
Chapter 3 - Results
Figure 3.13. Quantification of CD3+ T cells in the hippocampus of Theilervirusinfected C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV
= expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.3.5
Quantification of Foxp3+ regulatory T cells in the hippocampus
Foxp3-specific signals were characterized by nuclear labelling of cells with
lymphocyte morphology (figure 3.14). Highest numbers of Foxp3+ cells were
observed at 3, 14, and 42 dpi in combined treated animals (group IV,
Treg _CD8 _TMEV).
Statistical analyses revealed a significantly higher amount of Foxp3 + Treg in the
hippocampus of combined treated mice (group IV, Treg _CD8 _TMEV) as
compared to Treg-expanded (group II, Treg _TMEV), CD8-depleted (group III,
CD8 _TMEV) and untreated virus-infected mice (group I, TMEV) at 3 dpi. At 14 and
42 dpi significantly higher numbers of Foxp3+ Treg were counted in the hippocampus
of combined treated mice (group IV, Treg _CD8 _TMEV) and CD8-depleted mice
(group III, CD8 _TMEV) compared to untreated virus-infected mice (group I, TMEV;
figure 3.15). Results of statistical analyses are listed in the annex (table 8.1.11).
Chapter 3 - Results
53
Figure 3.14. Foxp3+ regulatory T cells in the hippocampus of a Theilervirus-infected
C57BL/6 mice following expansion of regulatory T cells by interleukin-2 complex
application and depletion of CD8+ T cells (combined treatment) at 14 days post
infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.15. Quantification of Foxp3+ regulatory T cells in the hippocampus of
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
3.3.6
Quantification of CD45R+ B cells in the hippocampus
CD45R+ B cells were observed in the hippocampus especially at 7 and 14 dpi.
Immunoreactivity was characterized by membrane bound labelling of cells with
lymphocyte morphology (figure 3.16).
54
Chapter 3 - Results
Statistical analyses showed a significant decrease of CD45R+ B cells in Tregexpanded mice (group II, Treg _TMEV) compared with combined treated mice
(group IV, Treg _CD8 _TMEV), CD8-depleted mice (group III, CD8 _TMEV) and
untreated virus-infected mice (group I, TMEV) at 3 dpi. At 14 dpi combined treated
mice (group IV, Treg _CD8 _TMEV) showed significantly higher numbers of
CD45R+ B cells as compared to untreated virus-infected mice (group I,TMEV; figure
3.17). Results of statistical analyses are listed in the annex (table 8.1.12).
Figure 3.16. CD45R+ B cells in the hippocampus of a Theilervirus-infected C57BL/6
mouse following expansion of regulatory T cells by interleukin-2 complex application
and depletion of CD8+ T cells (combined treatment) at 14 days post infection. Figure
A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.17. CD45R+ B cell in the brain of Theilervirus-infected C57BL/6 mice with
different treatments (TMEV = no treatment; Treg _TMEV = expansion of regulatory T
cells (Treg) by interleukin (IL)-2 immune complexes; Treg _CD8 _TMEV =
expansion of Treg by IL-2 immune complexes and antibody-mediated CD8depletion). Box and whisker plots display median and quartiles together with
minimum and maximum values. Significant differences (p 0.05) are labeled with an
asterisk.
Chapter 3 - Results
3.3.7
55
Quantification of CD107b+ microglia/macrophages in the hippocampus
CD107b-specific immunoreactivity was characterized by membrane bound staining of
cells with macrophage or microglial morphology, respectively (figure 3.18).
Statistical
analyses
revealed
a
significant
increase
of
CD107b +
microglia/macrophages in combined treated mice (group IV, Treg _CD8 _TMEV)
as compared to CD8-depleted mice (group III, CD8 _TMEV) at 3 dpi. At 14 dpi
combined treated mice (group IV, Treg _CD8 _TMEV) showed significantly higher
numbers of CD107b+ cells compared to untreated virus-infected mice (group I,
TMEV; figure 3.19). Results of statistical analyses are listed in the annex (table
8.1.13).
Figure 3.18. CD107b+ microglia/macrophages in the hippocampus of a Theilervirusinfected C57BL/6 mouse following expansion of regulatory T cells by interleukin-2
complex application and depletion of CD8+ T cells (combined treatment) at 14 days
post infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
56
Chapter 3 - Results
Figure 3.19. CD107b+ microglia/macrophages in the hippocampus of Theilervirusinfected C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV
= expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.4
Effects of regulatory T cell expansion and depletion of CD8+ T cells in the
spinal cord
3.4.1 Virus load quantification in the spinal cord by immunohistochemistry
The numbers of Theilervirus-infected cells were determined in the spinal cord by
immunohistochemistry (figure 3.20).
Statistical analyses revealed significantly higher numbers of infected cells in
combined treated mice (group IV, Treg _CD8 _TMEV) and CD8-depleted mice
(group III, CD8 _TMEV) at 14 dpi compared with untreated mice (group I, TMEV).
At 42 dpi, only combined treated mice (group IV, Treg _CD8 _TMEV) showed
significantly increased numbers of infected cells compared to untreated mice (group
I, TMEV) and Treg-expanded mice (group II, Treg _TMEV), indicative of prolonged
virus infection as an assumed consequence of impaired antiviral immunity in
C57BL/6 mice following treatment (figure 3.21). Results of statistical analyses are
listed in the annex (table 8.1.14).
Chapter 3 - Results
57
Figure 3.20: Theilervirus-infected cells (arrow) in the spinal cord of a C57BL/6 mouse
following expansion of regulatory T cells by interleukin-2 complex application and
depletion of CD8+ T cells (combined treatment) at 14 days post infection. Figure A
(Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.21: Quantification of infected cells in the spinal cord of Theilervirus-infected
C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV =
expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
58
3.4.2
Chapter 3 - Results
Histological examination of the spinal cord
Transversal sections of formalin-fixed, paraffin-embedded spinal cord tissue were
HE-stained for histological examination and scored as described in materials and
methods. Histological lesions, characterized by mild hypercellularity in the spinal cord
grey matter were first observed in untreated mice (group I, TMEV) and combined
treated mice (group IV, Treg _CD8 _TMEV) at 3 dpi. At 42 dpi, inflammatory
responses were observed predominately in the spinal cord white matter of CD8depleted mice (group III, CD8 _TMEV) and combined treated mice (group IV,
Treg _CD8 _TMEV; figure 3.22).
Statistical significant differences were observed at 14 dpi between combined treated
mice (group IV, Treg _CD8 _TMEV) and untreated mice (group I, TMEV). At 42
dpi, significantly increased scores were observed in CD8-depleted (group III,
CD8 _TMEV) and combined treated mice (group IV, Treg _CD8 _TMEV)
compared to untreated mice (group I, TMEV) and Treg-expanded mice (group II,
Treg _TMEV), respectively. Results of statistical analyses are listed in the annex
(table 8.1.15).
Figure 3.22: Inflammatory responses (arrow) in the spinal cord of a Theilervirusinfected C57BL/6 mouse following expansion of regulatory T cells by interleukin-2
complex application and depletion of CD8+ T cells (combined treatment) at 14 days
post infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Chapter 3 - Results
59
Figure 3.23: Quantification of inflammatory responses in the spinal cord of
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
3.4.3
Myelin basic protein expression
In order to determine myelin changes associated with leukomyelitis and prolonged
virus infection, respectively, myelin basic protein expression was quantified by
immunohistochemistry and densiometric analyses. Mild loss of myelin basic protein
was first observed at 14 dpi in CD8-depleted mice (group III, CD8 _TMEV). At 42
dpi, foci of demyelination were found in CD8-depleted and combined treated mice
(group
III,
CD8 _TMEV,
group
IV,
Treg _CD8 _TMEV)
associated
with
inflammation in the spinal cord white matter (figure 3.24).
Statistical analyses revealed a significant loss of myelin in the spinal cord of CD8depleted mice (group III, CD8 _TMEV) mice at 14 and 42 dpi compared to untreated
mice (group I, TMEV) as well as in combined treated mice (group IV,
Treg _CD8 _TMEV) at 42 dpi (figure 3.25). Results of statistical analyses are listed
in the annex (table 8.1.16).
60
Chapter 3 - Results
Figure 3.24: Loss of myelin basic protein expression (arrow) in the spinal cord of a
Theilervirus-infected C57BL/6 mouse following expansion of regulatory T cells by
interleukin-2 complex application and depletion of CD8+ T cells (combined treatment)
at 42 days post infection. Figure A (Scale bar = 200µm), Figure B (Scale bar =
20µm).
Figure 3.25: Quantification of myelin basic protein expression in the spinal cord of
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
3.4.4
Axonal β-amyloid precursor protein expression
Immunohistochemistry for the detection of β-amyloid precursor protein (β-APP) was
used to detect axonal damage associated with prolonged spinal cord infection of
C57BL/6 mice. Few β-APP-expressing swollen axons, characterized by intraaxonal
Chapter 3 - Results
61
accumulation of a finely granular precipitate were first detected in combined treated
mice (group IV, Treg _CD8 _TMEV) at 14 dpi. Indicative of ongoing axonal
damage, highest numbers of β-APP-expressing axons were observed in the spinal
cord white matter of combined treated mice (group IV, Treg _CD8 _TMEV) at 42
dpi (figure 3.26). No β-APP-accumulation was found at earlier time points (3 and 7
dpi) in any group (figure 3.27).
Statistical analyses revealed a significant increase of β-APP+ axons in combined
treated mice (group IV, Treg _CD8 _TMEV) at 42 dpi (Figure 3.31). The number of
individual immunoreactive axons at 14 dpi observed in combined treated mice
(group IV, Treg _CD8 _TMEV) did not reach the level of significance (figure 3.31).
Results of statistical analyses are listed in the annex (table 8.1.17).
Figure 3.26: β-amyloid precursor protein expression (arrows) in the spinal cord of a
Theilervirus-infected C57BL/6 mouse following expansion of regulatory T cells by
interleukin-2 complex application and depletion of CD8+ T cells (combined treatment)
at 42 days post infection. Figure A (Scale bar = 50µm), Figure B (Scale bar = 20µm).
62
Chapter 3 - Results
Figure 3.27: β-amyloid precursor protein expression in the spinal cord of Theilervirusinfected C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV
= expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.4.5
Quantification of CD3+ T cells in the spinal cord
Infiltrations of CD3+ T cell were observed during the entire observation period.
Immunoreactivity was characterized by a predominantly membrane bound staining of
cells with lymphocyte morphology (Figure 3.28). Highest numbers were found in
Treg-expanded mice (group II, Treg _TMEV) and CD8-depleted infected mice
(group III, CD8 _TMEV) at 14 dpi. Prominent CD3-infiltration was observed at 42 dpi
in CD8-depleted mice (group III, CD8 _TMEV) and combined treated mice (group
IV, Treg _CD8 _TMEV), while only very few immunopositive cells were found in
untreated mice (group I, TMEV) and Treg-expanded mice (group II, Treg _TMEV)
at this time point. While infiltrates were predominantly located in the spinal cord grey
matter at 3 and 7 dpi, infiltration at 42 dpi was found in the spinal cord white matter
(figure 3.28).
Statistical analyses revealed a significant increase of CD3+ T cells in combined
treated mice (group IV, Treg _CD8 _TMEV) compared to untreated infected mice
(group I, TMEV) and CD8-depleted mice (group III, CD8 _TMEV) at 3 dpi. At 7 dpi
a significant reduction of CD3+ T cells was found in Treg-expanded mice (group II,
Treg _TMEV) and combined treated mice (group IV, Treg _CD8 _TMEV)
Chapter 3 - Results
compared
to
untreated
63
mice
(group
I,
TMEV)
following
TMEV-infection.
+
Subsequently, at 14 dpi, significant elevation of CD3 T cells was observed in
combined treated mice (group IV, Treg _CD8 _TMEV) compared to untreated mice
(group I, TMEV) and Treg-expanded infected mice (group II, Treg _TMEV). At 42
dpi, significantly elevated CD3+ T cell numbers were detected in combined treated
mice (group IV, Treg _CD8 _TMEV) compared to untreated mice (group I, TMEV).
Similarly, compared to untreated mice (group I, TMEV) and Treg-expanded mice
(group II, Treg _TMEV), a significant increase of CD3-immunoreactivity was found
following CD8-depletion (group III, CD8 _TMEV) at this time point, indicative of
prolonged T cell responses in the spinal cord (figure 3.29 ). Results of statistical
analyses are listed in the annex (table 8.1.18).
Figure 3.28: CD3+ T cells (arrow) in the spinal cord of a Theilervirus-infected
C57BL/6 mouse following expansion of regulatory T cells by interleukin-2 complex
application and depletion of CD8+ T cells (combined treatment) at 42 days post
infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
64
Chapter 3 - Results
Figure 3.29: Quantification of CD3+ T cells in the spinal cord of Theilervirus-infected
C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV =
expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.4.6
Quantification of Foxp3+ regulatory T cells in the spinal cord
In parallel with peripheral Treg-expansion demonstrated by flow cytometry, highest
number of Foxp3+ cells were observed at 3 dpi in animals following IL-2 immune
complex treatment (group II, Treg _TMEV, group IV, Treg _CD8 _TMEV).
Subsequently (14 and 42 dpi), probably as a consequence of prolonged spinal cord
inflammation an increase of Foxp3+ Treg was observed in CD8-depleted mice with
and
without
Treg-expansion
(group
III,
CD8 _TMEV,
group
IV,
Treg _CD8 _TMEV). Foxp3-specific signals were characterized by nuclear labelling
of cells with lymphocyte morphology (figure 3.30).
Statistical analyses showed an early significant increase of Foxp3+ Treg in the spinal
cord of IL-2 immune complex treated mice with and without CD8-depletion (group II,
Treg _TMEV, group IV, Treg _CD8 _TMEV) compared to untreated mice (group I,
TMEV) and CD8-depleted (group III, CD8 _TMEV) mice, respectively, at 3 dpi. At 7
dpi, significantly higher numbers were observed only in Treg-expanded mice (group
II,
Treg _TMEV).
At
14
dpi,
combined
treated
animals
(group
IV,
Treg _CD8 _TMEV) showed significantly higher numbers of Foxp3 + Treg compared
Chapter 3 - Results
65
to untreated mice (group I, TMEV) and Treg-expanded mice (group II,
Treg _TMEV),
while
at
42
dpi,
combined
treated
mice
(group
IV,
Treg _CD8 _TMEV) and CD8-depleted mice (group III, CD8 _TMEV) exhibited
significantly elevated values compared to untreated mice (group I, TMEV) (figure
3.31). Results of statistical analyses are listed in the annex (table 8.1.19).
Figure 3.30: Foxp3+ T cells (arrow) in the spinal cord of a Theilervirus-infected
C57BL/6 mouse following expansion of regulatory T cells by interleukin-2 complex
application and depletion of CD8+ T cells (combined treatment) at 42 days post
infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Figure 3.31: Quantification of Foxp3+ regulatory T cells in the spinal cord of
Theilervirus-infected C57BL/6 mice with different treatments (TMEV = no treatment;
Treg _TMEV = expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune
complexes; Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes
and antibody-mediated CD8-depletion). Box and whisker plots display median and
quartiles together with minimum and maximum values. Significant differences
(p 0.05) are labeled with an asterisk.
66
3.4.7
Chapter 3 - Results
Quantification of CD45R+ B cells in the spinal cord
Spinal cord infiltration of CD45R+ B cells was observed during the entire observation
period, predominantly in CD8-depleted mice (group III, CD8 _TMEV) and combined
treated mice (group IV, Treg _CD8 _TMEV) at 14 and 42 dpi. CD45R-specific
immunoreactivity was characterized by membrane bound labelling of cells with
lymphocyte morphology (figure 3.33).
Statistical analyses revealed a significant decrease of CD45R+ B cells in combined
treated mice (group IV, Treg _CD8 _TMEV) compared to Treg-expanded mice
(group II, Treg _TMEV) at 3 dpi. At 7 dpi, a significant reduction of CD45R + B cells
was found in combined treated mice (group IV, Treg _CD8 _TMEV) compared to
untreated virus-infected mice (group I, TMEV). At later time points (14 and 42 dpi)
combined treated mice (group IV, Treg _CD8 _TMEV) exhibited significantly higher
numbers of CD45R+ B cells compared to untreated virus-infected mice (group I,
TMEV; figure 3.33). Results of statistical analyses are listed in the annex (table
8.1.20).
Figure 3.32: CD45R+ B cells (arrow) in the spinal cord of a Theilervirus-infected
C57BL/6 mouse following expansion of regulatory T cells by interleukin-2 complex
application and depletion of CD8+ T cells (combined treatment) at 14 days post
infection. Figure A (Scale bar = 200µm), Figure B (Scale bar = 20µm).
Chapter 3 - Results
67
Figure 3.33: CD45R+ B cell in the spinal cord of Theilervirus-infected C57BL/6 mice
with different treatments (TMEV = no treatment; Treg _TMEV = expansion of
regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk.
3.4.8
Quantification of CD107b+ microglia/macrophages in the spinal cord
Immunohistochemistry was performed to quantify the amount of CD107b +
microglia/macrophages in spinal cord lesions. CD107b-specific immunoreactivity,
characterized by membrane bound staining, was first detected in CD8-depleted mice
(group III, CD8 _TMEV) at 3 dpi. At 14 and 42 dpi, CD107b+ cell were detected
predominantly in in combined treated mice (group IV, Treg _CD8 _TMEV) and
CD8-depleted
mice
(group
III,
CD8 _TMEV),
indicative
of
long
lasting
neuroinflammation in these treatment groups (figure 3.34).
Statistical analyses revealed significantly higher numbers of CD107b + cell in CD8depleted mice (group III, CD8 _TMEV) compared to Treg-expanded mice (group II,
Treg _TMEV) and untreated virus-infected mice (group I, TMEV) at 3 dpi. At 14 dpi,
combined treated mice (group IV, Treg _CD8 _TMEV) exhibited significantly higher
numbers of CD107b+ cells compared to all other groups. At 42 dpi, combined treated
mice (group IV, Treg _CD8 _TMEV) had significantly higher numbers of CD107b+
cells compared with Treg-expanded mice (group II, Treg _TMEV) and untreated
virus-infected mice (group I, TMEV; figure 3.35). Similarly, CD8-depleted mice
(CD8 _TMEV) exhibited significantly increased numbers of CD107b+ cells compared
68
Chapter 3 - Results
to untreated virus-infected mice (group I, TMEV) at 42 dpi. Results of statistical
analyses are listed in the annex (table 8.1.21).
Figure 3.34: CD107b+ microglia/macrophages (arrow) in the spinal cord of a
Theilervirus-infected C57BL/6 mouse following expansion of regulatory T cells by
interleukin-2 complex application and depletion of CD8+ T cells (combined treatment)
at 42 days post infection. Figure A (Scale bar = 200µm), Figure B (Scale bar =
20µm).
Figure 3.35: CD107b+ microglia/macrophages in the spinal cord of Theilervirusinfected C57BL/6 mice with different treatments (TMEV = no treatment; Treg _TMEV
= expansion of regulatory T cells (Treg) by interleukin (IL)-2 immune complexes;
Treg _CD8 _TMEV = expansion of Treg by IL-2 immune complexes and antibodymediated CD8-depletion). Box and whisker plots display median and quartiles
together with minimum and maximum values. Significant differences (p 0.05) are
labeled with an asterisk
Chapter 4 - Discussion
Chapter 4 Discussion
69
70
Chapter 4 - Discussion
4. Discussion
The aim of the present study was to investigate the role of Treg during Theiler murine
encephalomyelitis virus (TMEV) infection in C57BL/6 mice. Treg play a key role in the
maintenance
of
immunological
tolerance
and
prevent
immunopathology
(SAKAGUCHI, 2003; SAKAGUCHI et al., 2006). However, in viral diseases Treg
can exhibit both beneficial effects by reducing immune mediated tissue damage and
detrimental effects due to their immunosuppressive properties, causing disease
exacerbation or viral persistence, respectively. Recently, rapid expansion of Treg has
been demonstrated in the brain of susceptible SJL mice but not in resistant C57BL/6
mice following TMEV-infection (HERDER et al., 2012; RICHARDS et al., 2011).
Functional inactivation of Treg by anti-CD25-antibodies prior to infection results in an
enhanced virus-specific immunity, reduced viral load and delayed disease
progression, while the adoptive transfer of Treg leads to disease exacerbation in
TMEV-infected SJL mice (MARTINEZ et al., 2014;
RICHARDS et al., 2011).
However, these treatments have failed to influence the disease course in resistant
mice strains (C57BL/6). Moreover, despite strong effector T cell responses and
enhanced induction of interferon (IFN)-γ-producing T cells in the absence of Treg no
augmented antiviral responses could be observed following genetic ablation of
Foxp3+ Treg in TMEV-infected transgenic DEREG mice (BAC-transgenic Foxp3
reporter mice), demonstrating the complexity of protective immune responses in
infectious CNS disorders (PRAJEETH et al., 2014).
Results of the present project demonstrate for the first time a synergistic effect of
Treg-expansion and CD8-depletion to efficiently reduce antiviral immune responses
in TMEV-infected C57BL/6 mice, which renders them susceptible to develop chronic
infection, leukomyelitis and myelin loss.
Chapter 4 - Discussion
4.1
71
Effect of interleukin-2 immune complex treatment and antibody mediated
CD8-depletion upon regulatory T cells in C57BL/6 mice
Interleukin-2 immune complexes (IL-2C) have been demonstrated to selectively
expand Foxp3+ Treg in mice for one to two weeks (WEBSTER et al., 2009).
Similarly, Foxp3+ Treg were transiently increased in IL-2C-treated animals in the
present survey as demonstrated by flow cytometry. Although the exact mechanism of
IL-2C-induced Treg-expansion in vivo remains unclear, an enhanced biological
activity of IL-2 by complex formation as a consequence of increasing the cytokine`s
half-life is proposed. The monoclonal IL-2-specific antibody JES6-1 is supposed to
bind an IL-2 site that is important for the interaction with CD122 but less crucial for
binding CD25, leading to a preferential activation of cells expressing the high affinity
IL-2 receptor (BOYMAN et al., 2006). Referring to this, Treg have been
demonstrated to constitutively express the high affinity IL-2 receptor, while CD8+ T
cells and natural killer cells express the intermediate affinity IL-2 receptor
(KAMIMURA and BEVAN, 2007).
The increased percentages of Foxp3+ Treg in blood and spleen samples of combined
treated animals were associated with an increased CD4/CD8 ratio in the present
experiment, probably due to Treg-mediated suppression of cytotoxic T cells.
Strikingly, also prolonged Treg-expansion has been observed following CD8depletion in these mice, indicative of an inhibitory effect of CD8+ T cells upon Treg or
bidirectional interaction of both T cell subsets, respectively. These peripheral
phenotypical changes resulted in an enhanced recruitment of lymphocytes to the
spinal cord of TMEV-infected C57BL/6 mice as described by immunohistochemistry.
In addition a decrease of cytotoxic T cells responses and increased suppressive
capacity by Foxp3+ Treg might have caused prolonged virus infection with enhanced
activity of microglia and macrophages. In contrast to this detrimental effect found in
72
Chapter 4 - Discussion
TMEV-infected C57BL/6 mice, therapeutic effects have been described in primary
autoimmune diseases. For instance, in vivo expansion of Treg with IL-2C induces
resistance to EAE and long-term acceptance of islet allografts in mice without
immunosuppression (BOYMAN et al., 2012; WEBSTER et al., 2009). Furthermore,
IL-2C-mediated Treg-expansion protects against renal injury in adriamycin-induced
nephropathy, a rodent model for proteinuric chronic kidney disease (POLHILL et al.,
2012).
4.2
Lesion development in the brain of Theilervirus-infected C57BL/6 mice
following interleukin-2 complex treatment and depletion of CD8+ T cells
Intracerebral infection of mice with the low virulent BeAn-strain of TMEV causes an
acute
virus-induced
polioencephalitis,
characterized
by
an
infiltration
of
macrophages, CD4+ T cells, CD8+ T cells, and B cells in the brain (GERHAUSERet
al., 2007; OLESZAK et al., 1995). In contrast to susceptible SJL mice, resistant
C57BL/6-mice eliminate the virus from the cerebral gray matter after the acute
phase by specific antiviral immunity (HERDER et al., 2012).
Similar to previous reports (KUMMERFELD et al., 2012), a predominant infection of
cerebral neurons was found in mice of all treatment groups during the early TME
phase. A variety of viruses, such as Borna disease virus, measles virus,
neuroadapted
Sindbis
virus,
herpes
virus,
rabies
virus
and
human
immunodeficiency virus (HIV) have the ability to infect and disturb hippocampal
neurons (BUENZ et al., 2006; KIMURA and GRIFFIN, 2003). Moreover, due to
their neurovirulent potential the possibility of subclinical hippocampal damage
following picornavirus infection (e.g. coxsackievirus) in human beings has to be
considered (BUENZ et al., 2006; MACLENNAN and SOLOMON, 2004). TMEVinfection of C57BL/6 mice has become a valuable model to investigate virusinduced hippocampal damage and infection-induced epilepsy, respectively (LIBBEY
Chapter 4 - Discussion
73
et al., 2008). In addition to acute seizures it has been shown that TMEV-infection
leads to an increased chronic seizure susceptibility associated with hippocampal
sclerosis, making TME of C57BL/6 mice also a model to study postinfection
hyperexcitability (SMEAL et al., 2012; STEWART et al., 2010a, 2010b). Changes
in the murine hippocampus are characterized by gliosis and perivascular infiltrates
with neuronal pyknosis and loss. In addition, hippocampal sclerosis is associated
with impaired cognitive ability, anxiety-like behavior and memory impairment of
TMEV-infected C57BL/6 mice (BUENZ et al., 2006; UMPIERRE et al., 2014).
Neuronal loss in C57BL/6 mice has been demonstrated to be consequence of
apoptosis predominately of non-infected cells, indicating that neuronal death during
acute picornavirus infection occurs in a non-cell-autonomous manner (bystander
injury) potentially triggered by pro-inflammatory mediators (BUENZ et al., 2009).
Accordingly, innate immune responses, especially driven by tumor necrosis factor
(TNF), IL-6 and the complement component C3 have been shown to contribute to
acute seizure development in TMEV-infected C57BL/6 mice, while expression of
transforming growth factor-β is supposed to have a protective effect (LIBBEY et al.,
2008; LIBBEY et al., 2010; KIRKMAN et al., 2010). At this, minocycline treatment
to inhibit monocyte recruitment into the CNS and to diminish microglia/macrophage
activation reduces the seizure frequency in C57BL/6 mice following TMEV-infection,
showing that both infiltrating innate immunity cells and resident CNS cells contribute
to the development of neurological symptoms (LIBBEY et al., 2011a, 2011c).
Among these cells, microglial cells have been shown to be the main source for TNF,
while infiltrating macrophages preferentially produce IL-6 (CUSICK et al., 2013).
The precise role of TNF in the pathogenesis of TME remains enigmatic, since
neuroprotective and neurodestructive effects have been described in different
infectious diseases (HERDER et al., 2012). The cytokine leads to an activation and
74
Chapter 4 - Discussion
recruitment of immune cells and therefore potentially enhances antiviral immune
responses in resistant C57BL/6 mice. TNF also exhibits protective function and
resolution of cerebral injury in resistant mouse strains infected with TMEV
(RODRIGUEZ et al., 2009). On the other hand, TNF has been shown to release
excitatory neurotransmitters able to cause neuronal damage and seizures in TMEVinfected C57BL/6 mice (KIRKMAN et al., 2010, LIBBEY et al., 2008). Other
proposed mechanisms of hippocampal injury by monocytes in TMEV-infected
C57BL/6 mice include the release of reactive oxygen species, blood-brain-barrierdegrading metalloproteinases, elastases, cathepsins and chemokines (HOWE et
al., 2012a). In contrast to C57BL/6 mice, overt injury of hippocampal neurons during
acute TMEV-infection is absent in SJL mice (HOWE et al., 2012a). Compared to
C57BL/6 mice, the number of CD45hiCD11b+Gr1+1A8- monocytes is reduced in SJL
mice, indicative of an attenuated acute inflammatory monocyte response to TMEV
in this mouse strain. Further evidence for inflammatory monocytes as key mediators
for hippocampal injury in TME has been shown by the induction of neuronal
damage and cognitive deficits in C57BL/6xSJL hybrid mice - which usually show
complete hippocampal preservation following acute picornavirus infection - by
adoptive transfer of C57BL/6 monocytes (HOWE et al., 2012a). Moreover, depletion
of inflammatory monocytes and neutrophils with Gr1-specific-antibodies results in
hippocampal protection and preservation of cognitive function showing that these
cells represent potential targets for novel therapeutic strategies to prevent virusinduced CNS pathology (HOWE et al., 2012b). Most of the above mentioned
studies have been performed using the Daniel`s strain of TMEV, which exhibits an
increased virulence compared to the BeAn-strain. However, hippocampal
neuropathology and virus load in BeAn-infected C57BL/6 mice have been
demonstrated to be similar to those in Daniel`s strain-infected C57BL/6 mice during
Chapter 4 - Discussion
75
the acute phase and both virus strains are cleared from the murine brain by 14 dpi
(LIBBEY et al., 2011b). Besides microglia/macrophages, brain-infiltrating CD8+ T
cells can either directly target neurons or lead to collateral neuronal damage as
demonstrated in a variety of inflammatory CNS disorders (MELZER et al., 2009).
However, in contrast to the therapeutic effect of CD8-depletion observed in primary
autoimmune disorders, CD8-depletion in investigated animals - predominately in
combination with Treg-expansion - fosters hippocampal damage by enhancing virus
infection. Since virus quantification by immunohistochemistry and real time PCR
revealed a prolonged brain infection up to 42 dpi in combined treated mice
associated with hippocampal inflammation, the present survey provides a base for
future studies upon the effect of sustained infection upon neuronal death.
Significant
infiltrations
of
CD3+
T
cells,
CD45R+
B
cells
and
CD107b+
microglia/macrophages were only observed in the brain till 14 dpi as observed in
previous studies (HERDER et al., 2012), while Foxp3+ Treg were found in the
hippocampus also during the late phase (42 dpi). Elevated numbers of braininfiltrating Foxp3+ Treg during the acute phase might cause reduced antiviral
immunity and prolonged virus infection, respectively. However, maintenance of these
cells together with reduced hippocampal damage present at 42 dpi might represent a
sequel of terminated inflammation and post-degenerative neuroregeneration,
respectively. Beneficial effects of Treg can be observed in degenerative CNS
disorders, since it has been demonstrated that Treg-depletion by anti-CD25antibodies results in an increased activation of microglia and T cells associated with
an up-regulation of TNF and IFN-γ causing clinical worsening (motor dysfunction)
and increase of infarct sizes in acute stroke models (LIESZ et al., 2009; REN et al.,
2011). Treg reduce secondary infarct growth following acute cerebral ischemia in
murine brain stroke models by limiting pro-inflammatory cytokine expression (TNF,
76
Chapter 4 - Discussion
IFN-γ and IL-1β) mediated by IL-10 and by reducing neutrophilic infiltration and
microglial responses (LIESZ et al., 2009). Noteworthy, also reduced numbers of Treg
during the early phase of canine spinal cord injury and canine distemper are
supposed to cause excessive glial activation and secretion of pro-inflammatory and
potentially neurotoxic cytokines (QESKA et al., 2013; SPITZBARTH et al., 2011). In
contrast to these protective effects also detrimental effects of Treg have been
described in degenerative CNS disorders, showing divergent and probably diseasephase specific functions of this cell population in CNS recovery (WALSH and
KIPNIS, 2011). For instance, adoptive transfer of Treg has been described to reduce
neuroprotective immunity, while Treg-depletion prevents neuronal damage in a
mouse model for spinal cord injury (KIPNIS et al. 2002; ZIV et al., 2006). Similarly,
disease phase-specific functions of Treg have to be considered in the pathogenesis
of TME.
4.3
Lesion development in the spinal cord of Theilervirus-infected C57BL/6
mice following interleukin-2 complex treatment and depletion of CD8+ T
cells
Low virulent TMEV strains, such as the BeAn strain, initially infect neurons and
astrocytes and cause acute polioencephalitis. The subsequent disease course
depends decisively upon the genetic background of the used mouse strain. Thus,
while the virus is eliminated in resistant C57BL/6 mice from the brain after the initial
phase, inadequate antiviral immune responses lead to prolonged virus infection and
demyelinating leukomyelitis in susceptible SJL mice (KUMMERFELD et al., 2012;
OLESZAK et al., 2004). Results of the present study revealed the occurrence of a
prolonged spinal cord infection together with leukomyelitis and loss of myelin basic
protein in TMEV-infected C57BL/6 mice following antibody mediated depletion of
CD8+ T cells and expansion of Foxp3+ Treg. In contrast, the pathogen was totally
Chapter 4 - Discussion
77
eliminated and spinal cord inflammation was terminated in untreated and Tregexpanded mice. Lesion initiation and progression in combined treated C57BL/6 mice
is supposed to be similar to the processes observed in SJL mice. In these
susceptible mouse strains a cell tropism switch of TMEV with preferential glial
infection of the spinal cord white matter might be a relevant prerequisite for the
development of myelin loss (LIPTON et al., 1995; LIPTON et al., 2005). TMEV has
been detected in macrophages/microglia during the persistent phase in SJL mice
infected with the BeAn strain (KUMMERFELD et al. 2012, LIPTON et al., 1995) and
Daniels strain (ROSSI et al., 1997). In addition, virus persistence has been described
in astrocytes or oligodendrocytes in susceptible mouse strains (ZHENG et al., 2001;
ZOECKLEIN et al., 2003). Viral dissemination within the spinal cord is based upon
continuous viral replication within glial cells and infection of microglia/macrophages
by phagocytosis of infectious material released from apoptotic cells (LIPTON et al.,
1995; SCHLITT et al., 2003). A similar mode of virus transmission is supposed in
the present experiment. In addition to myelin loss, mild but significant axonal damage
was
observed
in
the
spinal
cord
as
demonstrated
by
β-APP-specific
immunohistochemistry. Axonal transport is supposed to be the primary mode of virus
transmission within the CNS grey matter during the acute TME phase and possibly
responsible for the spread to the white matter (BRAHIC and ROUSSARIE, 2009;
KUMMERFELD et al., 2012). Dysregulation of the axonal transport machinery and
impairment of neurofilament phosphorylation and protein metabolism might represent
an axonal self-destruction program to prevent virus spread, which contributes to
clinical disability as observed in MS patients (KREUTZER et al., 2012).
Viral antigen-induced delayed-type hypersensitivity and myelin-specific autoimmunity
induce inflammatory demyelination predominantly in the spinal cord of susceptible
mouse strains, resembling lesions in the chronic progressive form of MS (OLESZAK
78
Chapter 4 - Discussion
et al., 2004). Likewise, demyelination in combined treated mice at 42 dpi is
associated
with
an
accumulation
of
CD3+
T
cells
and
CD107b+
macrophages/microglia, suggestive of T cell-mediated immunopathology. Similar to
these findings, CD107b+ cells are associated with myelin loss in the brain stem and
spinal cord white matter of TMEV-infected SJL mice (KUMMERFELD et al., 2012).
Microglia and CNS-infiltrating macrophages play a central role in the pathogenesis of
TMEV-induced demyelination. For instance, the cells represent targets for viral
persistence during the chronic disease phase (ROSSI et al., 1997) and contribute to
myelin damage by the release of myelinotoxic factors (bystander demyelination),
delayed-type hypersensitivity reaction and induction of myelin-specific autoimmunity
in susceptible mice (LIUZZI et al. 1995). Microglia cause myelin damage also in EAE
and cuprizone-induced demyelination models, respectively (LIU et al., 2013;
SKRIPULETZ et al., 2010a). Moreover, while progressive demyelination in MS is a
consequence of T cell-mediated immunopathology, microglial activation can be
observed in predemyelinating lesions of MS patients (MARIK et al., 2007) supporting
the hypothesis that innate immunity represents an initiating factor in myelin loss
disorders. Interestingly, despite a reduced myelin degrading proteolytic capacity of
brain microglia and macrophages in C57BL/6 mice described by others (LIUZZI et
al., 1995), prolonged inflammation is able to induce myelin loss in this resistant
mouse strain as shown in the present study. Referring to this, sustained TMEVinfection
in
the
spinal
cord
of
SJL
mice
causes
M1-polarization
of
macrophages/microglia with pro-inflammatory and myelinotoxic properties (HERDER
et al., 2014).Topographical differences of glial functionality, such as the capacity to
release reactive oxygen species, might explain the observed variable sensitivities of
the brain and spinal cord white matter to develop myelin loss following virus infection
(KUMMERFELD et al., 2012; ENSINGER et al. 2010; GUDI et al., 2009;
Chapter 4 - Discussion
79
SKRIPULETZ et al., 2008; SKRIPULETZ et al., 2010b). In addition to T cell
responses, mounting CD45R+ B cell responses have been observed in combined
treated mice. In agreement with this, gene expression analyses of lymphoid organs
and spinal cord showed an activation of B cell immune responses during the early
infection phase in TMEV-infected SJL mice, which potentially triggers local humoral
immune responses during the progressive demyelinating phase (NAVARRETETALLONI et al., 2010; ULRICH et al., 2010). Interestingly, according to the currently
discussed role of B cells and autoantibodies in the pathogenesis of MS (CROSS and
WAUBANT, 2011), TMEV-specific immunoglobulins accumulate in the CNS of
infected animals (LIPTON et al., 1978; TSUNODA and FUJINAMI, 1996), which
have the ability to cross-react with myelin components and potentially act as
demyelinating antibodies (YAMADA et al., 1990). Thus, beside its assumed impact
on protective immunity, CNS infiltration of CD45R+ B cells might represent an
initiating event for plasma cell differentiation and intrathecal immunoglobulin
production with subsequent myelin loss as described in TME for susceptible mouse
strains (PACHNER et al., 2007).
CNS-infiltrating Foxp3+ Treg might contribute to reduced antiviral immunity during
early and late TME phases. However, Treg also play a central role for immune
tolerances in the CNS and have the ability to dampen immune mediated tissue
damage. MS patients are supposed to have functional defects of Treg probably as a
consequence of reduced levels of Foxp3 mRNA and protein. Furthermore, disturbed
thymic generation of Treg and expansion of memory Treg might lead to an impaired
immune homeostasis and hence favor the progression of autoimmunity in MS
patients (LOWTHER and HAFLER, 2012). In agreement with this, in vivo expansion
of Treg and the adoptive transfer of in vitro expanded Treg reduce the severity of
demyelination in EAE (KORN et al., 2007; JEE et al., 2007). Thus, a dual function of
80
Chapter 4 - Discussion
Treg in the pathogenesis of TME, especially during the chronic demyelinating phase
is not unlikely and needs to be investigated in future studies.
4.4
Interaction between regulatory T cells and cytotoxic CD8+ T cells
Results of the present survey demonstrate a synergistic effect of Treg-expansion and
CD8-depletion to reduce antiviral immunity in resistant C57BL/6 mice. Foxp3 + Treg
efficiently control the homeostasis of CD8+ T cells and modulate CD8+ T cell effector
differentiation in several infectious, autoimmune and neoplastic diseases (MCNALLY
et al., 2011). Accordingly, an inappropriate Treg-to-CD8+ T cell ratio during the acute
phase is supposed to contribute to virus persistence and TMEV-induced
demyelination in susceptible mouse strains (RICHARDS et al., 2011).
Selective depletion of Foxp3+ Treg in transgenic DEREG mice leads to an increased
frequency of CD44+CD8+ and granzyme B+CD8+ T cells, indicative of cytotoxic T cell
activation, which enhances antitumor immunity in animal melanoma models
(KLAGES et al., 2010). Treg also suppress CD8+ T cell proliferation and interferon-γ
production as well as the cytolytic activity of CD8+ T cells by decreasing their
expression of IL-2 and CD25 (alpha chain of the IL-2 receptor). This reduced IL-2
responsiveness of cytotoxic cells is mediated by direct T-T cell interaction in the
absence of antigen presenting cells (PICCIRILLO and SHEVACH, 2001). Besides
suppressing IL-2 production of antigen-specific CD8+ T cells, Treg have been
demonstrated to limit the cytokine’s bioavailability, which additionally disturbs CD8+ T
cell effector differentiation (KASTENMULLER et al., 2011). Absorption of IL-2 by
Foxp3+ Treg is caused by their high affinity IL-2 receptors (MCNALLY et al., 2014).
Furthermore, a regulatory loop exists between Treg and effector CD8 + T cells, where
IL-2 production during T cell differentiation promotes Treg-expansion, demonstrating
the critical role of the molecule for Treg-mediated suppression of cytotoxicity
Chapter 4 - Discussion
81
(MCNALLY et al., 2011). In agreement with this, a prolonged disturbed CD4/CD8
ratio was found by flow cytometry in the present study, which might be a
consequence of enhanced IL-2 consumption. Subsequent reduced availability of the
cytokine might have limited the expansion of CD8+ T cells in combined treated
C57BL/6 mice.
Recent reports suggest that Treg preferentially inhibit the priming of self-reactive,
potentially autoagressive CD8+ T cells, while sparing T cell responses to nonself
antigens. This has been demonstrated by Treg-depletion in mice, leading to an
expansion of low-avidity CD8+ T cells by an enhanced production of chemokines,
which stabilizes the interaction between these T cells and dendritic cells (PACE et
al., 2012). In agreement with this, Treg-depletion in mice using a viral vector system
(modified vaccinia virus Ankara) has been shown to prevent effector differentiation
but not memory CD8+ T cell responses or rapid recall responses upon pathogen reexposure. These studies demonstrate the difficulties of Treg-modification for
prophylactic vaccinations (KASTENMULLER et al., 2011). However, genetic ablation
of Treg in DEREG mice combined with antibody-mediated blockade of inhibitory
receptors (PD-1 ligand, TIM-3) reactivates exhausted CD8+ T cells and efficiently
reduce the chronic load of friend retrovirus (DIETZ et al., 2013). Furthermore, Tregdepletion in friend retrovirus-infected C57BL/6 mice causes a significant increase of
virus-specific CD8+ T cells associated with an enhanced cytotoxicity, including
increased granzyme and CD107a expression (ZELINSKYY et al., 2009).
82
Chapter 4 - Discussion
4.5
General aspects of regulatory T cells in infectious disorders of the
central nervous system
Several pathogens have evolved strategies to modulate host immune responses,
thereby promoting immune evasion and persistence. For instance, Japanese
encephalitis virus infects dendritic cells, which represents a mechanism of virus
delivery to the CNS. Interestingly, Japanese encephalitis virus also modulates the
maturation and antigen presenting capacity of dendritic cells, leading to an expansion
of Treg and reduction of antiviral immune responses (CAO et al., 2011). Thus, Treg
potentially have dual functions in CNS disorders, causing beneficial effects by the
reduction of immune mediated tissue damage as well as detrimental effects by
reducing protective immune responses.
In TME, an increased amount of Treg together with an elevated IL-10 production
reduce antiviral immunity during the acute phase which leads to delayed virus
elimination in susceptible SJL mice (HERDER et al., 2012; RICHARDS et al., 2011).
As described above, strong antiviral CD8-mediated cytotoxicity leads to virus
elimination in C57BL/6 mice. In contrast, SJL mice show reduced CD8 + T cells
responses and increased numbers of Foxp3+ Treg in the CNS, which might lead to
Treg-mediated dampening of virus-specific CD8+ T cells (RICHARDS et al., 2011). In
agreement with this, results of the present study show that Treg-expansion together
with cytotoxic T cell ablation has the ability to reduce antiviral immunity in resistant
C57BL/6 mice. Similarly, Treg contribute to impaired virus-specific responses also in
the Friend retrovirus mouse model (ZELINSKYY et al., 2009) and experimental
herpesvirus infection of mice by inhibiting virus-specific CD8+ T cells (TOKA et al.,
2004; FERNANDEZ et al., 2008). Expansion of CD4+CD25+Foxp3+ Treg by
superagonistic CD28 antibodies (clone D665) increases virus replication and the
number of infected neurons in measles virus-infected mice, while depletion of Foxp3
Chapter 4 - Discussion
83
in measles virus-infected DEREG mice decreases the number of infected neurons by
enhancing antiviral cytotoxicity (REUTER et al., 2012). In contrast to these studies,
others have demonstrated that depletion of Treg leads to an increased spread of
experimental herpes simplex virus to the murine CNS, indicative of a protective role
of Treg in this infectious model (LUND et al., 2008). These discrepancies, which
might be based on the experimental study design (e.g. time of infection, virus strain,
infectious dose, etc.), demonstrate the complexity of Treg function in infectious CNS
disorders. Besides their impact upon pathogen-specific immunity, Treg have the
ability to dampen immune responses to prevent collateral tissue damage in infectious
diseases. For example, in coronavirus-infected mice, the adoptive transfer of Treg
decreases immune mediated damage by limiting T cell proliferation, chemokine
production, cytokine production and dendritic cell activation (TRANDEM et al., 2010).
Treg cause beneficial effects also by inducing apoptosis of virus-infected bone
marrow macrophages and by reducing virus replication in murine models for HIVassociated
neurodegeneration.
Moreover,
Treg
decrease
astrogliosis
and
microgliosis and increase the expression of brain-derived neurotrophic factor and
glial cell line-derived neurotrophic factor which results in neuroprotection following
retrovirus infection (LIU et al., 2009). They also change the polarization of HIVinfected macrophages from a pro-inflammatory and neurodegenerative M1phenotype into an anti-inflammatory and neuroprotective M2-phenotype, by downregulating iNOS and up-regulating arginase-1 (HUANG et al., 2010).
84
4.6
Chapter 4 - Discussion
Conclusion
The present study confirms the central role of antiviral CD8-mediated cytotoxicity for
TMEV-elimination in resistant C57BL/6 mice (DRESCHER et al., 2000). Tregexpansion exert profound inhibitory responses only in an CD8-reduced environment,
which is in good agreement with previous reports showing that simple manipulation of
the Treg-compartment has only minor effect upon the TME disease course in
resistant mouse strains (MARTINEZ et al., 2014; PRAJEETH et al., 2014). Results
also supports the hypothesis that in addtion to the well described inhibitory effect of
Treg upon CD8+ T cells (GÖBEL et al., 2012), regulatory effects of cytotoxic cells
upon Treg or bidirectional interactions of the two T cell subsets, respectively, occur in
infectious CNS disoders. C57BL/6 mice are widely used for genetic modification and
several transgenic mice with this genetic background have been established for the
use as animal models for human diseases. Thus, the established treatment protocol
to induce chronic TME in C57BL/6 mice represents a base for future studies using
transgenic mice to investigate the pathogenesis of virus-induced demyelination and
immunopathology in more detail.
Summary
85
5. Summary
Muhammad Akram Khan
Expansion of regulatory T cells in Theiler’s murine encephalomyelitis virusinfected C57BL/6 mice.
Multiple sclerosis (MS), one of the most frequent central nervous system (CNS)
diseases in young adults, is a chronic demyelinating disease of unknown etiology
and possibly multifactorial causes. Due to clinical and pathological similarities,
Theiler’s murine encephalomyelitis (TME) represents a commonly used infectious
animal model for the chronic-progressive form of human MS. Inadequate viral
clearance in susceptible SJL mice leads to persistent infection of the CNS and
immune mediated spinal cord demyelination. In contrast resistant C57BL/6 mice
eliminate the virus from the CNS by specific cellular immunity, including effective
CD8-mediated cytotoxicity. Previous studies have demonstrated that regulatory T
cells (Treg) reduce antiviral immune responses in TME in susceptible mice strains.
However, the function of Treg in resistant C57BL/6 mice remains undetermined.
Thus the aim of the present study was to investigate the impact of in vivo-expansion
of Treg by interleukin-2 immune complexes (Treg-expansion) and antibodymediated CD8+ T cell depletion (CD8-depletion) upon TME virus-induced disease
progression in C57BL/6 mice to get further insights into role of Treg and their
interaction with other leukocyte subsets in persistent viral infections.
Results of the present study confirmed the central role of antiviral cytotoxicity for TME
virus elimination in resistant C57BL/6 mice. In agreement with previous reports
simple manipulation of the Treg-compartment showed only minor effect upon the
disease course in resistant mice, though Treg-expansion exerted profound inhibitory
responses in an CD8-reduced environment. Flow cytometric analyses revealed an
86
Summary
increased percentage of Foxp3+ Treg in the blood and spleen of C57BL/6 mice
following Treg-expansion and CD8-depletion (combined treated animals) associated
with an increased CD4/CD8 ratio. Furthermore, a prolonged effect of Treg-expansion
was observed in combined treated mice, indicative of an inhibitory effect of CD8 + T
cells upon Treg. Peripheral phenotypical changes were associated with an enhanced
recruitment of lymphocytes to the brain and spinal cord of TME virus-infected
C57BL/6 mice as determined by histology. Real time polymerase chain reaction
confirmed chronic brain infection at 42 days post infection (dpi) in combined treated
C57BL/6
mice.
Immunohistochemistry revealed
a
predominent
infection of
hippocampal neurons and significant infiltrations of CD3+ T cells, CD45R+ B cells and
CD107b+ microglia/macrophages till 14 dpi in the cerebrum. Foxp3 + Treg were found
in the hippocampus also during the late phase (42 dpi), potentially attributed to
termination of inflammatory responses. In the spinal cord CD8-depletion and
combined treatment led to prolonged infection and leukomyelitis in mice.
Immunohistochemistry revealed a significant loss of myelin basic protein expression
at 42 dpi in combined treated mice. Moreover, spinal cord demyelination was
associated with an increase of β-amyloid precursor protein-postitive axons
predominetely in combined treated mice, indicative of axonal damage. Inflammatory
responses in the spinal cord white matter were dominated by CD3+ T cells and
CD107b+ macrophages/microglia, suggestive of T cell-mediated immunopathology. In
addition, significant numbers of Foxp3+ Treg and CD45R+ B cells were found in the
spinal cord white matter during the late phase in combined treated C57BL/6 mice.
In conclusion, the present project demonstrates synergistic effects of combined Tregexpansion and CD8-depletion to efficiently reduce antiviral immune responses in
TME virus-infected C57BL/6 mice, which renders them susceptible to develop
chronic infection, leukomyelitis and myelin loss. In addtion to the well described
Summary
87
inhibitory effect of Treg upon CD8+ T cells, also regulatory effects of cytotoxic cells
upon Treg or bidirectional interaction of these two T cell subsets, respectively, may
occur in infectious CNS disoders. C57BL/6 mice are widely used for genetic
modification and several transgenic mice with this genetic background have been
established for the use as animal models. Thus, findings will potentially support the
development of novel therapeutic strategies for chronic inflammatory disorders and
provide a base for future studies upon virus-induced immunopathology using
transgenic mice.
88
Zusammenfassung
89
6. Zusammenfassung
Muhammad Akram Khan
Expansion von regulatorischen T-Zellen in Theilervirus-infizierten C57BL/6Mäusen.
Bei der Multiplen Sklerose (MS) des Menschen handelt es sich um eine der
häufigsten Erkrankungen des zentralen Nervensystems (ZNS). Die experimentelle
Theilersche murine Enzephalomyelitis (TME) zeigt deutliche Parallelen zur
chronisch-progressiven Form der MS und stellt daher ein anerkanntes Tiermodell zur
Untersuchung von Entmarkungsprozessen dar. Nach einer akuten Infektionsphase
im Gehirn wird das Virus in resistenten C57BL/6-Mäusen vollständig eliminiert,
wohingegen eine unzureichende antivirale Immunantwort in empfänglichen SJLMäusen eine Viruspersistenz bedingt. Zytotoxische CD8+ T-Zellen spielen eine
entscheidende
Rolle
für
die
effiziente
antivirale
Immunität
in
resistenten
Mäusestämmen. Weiterhin zeigen verschiedene Studien einen hemmenden Einfluss
von regulatorischen T-Zellen (Treg) auf die TME Virus-spezifische Immunität bei
empfänglichen Mäusen. Allerdings ist die Bedeutung von Treg in C57BL/6-Mäusen
bei der TME bislang unklar. Ziel der Arbeit war daher den Effekt einer Expansion von
Treg mittels Interleukin-2
Immunkomplexen
und
einer antikörpervermittelten
Depletion von CD8+ T-Zellen in vivo auf den Verlauf der TME in C57BL/6-Mäusen
näher zu untersuchen und Einblicke in die Interaktion von Treg mit anderen
Leukozyten-Populationen bei chronischen neurologischen Krankheiten zu geben.
In der Studie konnte die zentrale Bedeutung von zytotoxischen T-Zellen für die
antivirale Immunität bei der TME bestätigt werden. In Übereinstimmung mit
vorherigen Berichten rief die einfache Manipulation von Treg nur marginale Effekte
bei infizierten Tieren hervor. In Kombination mit einer CD8-Depletion konnten
90
Zusammenfassung
allerdings deutliche Effekte der Treg-Expansion auf die antivirale Immunität in TME
Virus-infizierten
C57BL/6-Mäusen
nachgewiesen
werden.
Mittels
Durchflusszytometrie konnte ein erhöhter Prozentsatz Foxp3 + Treg im Blut und in der
Milz in Verbindung mit einer erhöhten CD4/CD8-Ratio in C57BL/6-Mäusen nach
CD8-Depletion und Treg-Expansion nachgewiesen werden. Die anhaltende TregExpansion durch diese kombinierte Behandlung spricht außerdem für einen
inhibitorischen Effekt von CD8+ T-Zellen auf Treg. Phänotypische Veränderungen in
der Peripherie waren mit einer verstärkten Infiltration von Leukozyten in Gehirn und
Rückenmark der Tiere vergesellschaftet. Mittels Echtzeit-Polymerase-Kettenreaktion
konnte eine chronische Infektion in Gehirnen bis zum 42. Versuchstag in kombiniert
behandelten Mäusen nachgewiesen werden. Immunhistologisch fand sich eine
signifikant erhöhte Anzahl an CD3+ T-Zellen, CD45R+ B-Zellen und CD107b+
Makrophagen/Mikroglia am 14. Versuchstag. Foxp3+ Treg konnten hingegen bis zum
Ende des Tierversuchs (42. Versuchstag) im Hippocampus von kombiniert
behandelten Mäusen nachgewiesen werden. Im Rückenmark führt die CD8Depletion sowie die kombinierte Behandlung zu einer reduzierten Viruselimination
und anhaltenden Leukomyelitis. Mittels Immunhistologie fand sich ein signifikanter
Verlust des Myelin-basischen Proteins am 42. Versuchstag in kombiniert behandelten
Tieren. Außerdem konnte eine signifikante Zunahme von degenerierten Axonen mit
akkumuliertem β-Amyloid-Vorläuferprotein in Entmarkungsherden der spinalen
weißen Substanz nachgewiesen werden. Als Ausdruck einer potentiellen Typ IVHypersensitivität konnte eine dominierende Ansammlung von CD3+ T-Zellen und
CD107b+ Makrophagen/Mikroglia im Rückenmark während der chronischen Phase
festgestellt werden. Darüber hinaus fanden sich Infiltrate von Foxp3+ Treg und
CD45R+ B-Zellen in spinalen Herden von C57BL/6-Mäusen nach kombinierter
Behandlung.
Zusammenfassung
91
Die Untersuchungen verdeutlichen das Vorhandensein von synergistischen Effekten
einer Treg-Expansion und CD8-Depletion, die zu einer effizienten Reduktion der
antiviralen Immunität in TME Virus-infizierten C57BL/6-Mäusen führen. Vergleichbar
mit empfänglichen SJL-Mäusen konnte durch das Behandlungsschema eine
chronische Infektion und Leukomyelitis mit Myelinverlust hervorgerufen werden.
Zusätzlich zu einer Hemmung von zytotoxischen T-Zellen durch Treg muss aufgrund
der Studienergebnisse außerdem ein regulatorischer Effekt von CD8 + T-Zellen auf
Treg bzw. eine bidirektionale Interaktion beider Zellpopulationen bei infektiösen ZNSKrankheiten angenommen werden. C57BL/6-Mäuse werden in der Forschung häufig
für genetische Modifikationen verwendet. Die kombinierte Behandlung ermöglicht
daher die Untersuchung von virusinduzierten immunpathologischen Prozessen bei
der TME in transgenen Mäusen mit genetischem C57BL/6-Hintergrund in
Folgeprojekten.
92
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118
Annex
119
8. Annex
8.1
Results of statistical analyses
Table 8.1.1 Clinical examination
3 dpi
TMEV
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 1.000
7 dpi
p = 1.000
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 1.000
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 1.000
42 dpi
p = 1.000
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 1.000
TMEV
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 1.000
p = 1.000
p = 1.000
p = 1.000
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 0.010↑
p = 1.000
p = 0.142
p = 0.109
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.2. Spleen weight
3 dpi
TMEV
Treg↑
TMEV
p = 0.000↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.750
Treg↑CD8↓
TMEV
p = 0.004↑
7 dpi
p
=
0.000↓
p = 0.039↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p = 1.000
TMEV
CD8↓
TMEV
p = 0.374
Treg↑CD8↓
TMEV
p = 0.532
42 dpi
p = 0.653
p = 0.707
Treg↑
TMEV
CD8↓
TMEV
p = 0.760
TMEV
Treg↑
TMEV
p = 0.068
CD8↓
TMEV
p = 0.224
Treg↑CD8↓
TMEV
p = 0.578
p
=
0.033↓
p = 0.100
p = 0.825
Treg↑
TMEV
p = 0.877
CD8↓
TMEV
p = 0.825
Treg↑CD8↓
TMEV
p = 0.900
p = 0.422
p = 0.707
p = 0.866
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
120
Annex
Table 8.1.3. Foxp3+ cells in the blood determined by flow cytometry
3 dpi
TMEV
Treg↑
TMEV
p
0.000↑
=
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p
=
0.812
p
=
0.002↓
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p = 0.142
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p
0.019↓
Treg↑
TMEV
CD8↓
TMEV
=
CD8↓
TMEV
p
=
0.517
p
=
0.050↑
TMEV
Treg↑CD8↓
TMEV
p = 0.034↑
42 dpi
p = 0.007↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
TMEV
Treg↑
TMEV
p
=
0.006↑
CD8↓
TMEV
p
=
0.035↓
p
=
0.014↑
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.286
p = 0.003↑
Treg↑
TMEV
p
=
0.003↓
CD8↓
TMEV
p
=
0.021↓
p
=
0.050↑
Treg↑CD8↓
TMEV
p = 0.156
p = 0.023↑
p = 0.570
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.4. CD4/CD8 ratio in the blood determined by flow cytometry
3 dpi
TMEV
Treg↑
TMEV
p = 0.509
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p
=
0.001↑
p
=
0.002↑
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p = 0.000↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.464
Treg↑
TMEV
p=
0.006↑
CD8↓
TMEV
p
=
0.001↑
p
=
0.014↑
TMEV
Treg↑CD8↓
TMEV
p = 0.000↑
42 dpi
p = 0.007↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.040↑
TMEV
Treg↑
TMEV
p
=
0.000↑
CD8↓
TMEV
p
=
0.001↑
p
=
0.002↑
Treg↑CD↓
TMEV
p
=
0.000↑
p
=
0.000↑
p
=
0.019↓
Treg↑
TMEV
p = 0.137
CD8↓
TMEV
p
=
0.003↑
p
=
0.014↑
Treg↑CD↓
TMEV
p
=
0.000↑
p
=
0.008↑
p
=
0.004↑
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Annex
121
Table 8.1.5. Foxp3+ cells in the spleen determined by flow cytometry
3 dpi
TMEV
Treg↑
TMEV
p=
0.000↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.135
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p = 0.002↓
p = 0.351
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p = 0.111
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.307
Treg↑CD8↓
TMEV
p = 0.758
42 dpi
p = 0.086
p = 0.394
Treg↑
TMEV
CD8↓
TMEV
p = 0.341
TMEV
Treg↑
TMEV
p
=
0.000↑
CD8↓
TMEV
p
=
0.010↓
p
=
0.002↓
Treg↑CD8↓
TMEV
p = 0.012↑
p = 0.021↓
p = 0.003↑
Treg↑
TMEV
p = 0.222
CD8↓
TMEV
p
=
0.926
p
=
0.140
Treg↑CD8↓
TMEV
p = 0.263
p = 0.023↓
p = 0.167
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.6. CD4/CD8 ratio in the spleen determined by flow cytometry
3 dpi
TMEV
Treg↑
TMEV
p
=
0.006↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p
=
0.001↑
p
=
0.002↑
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p = 0.000↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.770
Treg↑
TMEV
p = 0.426
CD8↓
TMEV
p
=
0.001↑
p
=
0.014↑
TMEV
Treg↑CD8↓
TMEV
p = 0.000↑
42 dpi
p = 0.007↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.143
TMEV
Treg↑
TMEV
p=
0.001↑
CD8↓
TMEV
p
=
0.001↑
p
=
0.002↑
Treg↑CD8↓
TMEV
p = 0.001↑
p = 0.008↑
p = 0.770
Treg↑
TMEV
p = 0.489
CD8↓
TMEV
p
=
0.001↑
p
=
0.014↑
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.008↑
p = 0.004↑
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
122
Annex
Table 8.1.7. Theilervirus RNA concentration in the brain determined by real
time polymerase chain reaction
3 dpi
TMEV
Treg↑
TMEV
p = 0.854
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.859
Treg↑CD8↓
TMEV
p = 0.288
7 dpi
p = 0.391
p = 0.131
Treg↑
TMEV
CD8↓
TMEV
p = 0.380
Treg↑
TMEV
p = 0.201
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.093
Treg↑CD8↓
TMEV
p = 0.000↑
42 dpi
p
=
0.025↑
p = 0.006↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
TMEV
Treg↑
TMEV
p = 0.124
CD8↓
TMEV
p = 0.377
Treg↑CD8↓
TMEV
p = 0.722
p
=
0.027↓
p = 0.033↓
p = 0.380
Treg↑
TMEV
p = 0.593
CD8↓
TMEV
p = 0.550
Treg↑CD8↓
TMEV
p = 0.004↑
p = 1.000
p = 0.039↑
p = 0.024↑
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.8. Amount of Theilervirus-infected cells in the brain determined by
immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.843
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.220
Treg↑CD8↓
TMEV
p = 0.575
7 dpi
p = 0.623
p = 0.593
Treg↑
TMEV
CD8↓
TMEV
p = 0.142
Treg↑
TMEV
p=
0.029↑
TMEV
CD8↓
TMEV
p = 0.811
Treg↑CD8↓
TMEV
p = 0.000↑
42 dpi
p = 0.080
p = 0.033↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.004↑
TMEV
Treg↑
TMEV
p = 0.055
CD8↓
TMEV
p
=
0.634
p
=
0.111
Treg↑CD8↓
TMEV
p = 0.133
p = 0.824
p = 0.056
Treg↑
TMEV
p
=
0.006↑
CD8↓
TMEV
p
=
1.000
p
=
0.094
Treg↑CD8↓
TMEV
p = 0.040↑
p = 0.583
p = 0.212
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Annex
123
Table 8.1.9. Severity of hippocampal inflammation in the hippocampus
determined by histology
3 dpi
TMEV
Treg↑
TMEV
p = 0.841
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.810
Treg↑CD8↓
TMEV
p = 0.537
7 dpi
p = 0.663
p = 0.751
Treg↑
TMEV
CD8↓
TMEV
p = 0.506
Treg↑
TMEV
p = 0.424
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.430
Treg↑CD8↓
TMEV
p = 0.004↑
42 dpi
p = 0.902
p = 0.088
Treg↑
TMEV
CD8↓
TMEV
p = 0.269
TMEV
Treg↑
TMEV
p = 0.260
CD8↓
TMEV
p
=
0.125
p
=
0.500
Treg↑CD8↓
TMEV
p = 0.176
p = 0.894
p = 0.508
Treg↑
TMEV
p = 0.869
CD8↓
TMEV
p
=
0.887
p
=
0.803
Treg↑CD8↓
TMEV
p = 0.044↑
p = 0.153
p = 0.187
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.10. Number of CD3+ T cells in the hippocampus determined by
immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p
=
0.032↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.859
Treg↑CD8↓
TMEV
p = 0.811
7 dpi
p = 0.006↓
p = 0.001↓
Treg↑
TMEV
CD8↓
TMEV
p = 0.661
Treg↑
TMEV
p = 0.365
TMEV
CD8↓
TMEV
p = 0.195
Treg↑CD8↓
TMEV
p = 0.034↑
42 dpi
p = 0.624
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 0.770
TMEV
Treg↑
TMEV
p
=
0.722
CD8↓
TMEV
p
=
0.277
p
=
0.164
Treg↑CD8↓
TMEV
p = 0.007↓
p = 0.024↓
p = 0.143
Treg↑
TMEV
p
=
0.589
CD8↓
TMEV
p
=
0.277
p
=
0.881
Treg↑CD8↓
TMEV
p = 0.525
p = 0.424
p = 0.569
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
124
Annex
Table 8.1.11. Number of Foxp3+ regulatory T cells in the hippocampus
determined by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.472
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.745
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p = 0.664
p = 0.016↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p = 0.263
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.041↑
Treg↑CD8↓
TMEV
p = 0.002↑
42 dpi
p = 1.000
p = 0.349
Treg↑
TMEV
CD8↓
TMEV
p = 0.272
TMEV
Treg↑
TMEV
p = 0.688
CD8↓
TMEV
p = 0.173
Treg↑CD8↓
TMEV
p = 0.346
p = 0.162
p = 0.228
p = 0.769
Treg↑
TMEV
p = 0.160
CD8↓
TMEV
p = 0.008↑
Treg↑CD8↓
TMEV
p = 0.020↑
p = 0.612
p = 0.562
p = 0.870
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.12. Number of CD45R+ B cells in the hippocampus determined by
immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p
=
0.004↓
p
0.002↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.537
=
Treg↑CD8↓
TMEV
p = 0.280
7 dpi
p = 0.035↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.088
Treg↑
TMEV
p=
0.553
TMEV
CD8↓
TMEV
p = 0.241
Treg↑CD8↓
TMEV
p = 0.023↑
42 dpi
p = 1.000
p = 0.394
Treg↑
TMEV
CD8↓
TMEV
p = 0.509
TMEV
Treg↑
TMEV
p = 0.209
CD8↓
TMEV
p
=
0.760
p
=
0.503
Treg↑CD8↓
TMEV
p = 0.284
p = 0.770
p = 0.660
Treg↑
TMEV
p = 0.126
CD8↓
TMEV
p
=
0.484
p
=
0.371
Treg↑CD8↓
TMEV
p = 0.564
p = 0.076
p = 0.315
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Annex
125
Table 8.1.13. Number of CD107b+ macrophages/microglia in the hippocampus
determined by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.605
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.145
Treg↑CD8↓
TMEV
p = 0.212
7 dpi
p = 0.547
p = 0.112
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p = 0.424
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.086
Treg↑CD8↓
TMEV
p = 0.000↑
42 dpi
p = 0.902
p = 0.234
Treg↑
TMEV
CD8↓
TMEV
p = 0.079
TMEV
Treg↑
TMEV
p = 0.457
CD8↓
TMEV
p
=
0.165
p
=
0.124
Treg↑CD8↓
TMEV
p = 0.184
p = 0.059
p = 0.883
Treg↑
TMEV
p = 0.704
CD8↓
TMEV
p
=
0.281
p
=
0.772
Treg↑CD8↓
TMEV
p = 0.211
p = 0.565
p = 0.774
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.14. Amount of Theilervirus-infected cells in the spinal cord
determined by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.933
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.088
Treg↑CD8↓
TMEV
p = 0.362
7 dpi
p = 0.255
p = 0.371
Treg↑
TMEV
CD8↓
TMEV
p = 0.063
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p
=
0.015↑
p = 0.180
TMEV
Treg↑CD8↓
TMEV
p = 0.001↑
42 dpi
p = 0.058
Treg↑
TMEV
CD8↓
TMEV
p = 0.394
TMEV
Treg↑
TMEV
p = 0.231
CD8↓
TMEV
p
=
0.189
p
=
0.770
Treg↑CD8↓
TMEV
p = 0.256
p = 0.101
p = 0.063
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p
=
0.094
p
=
0.371
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.039↑
p = 0.056
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
126
Annex
Table 8.1.15. Severity of spinal cord inflammation determined by histology
3 dpi
TMEV
Treg↑
TMEV
p = 0.480
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.617
Treg↑CD8↓
TMEV
p = 0.494
7 dpi
p = 1.000
p = 0.264
Treg↑
TMEV
CD8↓
TMEV
p = 0.429
Treg↑
TMEV
p = 0.170
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.211
Treg↑CD8↓
TMEV
p = 0.007↑
42 dpi
p = 0.899
p = 0.217
Treg↑
TMEV
CD8↓
TMEV
p = 0.499
TMEV
Treg↑
TMEV
p
=
0.677
CD8↓
TMEV
p
=
0.750
p
=
1.000
Treg↑CD8↓
TMEV
p = 0.435
p = 0.680
p = 0.726
Treg↑
TMEV
p
=
1.000
CD8↓
TMEV
p
=
0.001↑
p
=
0.016↑
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.010↑
p = 0.204
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.16. Quantification of myelin basic protein expression in spinal cord
by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 1.000
7 dpi
p = 1.000
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 1.000
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p
=
0.015↑
p = 0.180
TMEV
Treg↑CD8↓
TMEV
p = 1.000
42 dpi
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 0.063
TMEV
Treg↑
TMEV
p
=
1.000
CD8↓
TMEV
p
=
1.000
p
=
1.000
Treg↑CD8↓
TMEV
p = 1.000
p = 1.000
p = 1.000
Treg↑
TMEV
p
=
0.593
CD8↓
TMEV
p
=
0.014↑
p
=
0.089
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.017↑
p = 0.462
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Annex
127
Table 8.1.17. Quantification of axonal β-amyloid precursor protein expression
in the spinal cord by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 1.000
7 dpi
p = 1.000
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
p = 1.000
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 1.000
Treg↑CD8↓
TMEV
p = 0.186
42 dpi
p = 1.000
p = 0.480
Treg↑
TMEV
CD8↓
TMEV
p = 0.429
TMEV
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p
=
1.000
p
=
1.000
Treg↑CD8↓
TMEV
p = 1.000
p = 1.000
p = 1.000
Treg↑
TMEV
p = 1.000
CD8↓
TMEV
p
=
1.000
p
=
1.000
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.007↑
p = 0.003↑
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.18. Number of CD3+ T cells in the spinal cord determined by
immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.506
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.859
Treg↑CD8↓
TMEV
p = 0.006↑
7 dpi
p = 0.540
p = 0.168
Treg↑
TMEV
CD8↓
TMEV
p = 0.048↑
Treg↑
TMEV
p = 0.671
TMEV
CD8↓
TMEV
p = 0.781
Treg↑CD8↓
TMEV
p = 0.014↑
42 dpi
p = 0.712
p = 0.050↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.306
TMEV
Treg↑
TMEV
p
=
0.039↓
CD8↓
TMEV
p
=
0.057
p
=
0.390
Treg↑CD8↓
TMEV
p = 0.004↓
p = 0.143
p = 0.569
Treg↑
TMEV
p
=
0.201
CD8↓
TMEV
p
=
0.002↑
p
=
0.014↑
Treg↑CD8↓
TMEV
p = 0.011↑
p = 0.131
p = 0.808
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
128
Annex
Table 8.1.19. Number of Foxp3+ regulatory T cells in the spinal cord determined
by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
P
=
0.001↑
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
CD8↓
TMEV
p = 0.360
Treg↑CD8↓
TMEV
p = 0.000↑
7 dpi
p
=
0.027↓
p = 0.056
Treg↑
TMEV
CD8↓
TMEV
p = 0.003↑
Treg↑
TMEV
p = 0.788
Treg↑
TMEV
CD8↓
TMEV
TMEV
CD8↓
TMEV
p = 0.510
Treg↑CD8↓
TMEV
p = 0.003↑
42 dpi
p = 0.455
p = 0.017↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.379
TMEV
Treg↑
TMEV
p
=
0.048↑
CD8↓
TMEV
p
=
0.943
p
=
0.138
Treg↑CD8↓
TMEV
p = 0.872
p = 0.064
p = 0.877
Treg↑
TMEV
p
=
0.281
CD8↓
TMEV
p
=
0.007↑
p
=
0.076
Treg↑CD8↓
TMEV
p = 0.010↑
p = 0.122
p = 0.459
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Table 8.1.20. Number of CD45R+ B cells in the spinal cord determined by
immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 0.079
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p = 0.552
Treg↑CD8↓
TMEV
p = 0.937
7 dpi
p = 0.447
p = 0.025↓
Treg↑
TMEV
CD8↓
TMEV
p = 0.548
Treg↑
TMEV
p = 0.657
TMEV
CD8↓
TMEV
p = 0.178
Treg↑CD8↓
TMEV
p = 0.025↑
42 dpi
p = 0.539
p = 0.088
Treg↑
TMEV
CD8↓
TMEV
p = 0.509
TMEV
Treg↑
TMEV
p
=
0.669
CD8↓
TMEV
p = 0.101
Treg↑CD8↓
TMEV
p = 0.007↓
p = 0.334
p = 0.143
p = 0.718
Treg↑
TMEV
p
=
0.790
CD8↓
TMEV
p = 0.550
Treg↑CD8↓
TMEV
p = 0.016↑
p = 0.752
p = 0.053
p = 0.150
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
Annex
129
Table 8.1.21. Number of CD107b+ microglia/macrophages in the spinal cord
determined by immunohistochemistry
3 dpi
TMEV
Treg↑
TMEV
p = 1.000
Treg↑
TMEV
CD8↓
TMEV
14 dpi
TMEV
Treg↑
TMEV
CD8↓
TMEV
CD8↓
TMEV
p
=
0.000↑
p
=
0.009↑
Treg↑CD8↓
TMEV
p = 0.099
7 dpi
p = 0.232
Treg↑
TMEV
CD8↓
TMEV
p = 0.146
Treg↑
TMEV
p = 0.437
TMEV
CD8↓
TMEV
p = 0.771
Treg↑CD8↓
TMEV
p = 0.007↑
42 dpi
p = 0.371
p = 0.029↑
Treg↑
TMEV
CD8↓
TMEV
p = 0.045↑
TMEV
Treg↑
TMEV
p
=
0.881
CD8↓
TMEV
p
=
0.699
p
=
0.637
Treg↑CD8↓
TMEV
p = 0.970
p = 0.904
p = 0.749
Treg↑
TMEV
p
=
1.000
CD8↓
TMEV
p
=
0.002↑
p
=
0.081
Treg↑CD8↓
TMEV
p = 0.000↑
p = 0.039↑
p = 0.246
Bold values display significant changes (p 0.05), ↑ = value in row significantly increased compared to value in column; ↓ = value
in row significantly decreased compared to value in column; dpi = days post infection; TMEV = Theiler`s murine
encephalomyelitis; Treg↑ = expansion of regulatory T cells; CD8↓ = depletion of CD8+ T cells
130
8.2
Annex
Materials used for animal infection
Theiler’s murine encephalitis virus BeAn strain II, passage 3, date: 03-11-11
Aesculap AG &Co KG, Tuttlingen, Germany
Scissors, BC603-R
Bayer Vital GmbH, Leverkusen, Germany
Bepanthen, 4145A
Hamilton, Bonaduz AG, Switzerland
Microliter syringe, 50ul, model 705
PAA laboratories GmbH, Pasching, Austria
Dulbecco’s eagle medium with L-Glutamin, E15-810
FCS, A15-101
Systec GmbH, Wettenberg, Germany
Autoclave, 3850 ELC
Sigma Aldrich, USA
Gentamicin, G1397
Terumo Europe N.V, Leuven, Belgium
Needles, 23 gauge
Tecniplast Germany GmbH, Hohenpeißenberg, Germany
Stainless steel frame Size: P, type 4541P, 4P02B700
Annex
131
Cage cup from Noryl, 4541P009
Kotschale from Noryl, 4501K389
Stainless Steel Feeder, 4401S960
Second level of Noryl, 4541P610
Water bottles 750 ml, ACBT0752
Potions flap with 30 ° curved nipple, ACCPS1246
Vertical stainless steel bottle holder, ACSAV309
8.3
Reagents, chemicals and antibodies
AbD Serotec, Oxford, UK
Rat Anti Mouse CD107b: Biotin, MCA2293B
BD Biosciences, Heidelberg, Germany
Biotin Rat Anti-Mouse CD45R/B220, 553085
BioXCell, West lebanon
Anti-CD8 antibody, Clone 53-6.72,
Carl Roth GmbH
Chloral hydrate, K318.2
Citric acid monohydrate, p.a., catalog no. 3958.1
Acetate, 4600.4
Ethanol, K928.2
2-Propanol, 9866.2
Nitrumchloride, P029.2
Nitriumchloridihydrogenphosphate-monohydrate, K300.2
Roticlear® , A5381
132
Rotilabo®- aluminium foil disk, mod. R80, E723.1
Roti® Histo kit II, catalog no. T160.2
Hydrogen peroxide, 8070.1
Chemicon International Inc, Temecula, CA, USA
Goat anti mouse, axonal damage (β-APP), MAB348
Rabbit anti-myelin basic protein (MBP) polyclonal, AB980
Dako Cytomation, Hamburg, Germany
Polyclonal rabbit, anti-CD3, A0452
Rabbit Anti-Cow Glial Fibrillary Acidic Protein (GFAP), Z0334
eBioscience, Inc., San Diego, CA, USA
Anti-mouse IL-2 functional grade purified, 16-7022, Clone JES6-1A12
Mouse IL-2 recombinant protein carrier-free, 34-8021
Fluka
Diminobenzidin-tetrahydrochloride, 32750
Harlan, Borchen, Germany
Female, 4 weeks old C57BL/6 mice.
Merck
Hematoxylin, 4305
Potassium aluminum sulfate 12-hydrate, P724.1
Sodium iodate, 6525
Merck Millipore,
Anti-myelin basic protein, Ab980
Annex
Annex
Menzel GmbH
Super Frost ® plus, J1800AMN2
Natu Tec, Frankfurt, Germany
Monoclonal rat Foxp3-specific antibody
Riedel de Haën, Seelze, Hannover, Germany
Potassium chloride, 911306
Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany
Rabbit Serum, R4505
Vector Laboratories, Burlingame
Biotinylated goat-anti-rabbit IgG
Vectastain Elite ABC Kit, PK-6100
Goat anti-rabbit biotinylated , BA-1000
8.3.1 Solutions for immunohistochemistry
3.3'-Diaminobenzidine tetrahydrochlorid solution (DAB)
Prepare shortly before use
0.1 g 3.3'-diaminobenzidine tetra hydrochloride
200 ml PBS
200 μl 30% hydrogen peroxide
1 M sodium hydroxide
40 g sodium hydroxide
1000 ml distilled water
Phosphate buffered saline (PBS)
133
134
40 g sodium chloride
8.97 g sodium hydrogen phosphate
5000 ml distilled water
With 1 M sodium hydroxide adjust pH value at 7.1
Citrate buffer, pH 6.0
2.1 g of citric acid monohydrate
1000 ml distilled water
With 1 M sodium hydroxide adjust the pH value at 6.0
8.3.4 Equipment and disposable items
B-BRAUN, AESCULAP AG/Germany
Surgical Blades, 4505155988
Syringes, Omnican® , 9161502
Carl Zeiss AG, Göttingen, Germany
Transmitted-light microscope
Carl Roth Gmbh+ CO.KG, Karlsruhe, Germany
Rotiprotect® Nitril glaves, catalog no. P777.1
Data weighing systems Inc., Elk Grove, USA
Precision balance Sartorius portable PT210
Gerhard Menzel Glasbearbeitungswerk GmbH & Co. KG, Braunschweig,
Germany
Super Frost® Plus microscopic slides, catalog no. 041300
Annex
Annex
Heraeus SEPATECH, Hannover, Germany
Centrifuge machine, 25-012228
Jürgens, Bremen, Germany
Magnetic stirrer Ika-Combimag RTC
Kendro Laboratory Products GmbH (formerly Heraeus), Langenselbold,
Germany
Heraeus drying oven UT 6
Leica Microsystems Nussloch GmbH, Nussloch, Germany
Automatic slide stainer, Leica ST 4040
Microtome, Leica RM2035
Medite Medizintechnik, Burgdorf, Germany
Automatic cover slipper, Promounter RCM 2000
Nerbe plus. GmbH Winsen/Luhe, Germany
Waste begs, 09.302-0020
Olympus Optical Co. (Europe) GmbH, Hamburg, Deutschland
Olympus BX-51 digital Microscope
SAKURA Finetek, Tokyo, Japan, Co, Ltd
Tissue-Tek , 4583
135
136
Thermo Electron Ltd., Dreieich, Germany
Shandon Coverplates®, catalog no. 72110013
Shandon Sequenza® Slide Racks catalog no. 7331017
W. Knittel Glasbearbeitungs GmbH, Braunschweig, Germany
Cover glass (24 x 50 mm)
GraphPad Software, Inc., La Jolla, USA
GraphPad Prism® Software Version 5.04
IBM, New York, USA
IBM® SPSS® Statistics Version 17
8.3.3 Materials used for RT-qPCR
Agilent Technologies, GB, UK
Tubes and lids, 401425
Agilent Technologies, Waldbronn, Germany
Stratagen Mx3005P qPCR system
BRAND, Germany
PCR-Tubes, 0.5ml, green, 781B13
B.D Bioscience, USA
Annex
Annex
BD FalconTM100um cell strainer, 352360
Brilliant III SYBER® MM, Waldbronn, Germany
Sybr Green, 600882
Reference dye, 1mM, 600530-53
Carl Roth Gmbh+ CO.KG, Karlsruhe, Germany
Ethanol 70% for RNA, 9065.2
Chloroform 99%, 3313.1
Eppendorf, AG, Hamburg, Germany
20ul Pipett, 00300733002
50-1000ul Pipett, 0030073304
200ul Pipett, 0030073266
0.5ml tubes, 0030121023
MVGH Biotech, AG, Ebersberg, GermanyNerbe plus. GmbH Winsen/Luhe,
Germany
Tips 10µl, 100µl, 1000µl
QIAGEN SCIENCES, Maryland, USA
QIAZOL Lysis reagent, 79306
QIAGEN GmbH, Hilden, Germany
RNeasy Mini kit 250, 74106
137
138
Annex
South Laboratory GmbH, Gauting, Germany
PCR Tissue Homogenizing kit, Omni TH220 homogenizer
Interchangeable tips, Omni Tip® Standard
8.5
Materials used for flow cytometry
BioXcell
Fcy-Block 2.4G2, (603-298-8564); 1 mg/ml in PBS 0.02% NaN3
BioLegend
Antibody aCD8 – APC, Clone SK1
BIOCHROM AG, Berlin, Germany
10 % FCS (50 ml), S 0415
eBioscience, Inc., San Diego, CA, USA
Foxp 3 Staining Buffer Set, 00-5523-00
Antibody, Foxp3 – PE, Clone FJK-16s
BD Horizont
Antibody aCD4 – HV450, Clone RPA-T4
Gibco, Darmstadt, Germany
Live-Dead-Staining
LIVE/DEAD Fixable Blue Dead Cell Stain Kit L23105
RPMI Medium 1640 (1x) (+ L-Glutamine), 500 ml
50 U/ml or 0.05mg/ml Pen/Strep (5 ml)
PBS pH 7.4 (1x), 500 ml
PBS/BSA 0.2% (10 ml 10% BSA in PBS)
Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany
Annex
Albumin from bovine serum (BSA), SIGMA-ALDRICH, SIGMA life Science
Cell count:
Dilution of the sample with TRYPAN BLUE SOLUTION (0.4%)
Trypan blue : PBS (1+2)
Counting chamber: Neubauer Improved
Jackson Immuno Research
Fixation/Permeabilization
Chrom Pure Rat IgG, whole molecule, 012-000-003
Erythrocyte-Lysing Buffer (ACK-Buffer)
0,01 M KHCO3
0,155 M NH4Cl
0,1 mM EDTA
pH auf 7,5 einstellen
Cytometer:
BD LSR-II SORP
Evaluation: FlowJo Data analyses soft ware
139
140
Acknowledgements
9. Acknowledgements
Finally, I would like to thank those people who supported and contributed
considerably in the completion of this PhD project.
Prof. Dr. Wolfgang Baumgärtner for providing me an opportunity to work under his
supervision and for his substantial support, invaluable advice and being a motivation
for me.
I would like to express my deepest gratitude to Prof. Dr. Andreas Beineke for his
open and friendly attitude and always willing to listen to my problems. Without his
consistent and illuminating instructions, this project would not have reached its
present form.
My profound regards are also for Dr. Vanessa Herder for her continuous support
and guidance. She always kept her door open with a friendly and helping attitude.
I am indebted to pay special thanks to Prof. Dr. Jochen Hühn and Dr. Rene Teich
from the Helmholtz Center for Infection Research, Braunschweig, for their
enthusiastic attitude and fruitful collaboration.
I would also like to thank the Department of Parasitology for their help and
permission to work with their laboratory equipment.
Special thanks to Bettina Buck, Petra Grünig, Kerstin Schöne and Caroline Schütz
from our department and special thanks to Kirsten Löhr from the Helmholtz Center for
Infection Research, Braunschweig for excellent technical support.
Many thanks to my colleagues Ann-Kathrin Uhde, Malgorzata Ciurkiewicz, Annika
Lehmbecker, Vanessa Pfankuche and Aimara Bello for always being cooperative
with me. I am thankful to Mr. Nadeem for constantly helping me with food from the
start till end of my work. Countless thanks to my friends Yimin Wang, Ning Zhang, Lin
Li and Cut Dahlia Iskandar for their nice company, suggestions and advices.
Acknowledgements
141
Numerous thanks to my brother Muhammad Imran Rahim and Touseef-ur-Rehman
who always stood with me since our bachelor studies until present.
I would like to say thanks to my family, my mother and my wife for their unconditional
support and encouragement during my studies.Thanks to my sweet son Muhammad
Zarar Ali Khan Niazi - I am really sorry that I missed your childhood.
Last but not the least, my father I am proud of you, we have done this.
Hannover 2015
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH
35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: [email protected] · Internet: www.dvg.de
MUHAMMAD AKRAM KHAN
ISBN 978-3-86345-246-9
Institut für Pathologie
Stiftung Tierärztliche Hochschule Hannover

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